Innate and adaptive immunity during SARS-CoV-2 infection: Biomolecular cellular markers and mechanisms

The coronavirus 2019 (COVID-19) pandemic was caused by a positive sense single-stranded RNA (ssRNA) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, other human coronaviruses (hCoVs) exist. Historical pandemics include smallpox and influenza, with efficacious therapeutics utilized to reduce overall disease burden through effectively targeting a competent host immune system response. The immune system is composed of primary/secondary lymphoid structures with initially eight types of immune cell types, and many other subtypes, traversing cell membranes utilizing cell signaling cascades that contribute towards clearance of pathogenic proteins. Other proteins discussed include cluster of differentiation (CD) markers, major histocompatibility complexes (MHC), pleiotropic interleukins (IL), and chemokines (CXC). The historical concepts of host immunity are the innate and adaptive immune systems. The adaptive immune system is represented by T cells, B cells, and antibodies. The innate immune system is represented by macrophages, neutrophils, dendritic cells, and the complement system. Other viruses can affect and regulate cell cycle progression for example, in cancers that include human papillomavirus (HPV: cervical carcinoma), Epstein-Barr virus (EBV: lymphoma), Hepatitis B and C (HB/HC: hepatocellular carcinoma) and human T cell Leukemia Virus-1 (T cell leukemia). Bacterial infections also increase the risk of developing cancer (e.g., Helicobacter pylori). Viral and bacterial factors can cause both morbidity and mortality alongside being transmitted within clinical and community settings through affecting a host immune response. Therefore, it is appropriate to contextualize advances in single cell sequencing in conjunction with other laboratory techniques allowing insights into immune cell characterization. These developments offer improved clarity and understanding that overlap with autoimmune conditions that could be affected by innate B cells (B1(+) or marginal zone cells) or adaptive T cell responses to SARS-CoV-2 infection and other pathologies. Thus, this review starts with an introduction into host respiratory infection before examining invaluable cellular messenger proteins and then individual immune cell markers.Biochem123 Ltd

Bruton’s tyrosine kinase inhibitors impair FcγRIIa-mediated platelet responses to bacteria in chronic lymphocytic leukaemia

Ibrutinib is highly effective in the treatment of chronic lymphocytic leukaemia (CLL), disrupting B cell receptor signalling through the inhibition of Bruton’s tyrosine kinase (Btk). However, ibrutinib has off-target effects, inhibiting platelet aggregation with associated haemorrhage. More recently, second generation Btk inhibitors have been developed to be more specific for Btk, including acalabrutinib. Still, acalabrutinib is similarly associated with a bleeding risk. Treatment of CLL with ibrutinib is also connected to an increased infection risk.Though platelets are known for their role in haemostasis, they additionally play a key role in innate immunity. Platelets can interact with bacteria through a variety of mechanisms, with one such mechanism being the FcγRIIa receptor, a low affinity IgG immune receptor that can mediate platelet aggregation, phagocytosis, and release of bactericidal substances. Btk, and potentially other Tec family kinases, is a key component of the FcγRIIa intracellular signalling pathway. However, the role of these kinases in FcγRIIa signalling in response to bacteria is unknown. Moreover, the effects of iBtks on platelet responses to bacteria have not been evaluated. This study aims to investigate the role of Btk in platelet FcγRIIa signalling in response to bacterial agonists, and how this is affected by ibrutinib.We show that ibrutinib and acalabrutinib inhibit healthy donor FcγRIIa-mediated platelet aggregation, alpha and dense granule release, in response to incubation with Staphylococcus aureus and Escherichia coli, and in response to FcγRIIa crosslinking with the anti-FcγRIIa monoclonal antibody IV.3. Moreover, we show platelets derived from ibrutinib-untreated CLL patients aggregate normally to bacteria in the presence of autologous plasma. However, platelets from ibrutinib-treated CLL patients have significantly inhibited aggregation, alpha granule release and bacteria scavenging. Platelet surface levels of FcγRIIa remained unchanged in both CLL groups, compared to healthy controls, however, levels of GPVI and αIIbβ3 were decreased in CLL samples regardless of ibrutinib therapy.In both healthy control and ibrutinib-untreated CLL platelets, phosphorylation of Btk at tyrosine 223 (a marker of Btk activation) was detected in response to FcγRIIa agonists including Staphylococcus aureus and Escherichia coli. In contrast, Btk phosphorylation at Y223 in response to bacteria was absent in ibrutinib-treated CLL platelets, and in healthy controls platelets inhibited by ibrutinib and acalabrutinib in vitro. We also show a significant decrease in Tec phosphorylation in healthy control platelets treated with ibrutinib, and in ibrutinibtreated CLL platelets. To determine if Btk was required for platelet bacteria responses, X-linked agammaglobulinaemia platelets, which lack a functional Btk, were exposed to bacteria and FcγRIIa-mediated platelet aggregation was observed, showing Btk to be unessential.To conclude, our data shows that iBtks impair FcγRIIa-mediated platelet responses to bacteria both in vitro and at therapeutic conditions, and this impairment may be a result of both Btk and Tec inhibition, though potential off-target effects on other kinases cannot be dismissed. The effect of iBtks on platelet immune responses may possibly contribute to increase infections observed in CLL

