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Chronic lymphocytic leukaemia (CLL) is the most common haematological malignancy in Australia and New Zealand (ANZ). Considerable changes to diagnostic and management algorithms have occurred within the last decade. The availability of next‐generation sequencing and measurable residual disease assessment by flow cytometry allow for advanced prognostication and response assessments. Novel therapies, including inhibitors of Bruton's tyrosine kinase (BTKi) and B‐cell lymphoma 2 (BCL2) inhibitors, have transformed the treatment landscape for both treatment‐naïve and relapsed/refractory disease, particularly for patients with high‐risk genetic aberrations. Recommendations regarding appropriate supportive management continue to evolve, and special considerations are required for patients with CLL with respect to the global SARS‐CoV‐2 pandemic. The unique funding and treatment environments in Australasia highlight the need for specific local guidance with respect to the investigation and management of CLL. This consensus practice statement was developed by a broadly representative group of ANZ experts in CLL with endorsement by peak haematology bodies, with a view to providing this standardised guidance. [ABSTRACT FROM AUTHOR]

Freshwater mussels are an ancient group of molluscs that are considered ecosystem engineers because of the many important functions they perform, including biofiltration and biodeposition. Associations between mussel declines and increased sedimentation are well documented, but the direct effects of suspended sediment (SS) concentrations on mussel functioning are poorly understood.This study investigated the effects of SS concentrations on the clearance and biodeposition rates of the Australian hyriid river mussel Alathyria jacksoni. Clearance rates of algae and sediment and biodeposition rates were measured in aquaria containing 3 L of aerated water for 1 hr, with six different concentrations of SS (bentonite clay: 0, 1, 10, 40, 100 and 300 mg/L). Clearance rates were estimated using flow cytometry and chlorophyll‐a with both methods giving similar results. Biodeposition rates were estimated by weighing the mussel biodeposits at the end of each trial.We found that SS concentrations did not have a significant effect on the clearance rates of algae, which ranged from 0.033 to 0.438 L mussel−1 hr−1, with an overall median of 0.118 L mussel−1 hr−1. By contrast, increasing SS concentrations had a significant effect on the clearance of sediment from the water column, with clearance rates peaking at 10 mg/L and declining thereafter. Clearance rates of suspended sediment ranged from 0.046 to 0.512 L mussel−1 hr−1, with an overall median of 0.182 L mussel−1 hr−1.We further detected a significant effect of SS concentration on the production of biodeposits, with inorganic and organic content in the biodeposits increasing as sediment concentrations increased. These results indicated that as SS concentrations increase, A. jacksoni rejects more inorganic and organic material in the form of pseudofaeces. This added maintenance work may result in an energetic cost to the mussel, and have potential consequences for growth, reproduction, and survival.Finally, population estimates of A. jacksoni in the lower Ovens River, Australia were combined with the clearance rates observed in the experiment, to estimate the potential impact mussel populations have in the clearance of total suspended matter from the open water of an unregulated south‐eastern Australian lowland river. A. jacksoni occurred patchily throughout the 35‐km section of the river surveyed, with an average overall mean and maximum density of 0.68, and 35 individuals/m, respectively. We estimate that under scenarios of 1 and 300 mg/L SS, the lower Ovens River mussel population can potentially clear up to 2–4 ML water/day, which equates to c. 1–2% of the average daily discharge in late summer, while transferring around 5.4–15.6 kg/day dry weight of organic material to the river bed.Increased SS concentrations negatively affect freshwater mussels around the world and many respond by reducing their clearance rates, but this was not the case for A. jacksoni. Instead, A. jacksoni increased production of inorganic and organic biodeposits rejecting more food particles in the form of pseudofaeces. For A. jacksoni and other species showing this response, the added energetic cost has the potential to impact growth rates, reproduction, and population dynamics. [ABSTRACT FROM AUTHOR]

Context: The legume shrub/tree leucaena (Leucaena leucocephala (Lam.) de Wit.) is highly regarded as a cattle fodder, although it is considered an environmental weed in many parts of Australia. Aims: We investigated the feasibility of developing a forage variety of triploid leucaena through interspecific hybridisation that is sterile (without seeds) as a strategy to mitigate its weed risk. Methods: A conventional breeding strategy was used to create triploid hybrids from hand-crosses between tetraploid and diploid species of leucaena. Molecular markers were developed to confirm successful crosses and flow cytometry was used to confirm the ploidy level. The plants are being evaluated for flowering behaviour, seed and herbage production across two environmentally diverse sites at Carnarvon and Kununurra in northern Western Australia. Key results: During 2018/19 and 2019/20, 2260 crosses between 45 different combinations of diploid females by tetraploid males and their reciprocals have created over 3000 putative triploid hybrid plants. This is the first time that triploids have successfully been created in Australia and 10 new parental combinations were created as a world-first. Conclusions: A cohort of sterile triploid plants has been identified and selections will be made based on their edible biomass productivity, insect tolerance, capacity for regrowth after cutting, nutritive value and plant chemistry (including anti-methanogenic responses). A sterile leucaena variety will have application not only in Australian grazing systems but also in other tropical regions of the world. Implications: A cost-effective technology for mass vegetative propagation of leucaena will be required for commercialisation of a sterile variety. The legume shrub/tree leucaena is highly regarded as a tropical cattle fodder, although it is considered an environmental weed in many parts of the world. In an Australian first, we report the successful creation of triploid leucaena germplasm through interspecific hybridisation. These give rise to sterile adults (without seeds), mitigating weed risk. Over 2000 hand-crosses between 45 different combinations of diploid and tetraploid leucaena species have created a cohort of sterile plants to evaluate for field performance. [ABSTRACT FROM AUTHOR]

