Deformation behaviour of stomatocyte, discocyte and echinocyte red blood cell morphologies during optical tweezers stretching.

The red blood cell (RBC) deformability is a critical aspect, and assessing the cell deformation characteristics is essential for better diagnostics of healthy and deteriorating RBCs. There is a need to explore the connection between the cell deformation characteristics, cell morphology, disease states, storage lesion and cell shape-transformation conditions for better diagnostics and treatments. A numerical approach inspired from the previous research for RBC morphology predictions and for analysis of RBC deformations is proposed for the first time, to investigate the deformation characteristics of different RBC morphologies. The present study investigates the deformability characteristics of stomatocyte, discocyte and echinocyte morphologies during optical tweezers stretching and provides the opportunity to study the combined contribution of cytoskeletal spectrin network and the lipid-bilayer during RBC deformation. The proposed numerical approach predicts agreeable deformation characteristics of the healthy discocyte with the analogous experimental observations and is extended to further investigate the deformation characteristics of stomatocyte and echinocyte morphologies. In particular, the computer simulations are performed to investigate the influence of direct stretching forces on different equilibrium cell morphologies on cell spectrin link extensions and cell elongation index, along with a parametric analysis on membrane shear modulus, spectrin link extensibility, bending modulus and RBC membrane-bead contact diameter. The results agree with the experimentally observed stiffer nature of stomatocyte and echinocyte with respect to a healthy discocyte at experimentally determined membrane characteristics and suggest the preservation of relevant morphological characteristics, changes in spectrin link densities and the primary contribution of cytoskeletal spectrin network on deformation behaviour of stomatocyte, discocyte and echinocyte morphologies during optical tweezers stretching deformation. The numerical approach presented here forms the foundation for investigations into deformation characteristics and recoverability of RBCs undergoing storage lesion.

Seroprevalence of antibodies to primate erythroparvovirus 1 (B19V) in Australia.

BACKGROUD: Primate erythroparvovirus 1 (B19V) is a globally ubiquitous DNA virus. Infection results in a variety of clinical presentations including erythema infectiosum in children and arthralgia in adults. There is limited understanding of the seroprevalence of B19V antibodies in the Australian population and therefore of population-wide immunity. This study aimed to investigate the seroprevalence of B19V antibodies in an Australian blood donor cohort, along with a cohort from a paediatric population. METHODS: Age/sex/geographical location stratified plasma samples (n = 2221) were collected from Australian blood donors. Samples were also sourced from paediatric patients (n = 223) in Queensland. All samples were screened for B19V IgG using an indirect- enzyme-linked immunosorbent assay. RESULTS: Overall, 57.90% (95% CI: 55.94%-59.85%) of samples tested positive for B19V IgG, with the national age-standardized seroprevalence of B19V exposure in Australians aged 0 to 79 years estimated to be 54.41%. Increasing age (p < 0.001) and state of residence (p < 0.001) were independently associated with B19V exposure in blood donors, with the highest rates in donors from Tasmania (71.88%, 95% CI: 66.95%-76.80%) and donors aged 65-80 years (78.41%, 95% CI: 74.11%-82.71%). A seroprevalence of 52.04% (95% CI: 47.92%-56.15%) was reported in women of child-bearing age (16 to 44 years). Sex was not associated with exposure in blood donors (p = 0.547) or in children (p = 0.261) screened in this study. CONCLUSIONS: This study highlights a clear association between B19V exposure and increasing age, with over half of the Australian population likely to be immune to this virus. Differences in seroprevalence were also observed in donors residing in different states, with a higher prevalence reported in those from the southern states. The finding is consistent with previous studies, with higher rates observed in countries with a higher latitude. This study provides much needed insight into the prevalence of B19V exposure in the Australian population, which has implications for public health as well as transfusion and transplantation safety in Australia.

Hepatitis E virus RNA in Australian blood donors: prevalence and risk assessment.

