Requirements for a Robust Animal Model to Investigate the Disease Mechanism of Autoimmune Complications Associated With ARF/RHD

The pathogenesis of Acute Rheumatic Fever/Rheumatic Heart Disease (ARF/RHD) and associated neurobehavioral complications including Sydenham's chorea (SC) is complex. Disease complications triggered by Group A streptococcal (GAS) infection are confined to human and determining the early events leading to pathology requires a robust animal model that reflects the hallmark features of the disease. However, modeling these conditions in a laboratory animal, of a uniquely human disease is challenging. Animal models including cattle, sheep, pig, dog, cat, guinea pigs rats and mice have been used extensively to dissect molecular mechanisms of the autoimmune inflammatory responses in ARF/RHD. Despite the characteristic limitations of some animal models, several rodent models have significantly contributed to better understanding of the fundamental mechanisms underpinning features of ARF/RHD. In the Lewis rat autoimmune valvulitis model the development of myocarditis and valvulitis with the infiltration of mononuclear cells along with generation of antibodies that cross-react with cardiac tissue proteins following exposure to GAS antigens were found to be similar to ARF/RHD. We have recently shown that Lewis rats injected with recombinant GAS antigens simultaneously developed cardiac and neurobehavioral changes. Since ARF/RHD is multifactorial in origin, an animal model which exhibit the characteristics of several of the cardinal diagnostic criteria observed in ARF/RHD, would be advantageous to determine the early immune responses to facilitate biomarker discovery as well as provide a suitable model to evaluate treatment options, safety and efficacy of vaccine candidates. This review focuses on some of the common small animals and their advantages and limitations

A mechanism for glu-plasminogen binding: an important aspect of the plasminogen activation cascade

The lysine-dependent, activation-resistant, conformation of glu-plasminogen is converted to an activation susceptible conformation after binding to cell-surface lysine residues. These lysine-dependent interactions of glu-plasminogen are mediated by its lysine binding sites (LBS\u27s). Thus, it is hypothesised that lysine residues from the cell surface plasminogen receptors compete with lysine residues from gluplasminogen for occupation of its LBS\u27s, thereby inducing a conformational change to glu-plasminogen. Previously, an α-enolase-related molecule was identified as a cell surface plasminogen receptor. Hence, the plasminogen binding characteristics of recombinant human α-enolase (r-α-enolase) were assessed. R-α-enolase bound glu-plasminogen in a lysine-dependent manner with an apparent Kd of 1.9 μM. This interaction [1] was dependent on the C-terminal lysine residue of r-α-enolase, [2] enhanced the activation rate of glu-plasminogen and [3] blocked α2-antiplasmin from binding glu-plasminogen. BIACORE kinetic analysis of the interaction suggested that the dissociation of glu-plasminogen from r-α-enolase was mediated by at least two components with apparent dissociation rate constants of kdl=4.7x10-2 s-1 andkd2=l.6x10-3 s-1.Global analysis of the interaction suggested that it was a two-state conformational change reaction, mediated by a concentration-dependent increase in the initial association rate. Intrinsic fluorescence spectroscopy confirmed that r-α-enolase induced a more open conformation of glu-plasminogen. Thus, the gene product of human ENOl encoded an authentic plasminogen binding protein and the binding of gluplasminogen to α-enolase is mediated by an initial lysine-dependent competition reaction that results in a conformational change to the zymogen. The binding of glu-plasminogen to the metastatic breast cancer cell line MDA-MB-231 in this study was: [1] lysine-dependent [2] low affinity (Kd = 1.8 μM), but high capacity (5.0x107 sites/cells) and [3] dependent on the viability status of the cells. Multiple plasminogen binding proteins may be responsible for localising glu-plasminogen to the cell surface. The MDA-MB-231 cells were capable of generating large amounts of plasmin compared to the non-metastatic breast cancer cell lines which did not have a high plasminogen binding capacity. Therefore, glu-plasminogen binds to the cell surface by a lysine-competitive, two-step binding event that results in a more open, activation-susceptible conformation. This competitive-lysine reaction mechanism of glu-plasminogen explains the relationship between binding, conformation and activation of gluplasminogen

Cell surface antigens of Mycoplasma species bovine group 7 bind and activate plasminogen

Mycoplasma species bovine group 7 bound plasminogen at the cell surface in a lysine-dependent manner. Cell-bound plasminogen was rapidly activated to plasmin by exogenous urokinase, and this activity was associated with plasminogen binding capacity. Binding assays using plasminogen modified with a trifunctional cross-linking agent revealed several binding proteins

Detection of Novel Biomarkers of Coeliac Disease at the Protein Level using both Fluoro-Immunohistochemistry and Conventional Immunohistochemistry