In recent times, numerous and significant technological and supportive changes have taken place in Australian transplantation. These changes are often deployed without the wider clinical community having a full understanding of what has brought about these changes and the impacts they have. Here, we aim to clarify the reasoning behind these changes and shed light on potential future endeavours to improve patient outcomes. [ABSTRACT FROM AUTHOR]

Background: Platelet hyperactivity has a crucial role in initiating vascular thrombosis and subsequent cardiovascular disease (CVD).Objective: This study aimed to assess the effect of anthocyanins (AC) on platelet aggregation and activation and lipid profile.Study Design: A total of 26 healthy participants consumed 320 mg of AC/day in the form of Medox® capsules for 28 days.Setting: This study was conducted in the laboratories of the School of Medical Sciences, Griffith University, Gold Coast, Australia.Participants: A total of 26 randomly recruited healthy 25- to 75-year-old participants completed this study.Primary Outcome Measures: Fasting blood samples were collected pre- and post-the intervention period to perform platelet activation studies by measuring platelet surface marker expression of CD41a and P-selectin, and platelet-monocyte aggregates in adenosine diphosphate (ADP) stimulated platelets. Platelet aggregation studies were performed by stimulating platelets with various agonists such as ADP, collagen and arachidonic acid. Full blood examination, coagulation and biochemistry profile analyses were also performed pre- and post-intervention. Flow cytometric analysis showed a significant effect of AC on the expression of P-selectin as measured by the platelet surface expression of CD62p.Results: There was a significant reduction of ADP-stimulated platelet aggregation. Hematologic analysis showed a significant reduction of mean platelet volume, mean cell hemoglobin, and mean cell hemoglobin concentration. Coagulation analysis demonstrated significant attenuation of fibrinogen level in the blood.Conclusion: This study showed inhibition of platelet activity, platelet aggregation and mean platelet volume (MPV). These results suggest that AC has a positive impact on attenuating platelet activity, which might minimize thrombotic risk. [ABSTRACT FROM AUTHOR]

Introduction: KLF1 is an essential transcriptional activator that drives erythropoiesis. KLF1 variants can result in the Inhibitor of Lutheran, or In(Lu), phenotype where red blood cells (RBCs) have reduced BCAM (LU) and CD44 (IN). Other RBC surface molecules also have changed expression; however, there is controversy in the literature regarding which are truly impacted. We aimed to investigate KLF1 variants in the Australian population.Study Design and Methods: In(Lu) samples were sourced through screening and through the RBC reference laboratory. Blood donor samples (8036) were screened to identify weakened/absent Lub antigen. Samples were genotyped by massively parallel sequencing, while surface carbohydrates and blood group molecules were assessed by flow cytometry. Hemoglobin (Hb) types were analyzed by high-performance liquid chromatography.Results: Four of 8036 donors were identified to be In(Lu), and two previously identified In(Lu) samples were provided from the RBC reference laboratory. Five different KLF1 variants were identified; two were novel: c.954G>C/p.Trp318Cys and c.421C>T/p.Arg141*. BCAM and CD44 were reduced in all samples, consistent with previous reports. As a group, In(Lu) RBCs had reduced CD35 (KN), ICAM4 (LW), and CD147 (OK), and demonstrated increased binding of lectins ECA and SNAI. One In(Lu) sample had elevated HbF and another elevated HbA2.Conclusion: Different KLF1 variants may potentially produce variable phenotypes. A framework for investigating KLF1 variants and their phenotypic impact has been provided. In the future, given available international databases, further testing algorithms (as advocated here) will allow for correlation of phenotype with genotype and therefore accurately document this variability between KLF1 variants. [ABSTRACT FROM AUTHOR]