BACKGROUND AND OBJECTIVES: Hepatitis E virus (HEV) is a known transfusion-transmissible agent. HEV infection has increased in prevalence in many developed nations with RNA detection in donors as high as 1 in 600. A high proportion of HEV infections are asymptomatic and therefore not interdicted by donor exclusion criteria. To manage the HEV transfusion-transmission (TT) risk some developed nations have implemented HEV RNA screening. In Australia, HEV is rarely notified; although locally acquired infections have been reported, and the burden of disease is unknown. The purpose of this study was to determine the frequency of HEV infection in Australian donors and associated TT risk. MATERIALS AND METHODS: Plasma samples (n = 74 131) were collected from whole blood donors during 2016 and screened for HEV RNA by transcription-mediated amplification (TMA) in pools of six. Individual TMA reactive samples were confirmed by RT-PCR and, if positive, viral load determined. Prevalence data from the study were used to model the HEV-TT risk. RESULTS: One sample in 74 131 (95% CI: 1 in 1 481 781 to 1 in 15 031) was confirmed positive for HEV RNA, with an estimated viral load of 180 IU/ml, which is below that typically associated with TT. Using a transmission-risk model, we estimated the risk of an adverse outcome associated with TT-HEV of approximately 1 in 3·5 million components transfused. CONCLUSION: Hepatitis E virus viremia is rare in Australia and lower than the published RNA prevalence estimates of other developed countries. The risk of TT-HEV adverse outcomes is negligible, and HEV RNA donor screening is not currently indicated.

Diverse and novel RHD variants in Australian blood donors with a weak D phenotype: implication for transfusion management.

BACKGROUND AND OBJECTIVES: Variant RHD genes associated with the weak D phenotype can result in complete or partial D-epitope expression on the red cell. This study examines the genetic classification in Australian blood donors with a weak D phenotype and correlates RHD variants associated with the weak D phenotype against D-epitope profile. MATERIALS AND METHODS: Following automated and manual serology, blood samples from donors reported as 'weak D' (n = 100) were RHD genotyped by a commercial SNP-typing platform and Sanger sequencing. Two commercial anti-D antibody kits were used for extended serological testing for D-epitope profiles. RESULTS: Three samples had wild-type RHD exonic sequences, and 97 samples had RHD variants. RHD*weak D type 1, RHD*weak D type 2 or RHD*weak D type 3 was detected in 75 donors. The remaining 22 samples exhibited 17 different RHD variants. One donor exhibited a novel RHD*c.939+3A>C lacking one D-epitope. Weak D types 1·1, 5, 15, 17 and 90 showed a partial D-epitope profile. CONCLUSION: The array of RHD variants detected in this study indicated diversity in the Australian donor population that needs to be accommodated for in future genotyping strategies.

The distribution of MNS hybrid glycophorins with Mur antigen expression in Chinese donors including identification of a novel GYP.Bun allele.

BACKGROUND AND OBJECTIVES: MNS hybrid glycophorins are identified by characteristic antigen profiles. One of these is the Mur antigen, which is expressed on red cell hybrid glycophorins of several phenotypes of the 'Miltenberger' series found predominantly in East Asian population. The aim of this study was to investigate the distribution of Mur-positive hybrid glycophorins and clarify the genetic basis in the donors from southern China. MATERIALS AND METHODS: Blood samples from 528 donors were collected for Mur antigen serological typing. Sequencing of GYPB pseudoexon 3 and MNS phenotyping were conducted in Mur-positive samples. The multiplex ligation-dependent probe amplification (MLPA) was used to confirm the zygosity of the GYP.Mur allele and determine the MNSs genotype. The expression of Mur antigen was evaluated by flow cytometry. RESULTS: Fifty-one Mur-positive samples were identified by serological testing. Sequencing analysis showed 50 donors (50/528, 9.5%) with the GYP.Mur allele (48 heterozygotes and two homozygotes), which were confirmed by the MLPA genotyping analysis, and one donor (1/528, 0.19%) with a novel GYP.Bun allele. Flow cytometry analysis revealed higher Mur antigen expression on GP.Mur (Mi.III) homozygotes than heterozygotes. For the GYP.Mur homozygotes, an incorrect 'N' positive typing with anti-N lectin was obtained. CONCLUSION: GP.Mur (Mi.III) is the main Mur-positive hybrid glycophorin in Guangzhou donors. The dosage effect of Mur antigen observed provides a basis for selecting the homozygous GP.Mur RBCs as the reagent cells to avoid neglecting weak antibodies. A separate GYP.Bun lineage found in the southern China provides evidence for further complexity in the MNS system.