Therapeutic IgY: Safe, Diverse, And Effective For Use Against Viral Targets

Passive antibody treatments are used to target infectious disease, toxins, venoms, and cancer antigens. Recently, there has been an increased interest in the use of avian-derived antibody treatments such as IgY. IgY is the primary serum antibody isotype present in the avian system, and IgY treatments have already been demonstrated to be effective against a variety of bacterial and viral infectious agents. There are two forms of IgY expressed in anseriformes birds, a full length IgY that is functionally similar to mammalian IgG, and an alternatively spliced IgY, IgY(ÃFc), that is comparable to the mammalian F(ab\u27)2 fragment. The difference in structure between IgY and mammalian IgG, prevents IgY from interacting with mammalian Fc receptors, complement, and other inflammatory factors. The phylogenetic distance between mammalian and avian species allows IgY to have a higher avidity for certain mammalian epitopes and a unique antibody repertoire is developed compared to mammals, further enhancing the therapeutic potential of IgY. Our current research is focused on developing goose IgY anti-viral treatments and ensuring the safety of these treatments in humans. The viral antigens of focus in this research are dengue virus type 2 (DENV-2) and the Andes virus (ANDV). In an attempt to generate monoclonal goose IgY antibodies, using modified mammalian hybridoma techniques, geese were immunized with DENV-2 antigen and blood was collected as a source of immune B-cells and fused with mammalian myeloma cells. Short lasting, virus- specific IgY producing hyrbidomas were created. To generate ANDV specific goose IgY antibodies geese were vaccinated with a DNA vaccine PWRG/AND-M, containing the full-length M genome segment of ANDV, via a needle-free device at two week intervals up to eight weeks and then at 12 weeks. One year later the same geese were booster vaccinated with either pWRG/AND(opt) or pWRG/AND(opt2) six times over a ten week time period. Average neutralizing titers of sera collected from geese six weeks after the primary vaccination was 10,000. Titers remained at this level for the one year in between vaccinations and then increased to nearly 100,000 after booster vaccination. Epitope mapping confirmed the specificity of the goose- derived antibodies and identified unique highly reactive epitopes. IgY from the initial vaccination recognized 11 epitopes across the M segment, and an additional 9 epitopes after booster vaccination. In vivo survival studies in a lethal challenge model of ANDV infection established the post-exposure treatment potential of the ANDV specific IgY. To test the safety of the anti-viral IgY treatments for use in humans in vivo and in vitro safety experiments were completed. In a single injection study, mice were injected with a single dose of IgY/IgY(ÃFc) or PBS, and in a multiple injection study, rabbits were injected with multiple doses of IgY/IgY(ÃFc), IgY(ÃFc), human immunoglobulin, or PBS. Organs were collected after injection, hematoxylin and eosin stained, and scored by a blinded pathologist for abnormal pathology and/or inflammation. There were no inflammatory manifestations in the organs from animals in either the single or multiple injection study receiving IgY/IgY(ÃFc) or IgY(ÃFc). PBMCs and neutrophils were isolated from fresh human blood and co-cultured with IgY/IgY(ÃFc), mammalian IgG, and other controls. Culture supernatants were collected at various time points and analyzed for the presence of IL-1â, TNF-á, IL-10, neutrophil elastase, and nitric oxide using kit-based assays. All assays reported less reactivity of goose IgY/IgY(ÃFc) with human PBMCs and neutrophils compared to mammalian IgG and positive control mitogens. These results further support the lack of reactivity of avian IgY in the mammalian system and the benefits of safely using IgY as a treatment in the mammalian system

