Virtual Reality for IMMunisation (VRIMM) pain in young children: Results of a randomised controlled trial in general practice.

BACKGROUND AND OBJECTIVES: Virtual reality (VR) may be useful for reducing needle-based pain and distress. Our objective was to compare VR against standard care for children undergoing routine four-year-old immunisations. METHOD: This was a randomised controlled superiority trial conducted in a single suburban general practice, comparing a VR sequence of an interactive marine adventure to standard care (parental comfort, distraction of child). Our primary outcome was self-rated pain scores (Faces Pain Scale - Revised and the poker chip tool). Secondary outcomes included observational ratings (visual analogue scales) of pain and distress from caregivers and an observing healthcare provider, and overall enjoyment of the immunisation experience for the parent and child. RESULTS: In all, 42 children received VR and 45 received standard care. There was no difference in the primary outcome, with median interquartile range self-rated pain scores of 2 (0-8) in the standard care group and 2 (0-6) in the VR group. Observer ratings of pain and distress, as well as ratings of overall enjoyment, favoured VR. There were no significant adverse events. DISCUSSION: VR was not superior to standard care for self-rated pain and distress in children aged four years receiving routine immunisations. However, parent and observer ratings of pain and distress and overall ratings of enjoyment all favoured VR.

The VRIMM study: Virtual Reality for IMMunisation pain in young children-protocol for a randomised controlled trial.

INTRODUCTION: Pain caused by routine immunisations is distressing to children, their parents and those administering injections. If poorly managed, it can lead to anxiety about future medical procedures, needle phobia and avoidance of future vaccinations and other medical treatment. Several strategies, such as distraction, are used to manage the distress associated with routine immunisations. Virtual reality (VR), a technology which transports users into an immersive 'virtual world', has been used to manage pain and distress in various settings such as burns dressing changes and dental treatments. In this study, we aim to compare the effectiveness of VR to standard care in a general practice setting as a distraction technique to reduce pain and distress in 4-year-old children receiving routine immunisations. METHODS AND ANALYSIS: The study is a randomised controlled clinical trial comparing VR with standard care in 100 children receiving routine 4-year-old vaccination. Children attending a single general practice in metropolitan Melbourne, Australia will be allocated using blocked randomisation to either VR or standard care. Children in the intervention group will receive VR intervention prior to vaccination in addition to standard care; the control group will receive standard care. The primary outcome is the difference in the child's self-rated pain scores between the VR intervention and control groups measured using The Faces Pain Scale-Revised. Secondary outcomes include another measure of self-rated pain (the Poker Chip Tool), parent/guardian and healthcare provider ratings of pain (standard 100 mm visual analogue scales) and adverse effects. ETHICS AND DISSEMINATION: Ethics approval has been obtained in Australia from the Royal Australian College of General Practitioners National Research and Evaluation Ethics Committee (NREEC 18-010). Recruitment commenced in July 2019. We plan to submit study findings for publication in a peer-reviewed journal and presentation at relevant conferences. TRIAL REGISTRATION NUMBER: ACTRN12618001363279.

Modulation of NF-kappaB activation in Theileria annulata-infected cloned cell lines is associated with detection of parasite-dependent IKK signalosomes and disruption of the actin cytoskeleton.

Apicomplexan parasites within the genus Theileria have the ability to induce continuous proliferation and prevent apoptosis of the infected bovine leukocyte. Protection against apoptosis involves constitutive activation of the bovine transcription factor NF-kappaB in a parasite-dependent manner. Activation of NF-kappaB is thought to involve recruitment of IKK signalosomes at the surface of the macroschizont stage of the parasite, and it has been postulated that additional host proteins with adaptor or scaffolding function may be involved in signalosome formation. In this study two clonal cell lines were identified that show marked differences in the level of activated NF-kappaB. Further characterization of these lines demonstrated that elevated levels of activated NF-kappaB correlated with increased resistance to cell death and detection of parasite-associated IKK signalosomes, supporting results of our previous studies. Evidence was also provided for the existence of host- and parasite-dependent NF-kappaB activation pathways that are influenced by the architecture of the actin cytoskeleton. Despite this influence, it appears that the primary event required for formation of the parasite-dependent IKK signalosome is likely to be an interaction between a signalosome component and a parasite-encoded surface ligand.