Duffy blood group phenotype-genotype correlations using high-resolution melting analysis PCR and microarray reveal complex cases including a new null FY*A allele: the role for sequencing in genotyping algorithms.

BACKGROUND AND OBJECTIVES: Duffy blood group phenotypes can be predicted by genotyping for single nucleotide polymorphisms (SNPs) responsible for the Fy(a) /Fy(b) polymorphism, for weak Fy(b) antigen, and for the red cell null Fy(a-b-) phenotype. This study correlates Duffy phenotype predictions with serotyping to assess the most reliable procedure for typing. MATERIALS AND METHODS: Samples, n = 155 (135 donors and 20 patients), were genotyped by high-resolution melt PCR and by microarray. Samples were in three serology groups: 1) Duffy patterns expected n = 79, 2) weak and equivocal Fy(b) patterns n = 29 and 3) Fy(a-b-) n = 47 (one with anti-Fy3 antibody). RESULTS: Discrepancies were observed for five samples. For two, SNP genotyping predicted weak Fy(b) expression discrepant with Fy(b-) (Group 1 and 3). For three, SNP genotyping predicted Fy(a) , discrepant with Fy(a-b-) (Group 3). DNA sequencing identified silencing mutations in these FY*A alleles. One was a novel FY*A 719delG. One, the sample with the anti-Fy3, was homozygous for a 14-bp deletion (FY*01N.02); a true null. CONCLUSION: Both the high-resolution melting analysis and SNP microarray assays were concordant and showed genotyping, as well as phenotyping, is essential to ensure 100% accuracy for Duffy blood group assignments. Sequencing is important to resolve phenotype/genotype conflicts which here identified alleles, one novel, that carry silencing mutations. The risk of alloimmunisation may be dependent on this zygosity status.

A novel FY*A allele with the 265T and 298A SNPs formerly associated exclusively with the FY*B allele and weak Fy(b) antigen expression: implication for genotyping interpretative algorithms.

BACKGROUND AND OBJECTIVES: An Australian Caucasian blood donor consistently presented a serology profile for the Duffy blood group as Fy(a+b+) with Fy(a) antigen expression weaker than other examples of Fy(a+b+) red cells. Molecular typing studies were performed to investigate the reason for the observed serology profile. MATERIAL AND METHODS: Blood group genotyping was performed using a commercial SNP microarray platform. Sanger sequencing was performed using primer sets to amplify across exons 1 and 2 of the FY gene and using allele-specific primers. RESULTS: The propositus was genotyped as FY*A/B, FY*X heterozygote that predicted the Fy(a+b+(w) ) phenotype. Sequencing identified the 265T and 298A variants on the FY*A allele. This link between FY*A allele and 265T was confirmed by allele-specific PCR. CONCLUSION: The reduced Fy(a) antigen reactivity is attributed to a FY*A allele-carrying 265T and 298A variants previously defined in combination only with the FY*B allele and associated with weak Fy(b) antigen expression. This novel allele should be considered in genotyping interpretative algorithms for generating a predicted phenotype.

Malaria antibody persistence correlates with duration of exposure.

BACKGROUND AND OBJECTIVES: In Australia, the risk of transfusion-transmitted malaria is managed through the identification of 'at-risk' donors, antibody screening enzyme-linked immunoassay (EIA) and, if reactive, exclusion from fresh blood component manufacture. Donor management depends on the duration of exposure in malarious regions (>6 months: 'Resident', <6 months: 'Visitor') or a history of malaria diagnosis. We analysed antibody testing and demographic data to investigate antibody persistence dynamics. To assess the yield from retesting 3 years after an initial EIA reactive result, we estimated the proportion of donors who would become non-reactive over this period. MATERIALS AND METHODS: Test results and demographic data from donors who were malaria EIA reactive were analysed. Time since possible exposure was estimated and antibody survival modelled. RESULTS: Among seroreverters, the time since last possible exposure was significantly shorter in 'Visitors' than in 'Residents'. The antibody survival modelling predicted 20% of previously EIA reactive 'Visitors', but only 2% of 'Residents' would become non-reactive within 3 years of their first reactive EIA. CONCLUSION: Antibody persistence in donors correlates with exposure category, with semi-immune 'Residents' maintaining detectable antibodies significantly longer than non-immune 'Visitors'.