Premise of research. The Australian members of the genus Craspedia (Asteraceae: Gnaphalieae) are known to contain polyploids, but only a very limited number of chromosome counts are available, and a systematic overview of ploidy levels and genome sizes is lacking. In the present study, we aimed to document chromosome number variation across the genus in Australia, test whether polyploids occur more often in alpine than in lowland environments, and test whether there is a proportional increase in genome size with polyploidy that would enable the use of flow cytometry as a proxy for chromosome counts in future studies. Methodology. We used flow cytometry to measure genome sizes (2C values) of 59 individual plants and obtained 45 chromosome counts from root tip squashes. Pivotal results. We found polyploidy to be common and documented intraspecific ploidy variation in several species, indicating multiple independent origins of polyploidy within the genus. Polyploidy was not more prevalent in high-elevation species than in lowland species. The highest ploidy levels cannot be differentiated on the basis of genome size. Conclusions. Occurrence in alpine habitats did not drive the evolution of polyploidy in Craspedia. Flow cytometry measurements of genome size cannot be used as a reliable proxy for the precise determination of higher ploidy levels across the genus. Patterns of genome evolution in Craspedia are complex, suggesting that detailed investigation will be necessary even for species-level studies or conservation management. [ABSTRACT FROM AUTHOR]

Circulating osteogenic progenitor (COP) cells are considered as surrogates of the mesenchymal repository in the body. In this study, we hypothesized that COP cells decrease with age and that lower levels of COP cells are associated with greater frailty and disability in older persons. Using well-established clinical criteria, we quantified physical performance and disability and stratified frailty in a random sample of community-dwelling individuals enrolled in the Nepean Osteoporosis and Frailty (NOF) Study (mean age 82.8; N = 77; 70% female; 27 nonfrail, 23 prefrail, and 27 frail). Percentage of COP cells was quantified by flow cytometry. Logistic regression models estimated the relationship between the percentage of COP cells and prevalent disability, poor physical performance, and frailty. We found that aging is associated with a significant decrease in COP cells (p < .001). Lower percentages of COP cells were associated with disability and poor physical performance (p < .001). Older adults with COP cells in the lower quartile were more likely to be frail (odds ratio 2.65, 95% confidence interval 2.72-3.15, p < .001). In conclusion, COP cells in the circulation decrease with age. Lower percentages of COP cells in late life are associated with prevalent frailty and disability. Further longitudinal studies are needed to understand COP cells as a risk stratifier, biomarker, or therapeutic target and to predict disability in frail older persons. [ABSTRACT FROM AUTHOR]

Coastlines, including estuaries, mudflats, and beaches, are particularly susceptible to plastic pollution, which can accumulate from both marine and terrestrial sources. While numerous studies have confirmed the presence of microplastics (1-5 mm) along coastlines, few have focused on very small particles (<1 μm) or quantified exposure within the organisms that inhabit these areas, such as shorebirds. Here, we quantified small plastics (200 nm-70 μm) in two resident shorebird species in Tasmania, and compared this to quantities found in the surrounding sediments in order to investigate the potential exposure and transfer of particles within these ecosystems. Analysis was performed using a combination of flow cytometry for quantification of micro- and nanoplastics (200 nm-70 μm), and μm-FT-IR for validation and polymer identification of particles >5.5 × 5.5 μm. Micro- and nano-plastics were detected in 100% of guano samples from surface-feeding Eastern Hooded Plovers (Thinornis cucullatus) and 90% of Australian Pied Oystercatcher (Haematopus longirostris) guano, a species that forages for coastal invertebrates at 60-90 mm depth, and 100% of beach sediments. Hooded Plover guano contained 32 × more plastics, on average, than Pied Oystercatcher guano. Interestingly, the abundance of plastic particles within sediments collected from shorebird foraging sites did not appear to have a significant effect on the number of plastics the birds had ingested, suggesting the difference between species is likely a result of other variables, such as prey selection. The results of this study highlight the importance of including techniques that provide quantitative data on the abundance and size of the smallest possible particle sizes, and demonstrate the significant proportion of small plastics that are 'missed' using standard analysis tools., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Karli Mylius reports financial support was provided by BirdLife Tasmania., (Copyright © 2022. Published by Elsevier Ltd.)

Plastic ingestion has been documented in a plethora of taxa. However, there is a significant gap in the detection of nano- and ultrafine particles due to size limitations of commonly used techniques. Using two Australian seabird species as case studies, the flesh-footed shearwater (FFSH) Ardenna carneipes and short-tailed shearwater (STSH) A. tenuirostris , we tested a novel approach of flow cytometry to quantify ingested particles <70 μm in the fecal precursor (guano; colon and cloacal contents) of both species. This method provided the first baseline data set for these species for plastics in the 200 nm-70 μm particle size ranges and detected a mean of 553.50 ± 91.21 and 350.70 ± 52.08 plastics (count/mg fecal precursor, wet mass) in STSH and FFSH, respectively, whereas Fourier transform infrared spectroscopy (FT-IR) provided accurate measurements of polymer compositions and quantities in the size range above 5.5 × 5.5 μm 2 . The abundance of nano- and ultrafine particles in the guano (count/mg) was not significantly different between species ( p -value = 0.051), suggesting that foraging distribution or prey items, but not species, may contribute to the consumption of small plastics. In addition, there was no correlation between macroplastics in the stomach compared to the fecal precursor, indicating that small particles are likely bioaccumulating (e.g., through shedding and digestive fragmentation) and/or being directly ingested. Combining flow cytometry with FT-IR provides a powerful quantitative and qualitative analysis tool for detecting particles orders of magnitude smaller than that are currently explored with wider applications across taxa and marine environments.

