Purification and Functional Analysis of BPIFA2

Short PLUNC 2, recently renamed BPIFA2, is predominantly expressed in the serous acinar cells and interlobular ducts of the major salivary glands and secreted abundantly into saliva. The original hypothesis that the structure of BPIFA2 is similar to that of the N-terminal of BPI and LBP led to the suggestion that it would also play a role in the innate immune defence of the oral cavity and upper airway. The function of BPIFA2 has not, however, been fully elucidated and thus the aim of this thesis was to develop a protocol for the purification of BPIFA2 from whole saliva, in its native form, to fully determine if it does have similar functions to BPI and LBP. Based on the current literature, a number of purification methods were assessed including precipitation, column chromatography and electrophoresis. Native polyacrylamide gel electrophoresis and electro-elution gave the highest yields of pure protein, which was then used in a variety of functional assays including binding, growth inhibition, bacterial killing, agglutination and biofilm disruption. A novelty of this study was that a range of bacteria were used including gram-positive and gram-negative bacteria and commensal and non-commensal oral bacteria. In addition, Der p 7, a dust mite allergen also shown to have structural similarities to the N-terminal domain of BPI and LBP, was used to develop an assay to examine the effect of BPIFA2 on the TLR-4 pathway in the presence of LPS. Although the allergen was initially used as a positive control for the assay system we were able to show for the first time that Der p 7 can mimic the action of LBP in the CD14-MD2-TLR4 pathway in response to gram-negative bacterial LPS. The most significant and novel finding of this thesis was the effect of BPIFA2 on gram-positive bacteria, particularly S. mutans, a known causative agent of dental caries. Reduced bacterial viability, increased agglutination and altered biofilm quality were all observed in the presence of BPIFA2. These results suggest a role for BPIFA2 in innate immunity, not against gram-negative bacteria as originally hypothesised, but against gram-positive bacteria

Heterospecific Anural Eavesdropping Cues

The ability to communicate within species is a trait utilized by every organism. Using cues conspecifically creates a better chance of survival for other members of the species and increases fitness overall. However, using cues heterospecifically also poses a great advantage as animals can eavesdrop on cues released by another species. Previous studies have recorded that eavesdropping is beneficial to prey species, such as squirrels reacting to bird calls and tadpoles reacting to visual and chemical cues to avoid predation. We asked how one local and one exotic species of frog would respond to cues emitted by another local species of frog, especially considering that the exotic frog has no natural predators. We hypothesized that Pseudacis cadaverina will react to the chemical cues released by an agitated Psuedacris regilla while Dendrobates tinctorius will not. For this experiment, data were collected on the reactions of the two species to P. regilla cues. The data was found to be non-normal and non-parametric, therefore a Wilcox test was run to determine if the control and experimental frogs had differing responses. We expected the control frogs to have no reaction since there should not have been a cue to respond to, but the p-values acquired for P. cadaverina, and D. tinctorius were less than .01. This could suggest our experimental design was flawed although the two species do appear to have different responses to the cues. Understanding how animals communicate and respond to potential danger can inform how we can best conserve these species

Characterisation and expression of SPLUNC2, the human orthologue of rodent parotid secretory protein

We recently described the Palate Lung Nasal Clone (PLUNC) family of proteins as an extended group of proteins expressed in the upper airways, nose and mouth. Little is known about these proteins, but they are secreted into the airway and nasal lining fluids and saliva where, due to their structural similarity with lipopolysaccharide-binding protein and bactericidal/permeability-increasing protein, they may play a role in the innate immune defence. We now describe the generation and characterisation of novel affinity-purified antibodies to SPLUNC2, and use them to determine the expression of this, the major salivary gland PLUNC. Western blotting showed that the antibodies identified a number of distinct protein bands in saliva, whilst immunohistochemical analysis demonstrated protein expression in serous cells of the major salivary glands and in the ductal lumens as well as in cells of minor mucosal glands. Antibodies directed against distinct epitopes of the protein yielded different staining patterns in both minor and major salivary glands. Using RT-PCR of tissues from the oral cavity, coupled with EST analysis, we showed that the gene undergoes alternative splicing using two 5' non-coding exons, suggesting that the gene is regulated by alternative promoters. Comprehensive RACE analysis using salivary gland RNA as template failed to identify any additional exons. Analysis of saliva showed that SPLUNC2 is subject to N-glycosylation. Thus, our study shows that multiple SPLUNC2 isoforms are found in the oral cavity and suggest that these proteins may be differentially regulated in distinct tissues where they may function in the innate immune response

Development of a transparent interactive decision interrogator to facilitate the decision-making process in health care.