Ontogeny of placental structural development and expression of the renin-angiotensin system and 11beta-HSD2 genes in the rabbit.

Common pregnancy complications are associated with impaired placental development. This study aimed to characterise the ontogeny of structural correlates of rabbit placental function, its expression of genes encoding components of the renin-angiotensin system (RAS), as well as 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) mRNA since these are known to be expressed by the placenta and are associated with pregnancy complications, including preeclampsia and intrauterine programming. Placentae were collected at gestational age (GA) 14, 21 and 28 (term=32 days). Gene expression was analysed using real time PCR and placental structures were quantified via image analyses. The volume densities and volumes of trophoblast, fetal capillaries, maternal blood space, surface density and surface area of trophoblast all progressively increased, while the arithmetic mean barrier thickness of trophoblast decreased across gestation. Maternal plasma renin activity (PRA) was positively correlated with volumes of trophoblast and maternal blood space, surface density and surface area of trophoblast. Placental renin mRNA declined ( downward arrow62%; P<0.01) across gestation and was negatively correlated with maternal PRA (GA0), fetal and placental weights, placental angiotensin type 1 and 2 receptors (AT(1)R and AT(2)R) mRNA and volume of trophoblast. AT(1)R mRNA expression was increased by 92% (P<0.001) across gestation. AT2R mRNA expression was approximately 81% (P<0.01) greater at GA14 compared to GA21. Placental 11beta-HSD2 mRNA expression was approximately 74% greater (P<0.01) at GA21 than GA14, but by GA28 was similar to that at GA14. These data show that changes in placental gene expression are associated with key events in placental and fetal development, indicating that the rabbit provides a good model for investigations of pregnancy perturbations that alter the RAS or programme the fetus.

Sex differences in postnatal growth and renal development in offspring of rabbit mothers with chronic secondary hypertension.

Previously, we demonstrated that adult blood pressure was increased in offspring of rabbit mothers with chronic secondary renal hypertension. Our study identified sex-specific differences in the programming of hypertension, with female, not male, offspring, having increased blood pressure at 30 wk of age. The aim of this study was to characterize the maternal hypertension during pregnancy to determine potential programming stimuli. Further, we examined the impact of chronic maternal hypertension on offspring birth weight, nephron number, and renal noradrenaline content (as an index of renal innervation density). Three groups of mothers and their offspring were studied: two-kidney, one-wrap (2K-1W, n = 9 mothers) hypertensive, two-kidney, two-wrap (2K-2W, n = 8) hypertensive, and a sham-operated group (n = 9). Mean arterial blood pressure was increased by approximately 20 mmHg throughout pregnancy in both hypertensive groups compared with sham mothers (P(G) < 0.001). Plasma renin activity (PRA; P(G) < 0.05) and aldosterone (P(G) < 0.05) levels were increased during gestation in the 2K-1W, but not the 2K-2W mothers. Birth weight was increased by approximately 20% in offspring of both groups of hypertensive mothers (P(T) < 0.001), though this was associated with a reduction in litter size. Renal noradrenaline content was increased ( approximately 40%, P < 0.05) at 5 wk of age in female 2K-1W offspring compared with sham offspring. Glomerular number was not reduced in female offspring of either group of hypertensive mothers; however, glomerular tuft volume was reduced in female 2K-2W offspring (P < 0.05), indicative of a reduction in glomerular filtration surface area. In conclusion, the two models of renal hypertension produced differential effects on the offspring. The impact of a stimulated maternal renin-angiotensin system in the 2K-1W model of hypertension may influence development of the renal sympathetic nerves and contribute to programming of adult hypertension.

Borna disease virus (BDV) infection in cats. A concise review based on current knowledge.