Immuunivajavuustilat, taudinaiheuttajat ja sukupuolierot ylähengitystiesairauksissa

In the first part of this thesis the association of different forms of sinonasal diseases and plasma concentrations of C3, C4, immunoglobulins, immunoglobulin G subclasses, C4A and C4B gene numbers were studied in 287 adult patients and 150 sex-matched adult controls. Patients were well characterized and stratified into groups using strict clinical criteria and females and males were also studied as separate groups. Severe primary antibody antibody deficiencies were rare in patients coming to sinonasal operations. Female patients had more recurrent sinusitis and other mucosal infections and males had more nasal polyposis. Upregulation of complement activity was seen in acute rhinosinusitis patients (high levels of plasma C3, C4, and complement classical pathway activity CH50) and male patients coming to sinonasal operations (high levels of plasma C3 and C4). In females, total and partial C4B deficiencies and lower levels of IgG1 and IgG3 were associated with rhinosinusitis leading to sinonasal operations. C4A deficiencies were found to predispose to severe chronic rhinosinusitis in females and males. In female patients with chronic or recurrent rhinosinusitis with nasal polyposis C4B deficiencies seem to predispose to the disease, but in males with a similar disease C4B deficiencies seem to be protective. This suggests a different pathophysiology between sexes in this form of sinonasal disease. In the second part of this thesis work 213 children coming to elective tonsillectomy were studied and compared with 155 randomly selected school children. An association with recurrent upper respiratory tract infections and hypersensitivity disorders was seen especially in children under 7 years of age. However, this association was not seen in levels of specific IgE to respiratory allergens in the same age group. Both symptomatic respiratory allergy and specific IgE to respiratory allergens became more common in boys than girls over 7 years of age. We were able to show that although both rhinoviruses and bacterial pathogens were found in the tonsils, no association between their presence and clinical forms of tonsillar disease was seen. The ability of GAS to bind complement regulators FH and C4BP did not differ between strains causing tonsillar diseases or septicemia, suggesting that other virulence mechanisms of the bacteria are more important.Väitöstutkimuksen ensimmäisessä osassa selvitettiin nenän ja sivuonteloiden tulehdusten ja polyyppitaudin yhteyttä komplementin C3- ja C4-tasoihin, vasta-aineiden ja niiden alaluokkien plasmapitoisuuksiin ja C4A- ja C4B geenilukumääriin tutkimalla 287 aikuista potilasta ja 150 aikuista kontrollihenkilöä. Potilaat jaettiin ryhmiin tiukkojen kliinisten kriteerien mukaan tutkien naiset ja miehet myös erillisinä ryhminään. Vakavat vasta-ainepuutokset olivat harvinaisia sivuonteloleikkauksiin tulevilla potilailla. Naisilla oli yleisemmin toistuvia sivuontelontulehduksia ja muita limakalvotulehduksia ja miehillä nenän polyyppitautia. Komplementin toiminnan lisääntymisen merkkejä oli havaittavissa äkillistä poskiontelontulehdusta sairastavilla potilailla (korkeat plasman C3, C4 ja komplementin klassisen tien aktivaation CH50 tasot) ja sivuonteloleikkaukseen tulevilla miehillä (korkeat plasman C3-, C4-tasot). Naisilla täydelliset ja osittaiset C4B-geenipuutokset ja matalat plasman IgG1- ja IgG2-tasot liittyivät leikkaukseen johtavaan sivuontelotautiin. C4A-geenipuutokset altistivat sekä miehiä että naisia vaikeille sivuontelontulehduksille. Kroonista tai toistuvaa sivuontelontulehdusta ja polyyppitautia samanaikaisesti sairastavilla naispotilailla C4B-puutokset vaikuttivat altistavan sairaudelle, mutta samalla tavalla sairastavilla miespotilailla samalla puutoksella oli suojaava vaikutus. Tämä viittaa siihen, että tämä sivuontelotaudin muoto kehittyy eri tavalla naisilla ja miehillä. Väitöstutkimuksen toisessa osassa tutkittiin 213 nielurisaleikkaukseen tulevaa lapsipotilasta verraten heitä 155 satunnaisesti valittuun koululaislapseen. Toistuvien ylähengitystieinfektioiden ja yliherkkyysoireiden välillä havaittiin yhteys alle 7-vuotiailla lapsilla. Tätä yhteyttä ei kuitenkaan ollut havaittavissa seerumin allergia-vasta-ainetutkimuksessa. Sekä oireinen hengitystieallergia että spesifisten vasta-aineiden määrä seerumissa lisääntyivät kouluiässä pojilla tyttöjä enemmän. Tutkimuksessa pystyttiin osoittamaan, että vaikka sekä rinoviruksia että tautia aiheuttavia bakteereja on osoitettavissa nielurisoissa, niiden läsnäololla ei ollut vaikutusta nielurisasairauden laatuun. Nielurisoista ja verenmyrkytyspotilailta eristettyjen A-streptokokkien kyvyllä sitoa komplementin säätelytekijöitä FH ja C4BP ei ollut vaikutusta näiden bakteerien aiheuttamien sairauksien kulkuun viitaten siihen, että muilla bakteerin virulenssiin vaikuttavilla tekijöillä on suurempi merkitys