Sarcostemma viminale (L.) R.Br, also known as caustic or milk bush, is a semi-succulent plant commonly found in the North West of Australia. Local Aboriginal populations have long used the milky white sap from this plant to treat skin cancers. An ethanol extract from S. viminale was tested by exposing the RAW264.7 cell line as an in vitro murine macrophage model, to the extract. Flow cytometric analysis was performed to determine if S. viminale skewed macrophages towards a pro-inflammatory or anti-inflammatory phenotype using a number of cell surface markers. Cell culture supernatants were also analysed by cytometric bead array to determine if S. viminale exposed macrophages produced pro-inflammatory or anti-inflammatory cytokines. After exposure to S. viminale, a significantly greater number of macrophages expressed pro-inflammatory major histocompatibility complex (MHC) class II molecules and significantly greater expression levels of the dendritic cell marker CD11c. Cytometric bead array analysis found that S. viminale induced significant amounts of the potent pro-inflammatory cytokine tumour necrosis factor (TNF) from macrophages. The markers CD40 and ICAM-1 were expressed but were not significantly different from the controls. Also, significantly higher expression of CX3CR1 indicated that macrophages were preparing to migrate. No anti-inflammatory cytokines were produced. No significant production of NO, IL-6, IFN-γ or IL-12 was found. These results demonstrate that S. viminale drives resting macrophages into a pro-inflammatory phenotype, reminiscent of activated immature dendritic cells. If this activation could be achieved in the peri-tumour environment, then S. viminale could be useful as an adjunct therapy for skin cancer. [ABSTRACT FROM AUTHOR]

AIM: To collate the expert opinion of Australian dairy practitioners on the relative importance of risk factors for subclinical mastitis in dairy cattle. METHODS: A modified Delphi technique was used to collect the data over two rounds. First, participants were asked to complete a survey involving ranking the level of importance of 42 risk factors on the incidence of subclinical mastitis on a scale from 1 to 10 for two categories of subclinical mastitis; contagious and environmental. After presenting and discussing the results of the first survey, the participants were asked to complete the same survey a second time. To rank the risk factors and identify the consensus amongst the participants, the median and total variation of answers were calculated and compared between the two surveys. RESULTS: The most important factors identified by the respondents after the second survey for contagious subclinical mastitis were: Teat Disinfection Post-milking, Management of High Cell Count Cows and Presence of Chronically Infected Mastitis Cows in the Herd. The most important factors for environmental subclinical mastitis were Cleanliness of the Environment and Technique of Teat Disinfection Post-milking. CONCLUSIONS: A movement toward consensus for the more important factors and a movement away from consensus for the less important factors in the second survey were observed. The most important factors for subclinical mastitis were found to be: teat disinfection post-milking, management of high cell count cows, presence of chronically infected mastitis cows in the herd and cleanliness of the environment. [ABSTRACT FROM AUTHOR]

Context: Euphorbia hirta L. (Euphorbiaceae) ( E. hirta) is a tree locally used as a traditional medicine in Africa and Australia to treat numerous diseases such as hypertension, respiratory ailments, tumors, and wounds, and it has reported antiallergic, antipyretic, anti-inflammatory activities, etc. Objective: This study evaluated the ability of fresh leaves of E. hirta ethanol extract to inhibit the intracellular tumor necrosis factor α (TNF-α) level in the synovial fluid and neutrophils in lipopolysaccharide (LPS)-induced inflamed rat knees. Materials and methods: Female Wister albino rats 140-160 g were used . E. hirta ethanol extract was given orally at 25, 50, 100, and 200 mg/kg, 2 h before an intra-articular (i.a.) injection of LPS. Two and three hours later, synovial fluid and neutrophils levels of intracellular TNF-α production were measured. Results: In the time course of the experiment, E. hirta maximum inhibition at 100 and 200 mg/kg (p.o.) dose showed 16.5 ± 1.34 and 14.4 ± 1.30% of synovial fluid, 4.26 ± 0.36 and 3.78 ± 0.29% of neutrophils levels of intracellular TNF-α productions at 2 h after LPS injection. LPS control displayed 22.97 ± 1.61 and 6.78 ± 0.34% of synovial fluid and neutrophils levels of intracellular TNF-α at 2 h after LPS injection. Intracellular TNF-α was also estimated at 3 h after LPS injection. Discussion and conclusion: The LPS-injected rat knee model gives a comparative study of acute anti-inflammatory responses. E. hirta inhibition of proinflammatory intracellular cytokine TNF-α production with LPS-induced inflamed rat knee is of great importance in defining the anti-arthritic potential of E. hirta. [ABSTRACT FROM AUTHOR]