Persistent viral infections of the central nervous system have been the subject of intense interest for decades. One of these viral agents has been identified as Borna disease virus (BDV) of the family Bornaviridae. There have been various reports that link BDV to staggering disease in cats, with symptoms that include ataxia and behavioural disorders, and the disease is often referred to as feline Borna disease. Serological and molecular detection of BDV has been reported at a higher prevalence in cats with neurological disorders in comparison to healthy cats. The transmission route(s) of BDV remain largely unknown, and the hypothesis that BDV is a zoonotic agent is yet to be proven. This review summarises the current knowledge on BDV infection in cats and discusses epidemiological aspects of infection.

No evidence of endemic Borna disease virus infection in Australian horses in contrast with endemic infection in other continents.

Borna disease virus (BDV) is a unique RNA virus that is a cause of neurological disease in horses, sheep and cats. The finding that BDV also infects humans has raised concern related to the impact of infection with this virus. The extent to which BDV may be endemic in geographical regions outside Europe is of interest in management of international movement of animals including horses. Sera from Australian horses (N = 553) sampled in Sydney, New South Wales (NSW), were analysed for BDV antigen, circulating immune complexes (CICs), and antibodies by monoclonal antibody-based ELISAs. One-tenth of the samples were investigated by further antibody tests, namely immunofluorescence (IFA) and a peptide ELISA, as well as for BDV RNA. The study revealed a very low frequency of serological markers that may be associated with exposure to BDV in Australian horses from NSW with a few sera (0.7%) displaying low range positive results in the CIC assay, and no detectable BDV RNA. This pattern is inconsistent with endemic BDV infection and strongly contrasts with the pattern of endemic infection, particularly in Europe.

Assessment of in vitro-generated platelet microparticles using a modified flow cytometric strategy.

Quantification of platelet microparticles (PMPs) may be a useful marker for the detection of in vivo platelet activation. Optimisation of flow cytometric methods for detection and quantification of PMPs has not been systemically evaluated. This study reports the optimisation of flow cytometric procedures for the detection of PMPs, the determination of limits of size detection using microbeads, and the characterisation of PMP generation by in vitro activation of platelets using collagen and adenosine 5' diphosphate (ADP). Fluorescent and plain microbeads proved useful for defining the limits of the flow cytometer in detecting PMPs. A systematic calibration of the forward scatter (FS) threshold parameter (size) of the flow cytometer using microbeads allowed for the detection of very small particles (down to 0.1 microm diameter). PMPs generated in vitro using ADP and collagen were reliably detected by flow cytometry using monoclonal antibodies (MAb) directed towards platelet surface membrane glycoproteins (Gp). The PMP events were detected in the FS low (i.e., small size events) and fluorescence (FL) high (i.e., platelet Gp MAb-labelled events) region. PMPs of different size profiles were observed for each of the agonists. Flow cytometry can be used as a tool in the assessment of PMPs. As detection of particles of this type is at the limit of resolution of flow cytometers, careful attention is required with the choice of platelet-specific MAb, isotype control, and optimisation of procedure setup and performance.

In vitro antiviral activity of the anthraquinone chrysophanic acid against poliovirus.

Chrysophanic acid (1,8-dihydroxy-3-methylanthraquinone), isolated from the Australian Aboriginal medicinal plant Dianella longifolia, has been found to inhibit the replication of poliovirus types 2 and 3 (Picornaviridae) in vitro. The compound inhibited poliovirus-induced cytopathic effects in BGM (Buffalo green monkey) kidney cells at a 50% effective concentration of 0.21 and 0.02 microgram/ml for poliovirus types 2 and 3, respectively. The compound inhibited an early stage in the viral replication cycle, but did not have an irreversible virucidal effect on poliovirus particles. Chrysophanic acid did not have significant antiviral activity against five other viruses tested: Coxsackievirus types A21 and B4, human rhinovirus type 2 (Picornaviridae), and the enveloped viruses Ross River virus (Togaviridae) and herpes simplex virus type 1 (Herpesviridae). Four structurally-related anthraquinones--rhein, 1,8-dihydroxyanthraquinone, emodin and aloe-emodin were also tested for activity against poliovirus type 3. None of the four compounds was as active as chrysophanic acid against the virus. The results suggested that two hydrophobic positions on the chrysophanic acid molecule (C-6 and the methyl group attached to C-3) were important for the compound's activity against poliovirus.

