Modulation of monocyte responses by progressive multiple sclerosis therapy.

<p>Multiple sclerosis (MS) is an immune-mediated neurodegenerative disorder that is distinguished by neuroinflammation and demyelination. MS is severely debilitating and remains the most common cause of disability arising from non-traumatic brain and CNS damage in adults. In its progressive phase there are no effective treatments, so new therapy options are an urgent research priority. Extensive work has been done on the role of the adaptive immune system in contributing to the disease pathology and on the effects of therapies targeting lymphocytes in relapsing-remitting MS. Fewer studies have examined innate immune cells in people with progressive MS. This thesis addresses that gap by profiling monocyte phenotype and function in response to new and repurposed drugs that may provide benefit in progressive MS. This was achieved by modelling the drugs’ effects in vitro using peripheral blood cells from people with progressive MS and healthy subjects. Clozapine is an atypical antipsychotic with broad receptor affinity that is primarily used to treat refractory schizophrenia. In addition to is antipsychotic action through dopamine receptor (DR) D2, its broad neuro-immune receptor affinity is thought to dampen inflammatory responses in the CNS. This thesis highlights clozapine’s anti-inflammatory effect by demonstrating a reduction in the expression of pro-inflammatory cytokines that are associated with MS pathology in treated monocytes. Clozapine also induced a significant increase in the expression of D1. We observed that D1 expression changes happened alongside alterations to immune cell activity and that MS participant monocytes were much more susceptible to DR expression changes compared to healthy people. Together this data substantiates clozapine as a potential treatment for progressive disease. MIS416 is a large, non-soluble microparticle suspension that induces nuclear factor kappa B (NFB) dependent cytokine induction. We show here that monocytes are key cytokine responder cells to MIS416 and explore the molecular mechanism by demonstrating its effects on transcription factor activity. Our data showing increased production of cytokines by MIS416 suggests a route of treatment efficacy through tolerisation mechanisms, and by reducing inflammation through upregulation of anti-inflammatory cytokines and negative feedback from pro-inflammatory cytokine release. Furthermore, we demonstrate how disease heterogeneity, phenotype, and genotype could significantly affect drug response outcomes in patients who received the drug as part of a phase 2 clinical trial. Much of this work was done using new spectral cytometer technology. Its use allowed for the novel approach that enabled the subtraction of autofluorescent noise from out data, and we demonstrate its efficient functioning, ease of use, and utility in acquiring high dimensional datasets. The resulting large dataset allowed us the opportunity to interrogate it using bioinformatics tools, and we show their utility as adjunct tools to conventional methods of gating and statistical analysis. These analyses help demonstrate that monocytes are a heterogenous immune cell subset that is functionally distinct in people with progressive MS when compared to monocytes from healthy individuals.</p>

Modulation of monocyte responses by progressive multiple sclerosis therapy.