Background. In Western Europe, North America, and Australia, large cohort collaborations have been able to estimate the short-term CD4 cell count-specific risk of AIDS or death in untreated human immunodeficiency virus (HIV)- infected adults with high CD4 cell counts. In sub-Saharan Africa, these CD4 cell count-specific estimates are scarce. Methods. From 1996 through 2006, we followed up 2 research cohorts of HIV-infected adults in Cote d'Ivoire. This included follow-up off antiretroviral therapy (ART) across the entire spectrum of CD4 cell counts before the ART era, and only in patients with CD4 cell counts >200 cells/lL once ART became available. Data were censored at ART initiation. We modeled the CD4 cell count decrease using an adjusted linear mixed model. CD4 cell count- specific rates of events were obtained by dividing the number of first events occurring in a given CD4 cell count stratum by the time spent in that stratum. Results. Eight hundred sixty patients were followed off ART over 2789 person-years (PY). In the ≥650, 500-649, 350-499, 200-349, 100-199, 50-99, and 0-49 cells/lL CD4 cell count strata, the rates of AIDS or death were 0.9, 1.7, 3.7, 10.4, 30.9, 60.8, and 99.9 events per 100 PY, respectively. In patients with CD4 cell counts ≥200 CD4 cells/lL, the most frequent AIDS-defining disease was tuberculosis (decreasing from 4.0 to 0.6 events per 100 PY for 200-349 and ≥650 cells/lL, respectively), and the most frequent HIV non-AIDS severe diseases were visceral bacterial diseases (decreasing from 9.1 to 3.6 events per 100 PY). Conclusions. Rates of AIDS or death, tuberculosis, and invasive bacterial diseases are substantial in patients with CD4 cell counts ≥200 cells/lL. Tuberculosis and bacterial diseases should be the most important outcomes in future trials of early ART in sub-Saharan Africa. [ABSTRACT FROM AUTHOR]

Abstract: Changes in oceanic circulation and physiochemical parameters due to climate change may alter the distribution, structure and function of marine microbial communities, thereby altering the action of the biological carbon pump. One area of current and predicted future change is the sub-Antarctic zone (SAZ) to the southeast of Tasmania, Australia, where a southward shift in westerly winds appears to be forcing warmer and macronutrient-poor subtropical waters into the sub-Antarctic zone (SAZ). We investigated the impact of these subtropical waters on the microbial community of the SAZ on the SAZ-Sense cruise during the austral summer of 2007. The abundance of pico- and nanoeukaryotic algae, cyanobacteria, heterotrophic nanoflagellates, bacteria and viruses was determined by flow cytometry at stations in the Polar Frontal Zone (PFZ), the SAZ and in Subtropical Zone (STZ). Using cluster and similarity profile analyses on integrated microbial abundances over the top 200m, we found that microbial communities located in the potential future SAZ to the southeast of Tasmania formed two distinct groups from those of the remainder of the SAZ and the PFZ. In the waters of the potential future SAZ, shallow mixed layers and increased iron concentrations elevated cyanobacterial, bacterial and viral abundances and increased percentage high DNA bacteria, resulting in communities similar to those of subtropical waters. Conversely, waters of the PFZ exhibited relatively low concentrations of autotrophic and heterotrophic microbes and viruses, indicative of the iron limitation in this region. A Distance Based Linear Model determined that salinity and nitrogen availability (nitrate, nitrite and ammonia concentrations) were the most influential environmental parameters over the survey, explaining 72% of the variation in microbial community structure. The microbial community of the potential future SAZ showed a shift away from particulate carbon export from the photic zone towards increased production by smaller cells, increased significance of the microbial loop and viral lysis. These changes would promote carbon recycling within the photic zone, thereby potentially decreasing the capacity of the future SAZ to absorb CO2. [Copyright &y& Elsevier]

Aim To assess associations between invasiveness, genome size and species traits in Australian Acacia species introduced outside their native range. Location Global. Methods Holoploid genome size was determined by flow cytometry for 92 species in Acacia subgenus Phyllodineae native to Australia. These data were used to test whether genome size was significantly different for invasive ( n = 21; species known to be established and spreading in foreign environments) and non-invasive ( n = 71; species that have been introduced to foreign environments but which are not known to be spreading) species. Data for five functional traits [seed mass, specific leaf area (SLA), relative growth rate (RGR), maximum height and dispersal mode] and three characteristics of native range (size, temperature range and precipitation range) were used to test for univariate and multivariate relationships between (1) invasiveness and traits and (2) genome size and traits. Results Genome size ranged from 1.20 to 2.13 pg/2C and was not significantly smaller in invasive compared with non-invasive acacias. However, invasive acacias were found to be taller and possess a larger native range size and a wider range of annual precipitation when compared with non-invasive acacias. Multivariate analyses revealed significant differences between invasive and non-invasive acacias (PERMANOVA; P < 0.01) driven largely by differences in native range characteristics. We detected a positive relationship between genome size and SLA ( P = 0.02) and elaiosome dispersal mode ( P < 0.01) in analyses across species, but these findings were not supported by evolutionary divergence analyses. Main conclusions Genome size variation does not underpin variation in traits associated with the invasive/non-invasive dichotomy in introduced acacias, probably because of the very small DNA values in the Acacia species studied. Acacias introduced into new environments are most likely to become invasive if they are tall shrubs or trees and are widely distributed in their native range. [ABSTRACT FROM AUTHOR]