Glioblastoma: multifaceted immunosuppression mediated through galectin family members

A thesis submitted to the University of Wolverhampton in partial fulfilment of the requirements for awarding the degree of Doctor of Philosophy.Glioblastoma (GBM) is an aggressive brain cancer with a near-uniformly lethal prognosis. Anti-tumoural immunity remains low due to a highly suppressive tumour microenvironment. This project set out to identify immunomodulatory proteins secreted by glioblastoma, describe the distribution of intratumoural immune cells, and characterise the effects of secreted immunomodulatory on adaptive immune effector cells. Mass spectrometry data identified galectin family members in the glioblastoma secretome. ELISA assays confirmed galectin presence in GBM-derived liquid samples; immunofluorescence confirmed their presence in tumour sections, as well as the presence of T-cells, B-cells and macrophages. Changes in cell surface marker expression and phagocytosis following exposure to recombinant proteins were assayed using flow cytometry and pHrodo-conjugated Escherichia coli respectively. B-cells and macrophages were predominantly found in tumour bulk (p=0.0053 and 0.0087) with T-cells located in the perivascular niche (p=0.0932). Additionally, 22% of tumours contained T-cell aggregations. Galectins 1, 3, 4 and 7 were also found in the majority of GBM assayed; immune checkpoints galectin-9 and PD-L1 were found in 100% and 50% of assayed tumours, respectively. The perivascular location of galectin-1 approached significance (p=0.0844), whereas other galectins showed a more equitable distribution. Consistent, though insignificant, downregulation of MHCII and phagocytosis was observed in pro-inflammatory adult macrophages exposed to GBM-relevant galectin-1 concentrations. These results suggested a T-cell-specific mechanism of suppression resulting in reduced infiltration, whereas B-cells and macrophages were able to effectively infiltrate GBM. Antigen presentation by macrophages may be inhibited by perivascular galectin-1, thus limiting T-cell retention; encounter with known immunosuppressive proteins such as galectins 1, 3, 9 and PD-L1 following effector extravasation may be a key mechanism through which GBM immunosuppression is mediated. These results posit the perivascular region as a uniquely immunosuppressive environment, and the presence of galectins as a potential key mediator of immunosuppression within glioblastoma.The Colin Oliphant Charitable Trus