Background: Coeliac Disease (CD) is an autoimmune enteropathy targeted against the harmless grain protein gliadin and affects around 1 in 300 people worldwide. The condition causes malabsorptive symptomology and the only current form of treatment is a strict. lifelong gluten-free diet. To date, the exact mechanisms of this autoimmune response have yet to be elucidated. Aims: We have developed and tested a fully-annotated and MIQE compliant 87-gene qRT-PCR array which was specifically designed to investigate the hypothesised immune pathways and processes of active CD. Using duodenal biopsy material from 34 patients with active CD, treated CD and healthy controls; this analysis showed a total of 25 genes which were significantly deferentially expressed between the grades of CD, with a total of 5 genes showing highly significant differential expression and significant linear relationships to the different grades of CD. The aim of this research therefore, was to investigate the expression and localisation of these live genes at the protein level: namely Interferon-y (IFNG), Interleukin 17 (IL17A), Interleukin 18 (IL18), B-lymphocyte Antigen CDI9 (CDI9) and Ectonuelcotide Pyrophosphatasc/Phosphodiesterase 3 (CD203c)

Enhancing understanding of foundation concepts in first year university STEM: evaluation of an asynchronous online interactive lesson

The transition to tertiary level learning requires students take responsibility for their own learning and independently synthesise conceptual knowledge specific to their discipline. We designed an interactive online lesson that aimed to build student engagement and foster self-regulation of understanding for a foundation concept in molecular biology in a first year general biology unit delivered to both on-campus and off-campus cohorts. Students demonstrated a high level of engagement following exposure to the lesson, and more attempted the molecular biology exam question, and subsequently recorded higher grades than in the previous year. The inclusion of an interactive online lesson has facilitated these first year science students to take responsibility for their own learning (through asynchronous engagement) and independently synthesise conceptual knowledge (improved summative outcomes). Development and application of online lessons for STEM disciplines that require students to synthesise discipline specific content has the potential to improve students' successful transition to university education

Post COVID Foundation Biology through Interactive Online Learning

The rapid migration of courses from blended teaching to a fully online learning environment during the COVID-19 pandemic required the timely development of new, online teaching resources and re-imagined strategies, to teach practical skills to university students. The move to deliver content that was fully online challenged the assumptions and conventions about the use of face-to-face classes, especially in STEM disciplines that rely on experiential learning. This critical review uses case studies to describe the innovations that were used to adapt undergraduate, first year (foundation) biology courses which were previously delivered using a blended learning pedagogy to a fully online format. In doing so, the opportunities to enhance traditionally practical based activities have also been considered. Future innovations for blended learning, however, require inherent properties and capacity of technologies to support aligned learning tasks. 
 Reflections on the crisis response to learning however, act as a catalyst for educational change towards more flexible models and practices in future interconnected learning environments

'Trichostrongylus colubriformis' induces IgE-independent CD13,CD164 and CD203c mediated activation of basophils in anin vitro intestinal epithelial cell co-culture model

Gastrointestinal nematodes pose a major risk to the farming of small ruminants worldwide. Infections are typically controlled by anthelmintics, however as resistance to anthelmintics increases, it is necessary that the mechanism of host responses are understood in order to develop alternative control options. It is hypothesised that basophils are involved in the initiation of an anti-parasite immune response, independent of IgE. In this study, the in vitro activation states of CD203c+ basophil-like KU812 cells were determined in the presence of 'Trichostrongylus colubriformis' parasitised HT29 epithelial cells with or without mucin. Cell surface expression of CD164, CD107a and CD13 antigens on gated CD203+ cells were determined and qRT-PCR was used to examine gene expression changes of IL33 (a Th2 cytokine) and the high affinity IgE receptor (FcεRIα) within the co-culture. When KU812 basophils encountered 'T. colubriformis' and/or mucin in a parasitised epithelium, the basophils increased cell surface expression of CD13 and CD164 antigens, independent of IgE. 'T. colubriformis' also increased the number of CD203c+ KU812 cells that expressed CD13 and CD164 antigens. These data support the in vivo observations of 'T. colubriformis' primary infections in guinea pigs and sheep

Activation of Basophils by Gastrointestinal Nematode Parasites

Gastrointestinal nematodes (GINs) pose a major risk to the farming of small ruminants worldwide. Typically GIN infections are controlled by anthelmintics, however the success of such programs are threatened by widespread emergence of anthelmintic resistance in parasite populations. Vaccine technology based on humoral antibody protection has been developed against 'Haemonchus contortus', however the mechanism of protection is ineffective for parasites such as 'Trichostrongylus colubriformis' that do not access the blood. The danger hypothesis suggests a link between tissue damage and the initiation of the correct immune response, theorising that an antigen must be presented in context of a danger signal. The host danger signals may provide the context of the immune response whilst the antigens provide the targets for the immune system. In the current study the activation state of basophils cultured with epithelial cells and 'T. colubriformis' larvae in the presence of mucin or Ivermectin was determined using a basophil specific activation marker (CD203). The expression and intensity of CD164, CD107a and CD13 in gated CD203+ cell populations was then determined. CD164 was upregulated only by contact with epithelial cells and CD107a showed no significant change. In the presence of only epithelial cells and 'T. colubriformis' but lacking mechanisms to limit worm motility, the mean fluorescence intensity of CD13 was significantly inhibited. These results suggest that 'T. colubriformis' movement may cause inhibition of basophil activation, whilst limiting worm motility allows an amplified immune response. Future studies will establish an immune cell-epithelial cell-parasite culture system allowing examination of cell-mediated immune responses at the site of infection to formulate a mucosal delivery vaccine