Apoptosis is one of the most critical forms of defense against cancer, and the induction of apoptosis by dietary polyphenols represents significant potential for cancer preventive activity. The present study examined polyphenols extracted from selected native Australian fruits-Illawarra plum (Podocarpus elatus Endl., Podocarpaceae), Kakadu plum (Terminalia ferdinandiana Exell, Combretaceae), muntries (Kunzea pomifera F. Muell., Myrtaceae), and native currant (Acrotriche depressa R.Br., Epacridaceae)-for antiproliferative activity against a panel of cancer and normal cell lines. Each fruit selectively inhibited the growth of cancer cell lines in a dose-dependent manner. The mechanism of growth inhibition of the human promyelocytic leukaemia cells (HL-60) was determined to be apoptosis by morphological assessment, DNA fragmentation, flow cytometry, and caspase-3 induction. Furthermore, Kakadu plum was found to activate caspase-7, -9, and poly (ADP-ribose) polymerase (PARP), suggesting it acts via the intrinsic apoptosis pathway. The same fruit also caused direct DNA damage in colon adenocarcinoma cells (HT-29) as detected using the cytokinesis-block micronucleus cytome (CBMN Cyt) assay. [ABSTRACT FROM AUTHOR]

Abstract: Marine sponges are key players in the transfer of carbon from the pelagic microbial food web into the benthos. Selective uptake of prokaryotic picoplankton (<2μm) by a demosponge (Callyspongia sp.), and carbon flux through this process, were examined for the first time in the oligotrophic coastal waters of southwestern Australia, where sponge abundance and biodiversity ranks among the highest in the world. Water sampling and flow rate measurements were conducted over five sampling occasions following the InEx method of Yahel et al. (2005), with heterotrophic bacteria and autotrophic Synechococcus cyanobacteria identified and enumerated by flow cytometry. Callyspongia sp. demonstrated high filtration efficiencies, particularly for high DNA (HDNA) bacteria (up to 85.3% in summer 2008) and Synechococcus (up to 91.1% in autumn 2007), however efficiency varied non-uniformly with time and food type (p <0.01). Overall filtration efficiency for Synechococcus (86.6±6.3%; mean ± s.d.) was always significantly higher (p <0.05) than for low DNA (LDNA) bacteria (40±17.2%), except during winter 2007 (p =0.14) when ambient Synechococcus concentrations were lowest. When compared to ambient abundances of the different food types, Callyspongia sp. exhibited consistently negative selectivity for LDNA bacteria and positive selectivity for Synechococcus, while HDNA bacteria was generally a neutral or positive selection. The total carbon removal rate (sum of all prokaryotic picoplankton cells), calculated on a per unit area basis, varied significantly with time (p <0.01), with lowest rates recorded during the winter (0.5±0.4–0.6±0.8mgCm−2 d−1) and highest values recorded in summer (3.5±1.9mgCm−2 d−1). These flux estimates quantify the role of a demosponge species in the ultimate fate of prokaryotic picoplankton within the nearshore food webs of southwestern Australia, and support the conclusion that sponges actively select food particles that optimise their nutritional intake. [Copyright &y& Elsevier]

Globally, fish populations are in decline from overfishing, habitat destruction and poor water quality. Recent mass fish deaths in Australia's Murray-Darling Basin highlight the need for improved conservation methods for endangered fish species. Cryopreservation of testicular tissue allows storage of early sperm precursor cells for use in generating new individuals via surrogacy. We describe successful isolation and cryopreservation of spermatogonia in an Australian rainbowfish. Testis histology showed rainbowfish spermatogonia are large (> 10 μm) and stain positive for Vasa, an early germ line-specific protein. Using size-based flow cytometry, testis cell suspensions were sorted through "A" (> 9 μm) and "B" gates (2-5 μm); the A gate produced significantly more Vasa-positive cells (45.0% ± 15.2%) than the "B" gate (0.0% ± 0.0%) and an unsorted control (22.9% ± 9.5%, p < 0.0001). The most successful cryoprotectant for "large cell" (> 9 μm) viability (72.6% ± 10.5%) comprised 1.3 M DMSO, 0.1 M trehalose and 1.5% BSA; cell viability was similar to fresh controls (78.8% ± 10.5%) and significantly better than other cryoprotectants (p < 0.0006). We have developed a protocol to cryopreserve rainbowfish testicular tissue and recover an enriched population of viable spermatogonia. This is the first step in developing a biobank of reproductive tissues for this family, and other Australian fish species, in the Australian Frozen Zoo.