Infection of bovine cells by the protozoan parasite Theileria annulata modulates expression of the ISGylation system.

The apicomplexan parasite, Theileria annulata, dedifferentiates and induces continuous division of infected bovine myeloid cells. Re-expression of differentiation markers and a loss of proliferation occur upon treatment with buparvaquone, implying that parasite factors actively maintain the altered status of the infected cell. The factors that induce this unique transformation event have not been identified. However, parasite polypeptides (TashAT family) that are located in the infected leucocyte nucleus have been postulated to function as modulators of host cell phenotype. In this study differential RNA display and proteomic analysis were used to identify altered mRNA and polypeptide expression profiles in a bovine macrophage cell line (BoMac) transfected with TashAT2. One of the genes identified by differential display was found to encode an ubiquitin-like protease (bUBP43) belonging to the UBP43 family. The bUBP43 gene and the gene encoding its ubiquitin-like substrate, bISG15, were expressed at a low level in T. annulata-infected cells. However, infected cells were refractory to induction of elevated bISG15 expression by lipopolysaccharide or type 1 interferons while TashAT2-transfected cells showed no induction when treated with camptothecin. Modulation of the ISGylation system may be of relevance to the establishment of the transformed infected host cell, as ISGylation is associated with resistance to intracellular infection by pathogens, stimulation of the immune response and terminal differentiation of leukaemic cells.

Alteration of host cell phenotype by Theileria annulata and Theileria parva: mining for manipulators in the parasite genomes.

The apicomplexan parasites Theileria annulata and Theileria parva cause severe lymphoproliferative disorders in cattle. Disease pathogenesis is linked to the ability of the parasite to transform the infected host cell (leukocyte) and induce uncontrolled proliferation. It is known that transformation involves parasite dependent perturbation of leukocyte signal transduction pathways that regulate apoptosis, division and gene expression, and there is evidence for the translocation of Theileria DNA binding proteins to the host cell nucleus. However, the parasite factors responsible for the inhibition of host cell apoptosis, or induction of host cell proliferation are unknown. The recent derivation of the complete genome sequence for both T. annulata and T. parva has provided a wealth of information that can be searched to identify molecules with the potential to subvert host cell regulatory pathways. This review summarizes current knowledge of the mechanisms used by Theileria parasites to transform the host cell, and highlights recent work that has mined the Theileria genomes to identify candidate manipulators of host cell phenotype.

Genome of the host-cell transforming parasite Theileria annulata compared with T. parva.

Theileria annulata and T. parva are closely related protozoan parasites that cause lymphoproliferative diseases of cattle. We sequenced the genome of T. annulata and compared it with that of T. parva to understand the mechanisms underlying transformation and tropism. Despite high conservation of gene sequences and synteny, the analysis reveals unequally expanded gene families and species-specific genes. We also identify divergent families of putative secreted polypeptides that may reduce immune recognition, candidate regulators of host-cell transformation, and a Theileria-specific protein domain [frequently associated in Theileria (FAINT)] present in a large number of secreted proteins.

A Theileria annulata DNA binding protein localized to the host cell nucleus alters the phenotype of a bovine macrophage cell line.

The apicomplexan parasite Theileria annulata is the only intracellular eukaryote that is known to induce the proliferation of mammalian cells. However, as the parasite undergoes stage differentiation, host cell proliferation is inhibited, and the leukocyte is eventually destroyed. We have isolated a parasite gene (SuAT1) encoding an AT hook DNA binding polypeptide that has a predicted signal peptide, PEST motifs, nuclear localization signals, and domains which indicate interaction with regulatory components of the higher eukaryotic cell cycle. The polypeptide is localized to the nuclei of macroschizont-infected cells and was detected at significant levels in cells that were undergoing parasite stage differentiation. Transfection of an uninfected transformed bovine macrophage cell line, BoMac, demonstrated that SuAT1 can modulate cellular morphology and alter the expression pattern of a cytoskeletal polypeptide in a manner similar to that found during the infection of leukocytes by the parasite. Our findings indicate that Theileria parasite molecules that are transported to the leukocyte nucleus have the potential to modulate the phenotype of infected cells.