Microsatellite instability markers in breast cancer: a review and study showing MSI was not detected at 'BAT 25' and 'BAT 26' microsatellite markers in early-onset breast cancer.

Microsatellite markers may provide evidence of faulty DNA mismatch repair (MMR) via the detection of microsatellite instability (MSI). The choice of microsatellite markers may impact on the MSI detection rate. In hereditary non-polyposis colon cancer (HNPCC), several informative microsatellite markers have been recommended. Two of these, BAT 25 and BAT 26, are quasi-homozygous, enabling analysis of tumour DNA in the absence of paired normal DNA. Sixty-six breast cancer patients under 45 years of age at diagnosis were examined for MSI at BAT 25 and BAT 26. Tumour DNA was extracted from paraffin-embedded tissue. No MSI was detected at the BAT 25 or BAT 26 loci. An additional five microsatellite markers, known to be informative for HNPCC, were examined for MSI in these patients. Apparently-normal profiles were achieved. A tabulated survey of 306 microsatellite markers used to detect MSI in breast cancer revealed that only 35.5% of markers detected MSI at an average rate of 2.9%. The MSI detection rate at the specific HNPCC markers varied from 0% to 10% in breast cancer, with D175250 and TP53 being the HNPCC markers most suitable for analysis of breast cancer. The size of the microsatellite marker's repeat unit did not impact on MSI detection rates. Compiled data from large studies (n > 100) revealed D115988 as the marker with the highest MSI detection rate. Genomic instability pathways of carcinogenesis, characterised by MMR defects and MSI, appear to play a role in the genesis of some breast cancer types.

Construction of infectious feline foamy virus genomes: cat antisera do not cross-neutralize feline foamy virus chimera with serotype-specific Env sequences.

Full-length genomes of the feline foamy virus (FFV or FeFV) isolate FUV were constructed. DNA clone pFeFV-7 stably directed the expression of infectious FFV progeny virus indistinguishable from wild-type, uncloned FFV isolate FUV. The env and bel 1 genes of pFeFV-7 were substituted for by corresponding sequences of the FFV serotype 951 since previous studies implicated a defined part of FFV Env protein as responsible for serotype-specific differences in serum neutralization (I. G. Winkler, R. M. Flügel, M. Löchelt, and R. L. P. Flower, 1998. Virology 247: 144-151). Recombinant virus derived from chimeric plasmid pFeFV-7/951 containing the hybrid env gene and the parental clone pFeFV-7 were used for neutralization studies. By means of a rapid titration assay for FFV infectivity, we show that progeny virus derived from plasmid pFeFV-7 was neutralized by FUV- but not by 951-specific antisera, whereas pFeFV-7/951-derived chimeric virus was neutralized by 951-specific antisera only. Both recombinant proviruses will be useful for repeated delivery of foreign genes for therapeutic gene applications into cats.

Epidemiology of feline foamy virus and feline immunodeficiency virus infections in domestic and feral cats: a seroepidemiological study.

Although foamy viruses (Spumaviruses) have repeatedly been isolated from both healthy and diseased cats, cattle, and primates, the primary mode of transmission of those common viruses remains undefined. A database of the feline foamy virus (FeFV) and feline immunodeficiency virus (FIV) antibody status, age, and sex of 389 domestic cats presented to veterinarians was assembled. A similar database for 66 feral (wild) cats was also assembled. That FeFV antibody status reflects infection was validated by PCR. Both FeFV and FIV infection rates were found to gradually increase with age, and over 70% of cats older than 9 years were seropositive for FeFV. In domestic cats, the prevalence of FeFV infection was similar in both sexes. In feral cats, FeFV infection was more prevalent in female cats than in male cats. Although both FeFV and FIV have been reported to be transmitted by biting, the patterns of infection observed are more consistent with an interpretation that transmission of these two retroviruses is not the same. The prevalence of FIV infection is highest in nondesexed male cats, the animals most likely to display aggressive behavior. The gradual increase in the proportion of FeFV-infected animals is consistent with transmission of foamy viruses by intimate social contact between animals and less commonly by aggressive behavior.