Background: Antigen-specific IgE binds the Fcε receptor I (FcεRI) expressed on several types of immune cells, including dendritic cells (DCs). Activation of FcεRI on DCs in atopics has been shown to modulate immune responses that potentially contribute to asthma development. However, the extent to which DC subsets differ in FcεRI expression between atopic children with or without asthma is currently not clear. This study aimed to analyse the expression of FcεRI on peripheral blood mononuclear cells (PBMCs) from atopic children with and without asthma, and non-atopic/non-asthmatic age-matched healthy controls., Methods: We performed multiparameter flow cytometry on PBMC from 391 children across three community cohorts and one clinical cohort based in Western Australia., Results: We confirmed expression of FcεRI on basophils, monocytes, plasmacytoid and conventional DCs, with higher proportions of all cell populations expressing FcεRI in atopic compared to non-atopic children. Further, we observed that levels of FcεRI expression were elevated across plasmacytoid and conventional DC as well as basophils in atopic asthmatic compared to atopic non-asthmatic children also after adjusting for serum IgE levels., Conclusion: Our data suggest that the expression pattern of FcεRI on DC and basophils differentiates asthmatic from non-asthmatic atopic children. Given the significant immune modulatory effects observed as a consequence of FcεRI expression, this altered expression pattern is likely to contribute to asthma pathology in children., (© 2019 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Objective: Bronchiectasis (BE) in children is common in some communities including Indigenous children in Australia. Relatively little is known about the nature of systemic inflammation in these children, especially the contribution of specific pro-inflammatory and cytotoxic lymphocyte subsets: T-cells, natural killer (NK) cells and NKT-like cells. We have shown that these cells produce increased cytotoxic (granzyme b and perforin) and inflammatory (IFNγ and TNFα) mediators in several adult chronic lung diseases and hypothesised that similar changes would be evident in children with BE., Methods: Intracellular cytotoxic mediators perforin and granzyme b and pro-inflammatory cytokines were measured in T cell subsets, NKT-like and NK cells from blood and bronchoalveolar samples from 12 children with BE and 10 aged-matched control children using flow cytometry., Results: There was a significant increase in the percentage of CD8+ T cells and T and NKT-like subsets expressing perforin/granzyme and IFNγ and TNFα in blood in BE compared with controls. There was a further increase in the percentage of pro-inflammatory cytotoxic T cells in Indigenous compared with non-Indigenous children. There was no change in any of these mediators in BAL., Conclusions: Childhood bronchiectasis is associated with increased systemic pro-inflammatory/cytotoxic lymphocytes in the peripheral blood. Future studies need to examine the extent to which elevated levels of pro-inflammatory cytotoxic cells predict future co-morbidities.

Some Pseudomonas aeruginosa strains including Australian Epidemic Strain-1 (AES-1 or AUS-01) cause persistent chronic infection in cystic fibrosis (CF) patients, with greater morbidity and mortality. Factors conferring persistence are largely unknown. Previously we analysed the transcriptomes of AES-1 grown in Luria broth, nematode growth medium for Caenorhabditis elegans assay (both aerobic) and artificial sputum medium (mainly hypoxic). Transcriptional comparisons included chronic AES-1 strains against PAO1 and acute AES-1 (AES-1R) against its chronic isogen (AES-1M), isolated 10.5 years apart from a CF patient and not eradicated in the meantime. Prominent amongst genes downregulated in AES-1M in all comparisons was homogentisate-1-2-dioxygenase (hmgA); an oxygen-dependent gene known to be mutationally deactivated in many chronic infection strains of P. aeruginosa. To investigate if hmgA downregulation and deactivation gave similar virulence persistence profiles, a hmgA mutant made in UCBPP-PA14 utilising RedS-recombinase and AES-1M were assessed in the C. elegans virulence assay, and the C57BL/6 mouse for pulmonary colonisation and TNF-α response. In C. elegans, hmgA deactivation resulted in significantly increased PA14 virulence while hmgA downregulation reduced AES-1M virulence. AES-1M was significantly more persistent in mouse lung and showed a significant increase in TNF-α (p<0.0001), sustained even with no detectable bacteria. PA14ΔhmgA did not show increased TNF-α. This study suggests that hmgA may have a role in P. aeruginosa persistence in chronic infection and the results provide a starting point for clarifying the role of hmgA in chronic AES-1.

Tasmanian devils (Sarcophilus harrisii) risk extinction from a contagious cancer, devil facial tumour disease (DFTD) in which the infectious agent is the tumor cell itself. Because devils are unable to produce an immune response against the tumor cells no devil has survived 'infection'. To promote an immune response we immunized healthy devils with killed DFTD tumor cells in the presence of adjuvants. Immune responses, including cytotoxicity and antibody production, were detected in five of the six devils. The incorporation of adjuvants that act via toll like receptors may provide additional signals to break 'immunological ignorance'. One of these devils was protected against a challenge with viable DFTD cells. This was a short-term protection as re-challenge one year later resulted in tumor growth. These results suggest that Tasmanian devils can generate immune responses against DFTD cells. With further optimization of immune stimulation it should be possible to protect Tasmanian devils against DFTD with an injectable vaccine., (Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.)