<p>Multiple sclerosis (MS) is an immune-mediated neurodegenerative disorder that is distinguished by neuroinflammation and demyelination. MS is severely debilitating and remains the most common cause of disability arising from non-traumatic brain and CNS damage in adults. In its progressive phase there are no effective treatments, so new therapy options are an urgent research priority. Extensive work has been done on the role of the adaptive immune system in contributing to the disease pathology and on the effects of therapies targeting lymphocytes in relapsing-remitting MS. Fewer studies have examined innate immune cells in people with progressive MS. This thesis addresses that gap by profiling monocyte phenotype and function in response to new and repurposed drugs that may provide benefit in progressive MS. This was achieved by modelling the drugs’ effects in vitro using peripheral blood cells from people with progressive MS and healthy subjects. Clozapine is an atypical antipsychotic with broad receptor affinity that is primarily used to treat refractory schizophrenia. In addition to is antipsychotic action through dopamine receptor (DR) D2, its broad neuro-immune receptor affinity is thought to dampen inflammatory responses in the CNS. This thesis highlights clozapine’s anti-inflammatory effect by demonstrating a reduction in the expression of pro-inflammatory cytokines that are associated with MS pathology in treated monocytes. Clozapine also induced a significant increase in the expression of D1. We observed that D1 expression changes happened alongside alterations to immune cell activity and that MS participant monocytes were much more susceptible to DR expression changes compared to healthy people. Together this data substantiates clozapine as a potential treatment for progressive disease. MIS416 is a large, non-soluble microparticle suspension that induces nuclear factor kappa B (NFB) dependent cytokine induction. We show here that monocytes are key cytokine responder cells to MIS416 and explore the molecular mechanism by demonstrating its effects on transcription factor activity. Our data showing increased production of cytokines by MIS416 suggests a route of treatment efficacy through tolerisation mechanisms, and by reducing inflammation through upregulation of anti-inflammatory cytokines and negative feedback from pro-inflammatory cytokine release. Furthermore, we demonstrate how disease heterogeneity, phenotype, and genotype could significantly affect drug response outcomes in patients who received the drug as part of a phase 2 clinical trial. Much of this work was done using new spectral cytometer technology. Its use allowed for the novel approach that enabled the subtraction of autofluorescent noise from out data, and we demonstrate its efficient functioning, ease of use, and utility in acquiring high dimensional datasets. The resulting large dataset allowed us the opportunity to interrogate it using bioinformatics tools, and we show their utility as adjunct tools to conventional methods of gating and statistical analysis. These analyses help demonstrate that monocytes are a heterogenous immune cell subset that is functionally distinct in people with progressive MS when compared to monocytes from healthy individuals.</p>

The Impact of ACE2 Polymorphisms on COVID-19 Disease: Susceptibility, Severity, and Therapy

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has currently spread worldwide, leading to high morbidity and mortality. As the putative receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2) is widely distributed in various tissues and organs of the human body. Simultaneously, ACE2 acts as the physiological counterbalance of ACE providing homeostatic regulation of circulating angiotensin II levels. Given that some ACE2 variants are known to cause an increase in the ligand-receptor affinity, their roles in acquisition, progression and severity of COVID-19 disease have aroused widespread concerns. Therefore, we summarized the latest literature and explored how ACE2 variants and epigenetic factors influence an individual’s susceptibility to SARS-CoV-2 infection and disease outcome in aspects of ethnicity, gender and age. Meanwhile, the possible mechanisms for these phenomena were discussed. Notably, recombinant human ACE2 and ACE2-derived peptides may have special benefits for combating SARS-CoV-2 variants and further studies are warranted to confirm their effects in later stages of the disease process. As the uncertainty regarding the severity and transmissibility of disease rises, a more in-depth understanding of the host genetics and functional characteristics of ACE2 variants will not only help explain individual clinical differences of the disease, but also contribute to providing effective measures to develop solutions and manage future outbreaks of SARS-CoV-2.

Synthesis, Characterization and In Vitro Evaluation of Hybrid Monomeric Peptides Suited for Multimodal Imaging by PET/OI: Extending the Concept of Charge–Cell Binding Correlation

In the context of hybrid multimodal imaging agents for gastrin releasing peptide receptor (GRPR) targeting, a correlation between the net charge and the receptor affinity of the agents was recently found. In particular, a decrease in in vitro GRPR binding affinity was observed in case of an increasing number of negative charges for dually labeled GRPR–specific peptide dimers suited for positron emission tomography and optical imaging (PET/OI). This adverse influence of anionic charges could be in part compensated by a higher valency of peptide multimerization. However, it remains unknown whether this adverse effect of anionic charges is limited to peptide multimers or if it is also found or even more pronounced when GRPR–specific peptide monomers are dually labeled with fluorescent dye and chelating agent/radionuclide. Moreover, it would be important to know if this effect is limited to GRPR–specific agents only or if these observations also apply to other dually labeled peptides binding to other receptor types. To address these questions, we synthesized hybrid labels, comprising a chelator, different fluorescent dyes carrying different net charges and a functional group for bioconjugation and introduced them into different peptides, specifically targeting the GRPR, the melanocortin–1 receptor (MC1R) and integrin αvβ3. The synthesized conjugates were evaluated with regard to their chemical, radiochemical, photophysical and receptor affinity properties. It was found that neither the 68Ga–radiolabeling nor the fluorescence characteristics of the dyes were altered by the conjugation of the MIUs to the peptides. Further, it was confirmed that the net number of anionic charges has a negative effect on the GRPR–binding affinity of the GRPR–targeting MIU–peptide monomer conjugates and that this same effect was also found to the same extent for the other receptor systems studied.