TashHN, a Theileria annulata encoded protein transported to the host nucleus displays an association with attenuation of parasite differentiation.

The intracellular apicomplexan parasite, Theileria annulata, manipulates its bovine host cell by over-riding the cells natural apoptotic response and inducing proliferation of the infected leukocyte. We have recently identified a T. annulata encoded family of polypeptides (TashATs) with characteristics that indicate that they are involved in control of host cell gene expression. Here we present data on another member of this family, TashHN, showing that it is located to the parasite and host cell nucleus. Immunoblot analysis demonstrated that, unlike TashAT2 and 3, TashHN displays three forms, the largest of which is enriched in the host nuclear fraction and appears to be phosphorylated. Northern and 5 prime race analyses identified multiple TashHN RNA species in infected cells that have retained the ability to differentiate. These transcripts showed subtly different kinetics, but all decreased during differentiation to the merozite, and two showed reduced levels prior to down-regulation of the other TashATs. In addition, analyses of multiple cell lines that have become severely attenuated in their potential to differentiate, indicated a substantial increase in TashHN expression, with host nuclear reactivity particularly enhanced.

A role for tertiary structure in the generation of antigenic diversity and molecular association of the Tams1 polypeptide in Theileria annulata.

The major merozoite-piroplasm surface antigen (mMPSA) of Theileria annulata, Tams1, is known to be antigenically diverse. The possession of variable N-linked glycosylation sites and removal of monoclonal antibody 5E1 reactivity by mild periodate treatment suggested, previously, that divergent epitopes may be conferred by secondary modification. This study has shown that monoclonal antibody 5E1 and polyspecific antisera raised against the native protein react against divergent amino acid epitopes that are dependent on a molecular conformation that is sensitive to periodate. Therefore, no experimental evidence exists to confirm the sequence prediction that Tams1 undergoes N-linked glycosylation. Data is also presented indicating that the conformation of the antigen results in presentation of divergent regions on the external surface of the molecule, while conserved regions are more likely to be internal and hidden. In addition, non-reducing SDS-PAGE analysis demonstrated that Tams1 can undergo molecular association to form homo-dimers, trimers and multimers. The potential influence of tertiary structure and inter-molecular association on Tams1 diversity and function is discussed.

Characterization of a polymorphic Theileria annulata surface protein (TaSP) closely related to PIM of Theileria parva: implications for use in diagnostic tests and subunit vaccines.

Theileria annulata is a tick-transmitted protozoan that causes tropical theileriosis, an often fatal leukoproliferative disorder of cattle. To characterize and identify parasite proteins suitable as diagnostic antigens and/or vaccine candidates, a cDNA clone encoding a macroschizont stage protein was isolated and characterized (here designated TaSP). The gene, present as a single copy within the parasite genome, is transcribed in the sporozoite and schizont stage and codes for a protein of about 315 amino acids, having a predicted molecular weight of 36 kDa. Allelic variants were found within single parasite isolates and between isolates originating from different geographical regions. The N-terminal part contains a predicted signal peptide and the C-terminal section encodes membrane-spanning regions. Comparison of a number of cDNA clones showed that both these sequence regions are conserved while the central region shows both size and amino acid sequence polymorphism. High identity of the N- and C-terminal regions with the polymorphic immunodominant molecule (PIM) of Theileria parva (identity of 93%), the existence of a central polymorphic region and two short introns within genomic clones suggest that the presented gene/protein may be the T. annulata homologue of PIM. However, the central region of TaSP has no significant identity with PIM, contains no repetitive peptide motifs and is shorter, resulting in a lower molecular weight. The existence of the predicted secretion signal peptide and membrane spanning regions suggest that TaSP is located at the parasite membrane.