BACKGROUND: Decisions about the use of new technologies in health care are often based on complex economic models. Decision makers frequently make informal judgments about evidence, uncertainty, and the assumptions that underpin these models. OBJECTIVES: Transparent interactive decision interrogator (TIDI) facilitates more formal critique of decision models by decision makers such as members of appraisal committees of the National Institute for Health and Clinical Excellence in the UK. By allowing them to run advanced statistical models under different scenarios in real time, TIDI can make the decision process more efficient and transparent, while avoiding limitations on pre-prepared analysis. METHODS: TIDI, programmed in Visual Basic for applications within Excel, provides an interface for controlling all components of a decision model developed in the appropriate software (e.g., meta-analysis in WinBUGS and the decision model in R) by linking software packages using RExcel and R2WinBUGS. TIDI's graphical controls allow the user to modify assumptions and to run the decision model, and results are returned to an Excel spreadsheet. A tool displaying tornado plots helps to evaluate the influence of individual parameters on the model outcomes, and an interactive meta-analysis module allows the user to select any combination of available studies, explore the impact of bias adjustment, and view results using forest plots. We demonstrate TIDI using an example of a decision model in antenatal care. CONCLUSION: Use of TIDI during the NICE appraisal of tumor necrosis factor-alpha inhibitors (in psoriatic arthritis) successfully demonstrated its ability to facilitate critiques of the decision models by decision makers

Human LPLUNC1 is a secreted product of goblet cells and minor glands of the respiratory and upper aerodigestive tracts

Long PLUNC1 (LPLUNC1, C20orf114) is a member of a family of poorly described proteins (PLUNCS) expressed in the upper respiratory tract and oral cavity, which may function in host defence. Although it is one of the most highly expressed genes in the upper airways and has been identified in sputum and nasal secretions by proteomic studies, localisation of LPLUNC1 protein has not yet been described. We developed affinity purified antibodies and localised the protein in tissues of the human respiratory tract, oro- and nasopharynx. We have complemented these studies with analysis of LPLUNC1 expression in primary human lung cell cultures and used Western blotting to study the protein in cell culture secretions and in BAL. LPLUNC1 is a product of a population of goblet cells in the airway epithelium and nasal passages and is also present in airway submucosal glands and minor glands of the oral and nasal cavities. The protein is not expressed in peripheral lung epithelial cells. LPLUNC1 is present in bronchoalveolar lavage fluid as two glycosylated isoforms and primary airway epithelial cells produce identical proteins as they undergo mucociliary differentiation. Our results suggest that LPLUNC1 is an abundant, secreted product of goblet cells and minor mucosal glands of the respiratory tract and oral cavity and suggest that the protein functions in the complex milieu that protects the mucosal surfaces in these locations

Para-infectious brain injury in COVID-19 persists at follow-up despite attenuated cytokine and autoantibody responses

To understand neurological complications of COVID-19 better both acutely and for recovery, we measured markers of brain injury, inflammatory mediators, and autoantibodies in 203 hospitalised participants; 111 with acute sera (1–11 days post-admission) and 92 convalescent sera (56 with COVID-19-associated neurological diagnoses). Here we show that compared to 60 uninfected controls, tTau, GFAP, NfL, and UCH-L1 are increased with COVID-19 infection at acute timepoints and NfL and GFAP are significantly higher in participants with neurological complications. Inflammatory mediators (IL-6, IL-12p40, HGF, M-CSF, CCL2, and IL-1RA) are associated with both altered consciousness and markers of brain injury. Autoantibodies are more common in COVID-19 than controls and some (including against MYL7, UCH-L1, and GRIN3B) are more frequent with altered consciousness. Additionally, convalescent participants with neurological complications show elevated GFAP and NfL, unrelated to attenuated systemic inflammatory mediators and to autoantibody responses. Overall, neurological complications of COVID-19 are associated with evidence of neuroglial injury in both acute and late disease and these correlate with dysregulated innate and adaptive immune responses acutely

HIV-1 Adaptation to Antigen Processing Results in Population-Level Immune Evasion and Affects Subtype Diversification

Summary: The recent HIV-1 vaccine failures highlight the need to better understand virus-host interactions. One key question is why CD8+ T cell responses to two HIV-Gag regions are uniquely associated with delayed disease progression only in patients expressing a few rare HLA class I variants when these regions encode epitopes presented by ∼30 more common HLA variants. By combining epitope processing and computational analyses of the two HIV subtypes responsible for ∼60% of worldwide infections, we identified a hitherto unrecognized adaptation to the antigen-processing machinery through substitutions at subtype-specific motifs. Multiple HLA variants presenting epitopes situated next to a given subtype-specific motif drive selection at this subtype-specific position, and epitope abundances correlate inversely with the HLA frequency distribution in affected populations. This adaptation reflects the sum of intrapatient adaptations, is predictable, facilitates viral subtype diversification, and increases global HIV diversity. Because low epitope abundance is associated with infrequent and weak T cell responses, this most likely results in both population-level immune evasion and inadequate responses in most people vaccinated with natural HIV-1 sequence constructs. Our results suggest that artificial sequence modifications at subtype-specific positions in vitro could refocus and reverse the poor immunogenicity of HIV proteins. : CD8+ T cell responses against HIV-1 effectively delay disease progression in a minority of patients with relatively rare HLA variants but are ineffective in most. Here, Tenzer et al. identify fundamental HIV-1 adaptation to the conserved human antigen-processing machinery that feeds epitopes to HLA. This adaptation occurs at subtype-specific motifs, facilitates subtype diversification, is predictable, and results in CD8 epitope abundances that correlate inversely with the HLA allele frequencies in affected populations. Thus, HIV vaccine immunogenicity might be increased by unnatural substitutions at subtype-specific motifs