SARS-CoV-2 susceptibility and ACE2 gene variations within diverse ethnic backgrounds

Background Host genetics play a major role in COVID-19 susceptibility and severity. Here, we analyse an ethnically diverse cohort of National Health Service (NHS) patients in the United Kingdom (UK) to assess the association between variants in the ACE2 locus and COVID-19 risk. Methods We analysed whole-genome sequencing (WGS) data of 6,274 participants who were tested for SARS-CoV-2 from the UK's 100,000 Genomes Project (100KGP) for the presence of ACE2 variants and expression quantitative trait loci (eQTLs). Findings We identified a splice site variant (rs2285666) associated with increased ACE2 expression with an overrepresentation in SARS-CoV-2 positive patients relative to 100KGP controls (p = .015), and in hospitalised European patients relative to outpatients in intra-ethnic comparisons (p = .029). We also compared the prevalence of 288 eQTLs, of which 23 were enriched in SARS-CoV-2 positive patients. The eQTL rs12006793 had the largest effect size (d = 0.91), which decreases ACE2 expression and is more prevalent in controls, thus potentially reducing risk of COVID-19. We identified three novel nonsynonymous variants predicted to alter ACE2 function, and showed that three variants (p.K26R, p.H378R, p.Y515N) alter receptor affinity for the viral Spike (S) protein. Variants p.K26R and p.N720D are more prevalent in the European population (p < .001), but Y497H is less prevalent compared to East Asians (p = .020). Interpretation Our results demonstrate that the spectrum of genetic variants in ACE2 may inform risk stratification of COVID-19 patients and could partially explain the differences in disease susceptibility and severity among different ethnic groups. Funding The 100KGP is funded by the National Institute for Health Research and NHS England. Funding was also obtained from Stanford University, Palo Alto.

A new class of 5-HT1A receptor antagonists with procognitive and antidepressant properties

Aims: 5-HT1A receptor antagonists constitute a potential group of drugs in the treatment of CNS diseases. The aim of this study was to search for new procognitive and antidepressant drugs among amide derivatives of aminoalkanoic acids with 5-HT1A receptor antagonistic properties. Materials & methods: Thirty-three amides were designed and evaluated in silico for their drug-likeness. The synthesized compounds were tested in vitro for their 5-HT1A receptor affinity and functional profile. Moreover, their selectivity over 5-HT7, 5-HT2A and D2 receptors and ability to inhibit phosphodiesterases were evaluated. Results: A selected 5-HT1A receptor antagonist 20 ( Ki = 35 nM, Kb = 4.9 nM) showed procognitive and antidepressant activity in vivo. Conclusion: Novel 5-HT1A receptor antagonists were discovered and shown as potential psychotropic drugs.

E484K and N501Y SARS-CoV 2 Spike Mutants Increase ACE2 Recognition but Reduce Affinity for Neutralizing Antibody

SARS-CoV2 mutants emerge as variants of concern (VOC) due to altered selection pressure and rapid replication kinetics. Among them, lineages B.1.1.7, B.1.351, and P.1 contain a key mutation N501Y. B.1.135 and P.1 lineages have another mutation, E484K. Here, we decode the effect of these two mutations on the host receptor, ACE2, and neutralizing antibody (B38) recognition. The gain in binding affinity for the N501Y RBD mutant to the ACE2 is attributed to improved π-π stacking and π-cation interactions. The enhanced receptor affinity of the E484K mutant is caused due to the formation of a specific hydrogen bond and salt-bridge interaction with Glu75 of ACE2. Notably, both the mutations reduce the binding affinity for B38 due to the loss of several hydrogen-bonding interactions. The insights obtained from the study are crucial to interpret the increased transmissibility and reduction in the neutralization efficacy of rapidly emerging SARS-CoV2 VOCs.