The functional properties, including antioxidant and chemopreventative capacities as well as the inhibitory effects on angiotensin-converting enzyme (ACE), α-glucosidase and pancreatic lipase, of three Australian-grown faba bean genotypes (Nura, Rossa and TF(Ic*As)*483/13) were investigated using an array of in vitro assays. Chromatograms of on-line post column derivatisation assay coupled with HPLC revealed the existence of active phenolics (hump) in the coloured genotypes, which was lacking in the white-coloured breeding line, TF(Ic*As)*483/13. Roasting reduced the phenolic content, and diminished antioxidant activity by 10–40 % as measured by the reagent-based assays (diphenylpicrylhydrazyl, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and oxygen radical absorbance capacity) in all genotypes. Cell culture-based antioxidant activity assay (cellular antioxidant activity) showed an increase of activity in the coloured genotypes after roasting. Faba bean extracts demonstrated cellular protection ability against H2O2-induced DNA damage (assessed using RAW264.7 cells), and inhibited the proliferation of all human cancer cell lines (BL13, AGS, Hep G2 and HT-29) evaluated. However, the effect of faba bean extracts on the non-transformed human cells (CCD-18Co) was negligible. Flow cytometric analyses showed that faba bean extracts successfully induced apoptosis of HL-60 (acute promyelocytic leukaemia) cells. The faba bean extracts also exhibited ACE, α-glucosidase and pancreatic lipase inhibitory activities. Overall, extracts from Nura (buff-coloured) and Rossa (red-coloured) were comparable, while TF(Ic*As)*483/13 (white-coloured) contained the lowest phenolic content and exhibited the least antioxidant and enzyme inhibition activities. These results are important to promote the utilisation of faba beans in human diets for various health benefits. [ABSTRACT FROM PUBLISHER]

Introduction: Impaired T cell function in sepsis is associated with poor outcome, but the mechanisms are unclear. In cancer, arginase-expressing myeloid derived suppressor cells (MDSCs) deplete arginine, impair T cell receptor CD3 zeta-chain expression and T cell function and are linked to poor clinical outcome, but their role during acute human infectious disease and in particular sepsis remains unknown. Hypoarginemia is prevalent in sepsis. This study aimed to determine whether neutrophils that co-purify with PBMC express arginase, and if arginine depletion constrains T cell CD3 zeta-chain expression and function in human sepsis., Methods: Using flow cytometry, cell culture, HPLC, arginase activity and mRNA detection, our study examined whether neutrophils, with reduced buoyant density isolated in the Ficoll interface, metabolise L-arginine and suppress T cell proliferation in sepsis. A total of 35 sepsis patients (23 with septic shock) and 12 hospital controls in a tertiary referral hospital in tropical Australia were evaluated., Results: Only sepsis patients had interphase neutrophils, neutrophils co-purifying with mononuclear cells (≤1.077 specific gravity). The percentage of interphase neutrophils in sepsis was proportional to sepsis severity and correlated with plasma IL-6 concentrations. Ex vivo, sepsis-derived interphase neutrophils expressed arginase, metabolised culture L-arginine and suppressed T cell proliferation and CD3 zeta-chain expression. In vivo, in septic shock there was a longitudinal inverse association between interphase neutrophil number and CD3 zeta-chain expression. Depletion or inhibition of interphase neutrophils in vitro restored zeta-chain expression and T cell function., Conclusions: For the first time during an acute human infection, interphase neutrophils that express arginase were found to circulate in sepsis, in proportion to disease severity. These neutrophil-MDSCs impair T cell CD3 zeta-chain expression and T cell function via L-arginine metabolism, and likely contribute to the T cell dysfunction seen in sepsis. Modulation of neutrophil-MDSC or their downstream effects warrant consideration as targets for novel adjunctive therapies in sepsis.

An independent pool of 16 incompatible live donor-recipient pairs registered at the Vienna transplant unit was applied to test whether virtual crossmatch allocation used in the Australian kidney paired donation (KPD) program reliably predicts negative crossmatches. High resolution HLA data were entered into the computer-matching algorithm and allocation was performed excluding any DSA>2000MFI. CDC and flow crossmatch data of recipients against any of the donors were available for 112 crossmatch combinations. The computer program identified 19 possible pairings in 2-way or 3-way chains in multiple combinations. The top ranked combination included one 3-way and two 2-way ABO-compatible chains. Where crossmatches were available all recipients were CDC crossmatch negative with the computer-matched donor. Excluding allocation of KPD donors in the presence of DSA>2000MFI had a negative predictive of 99.9% for CDC and 96.4% for flow crossmatch. In the 12 pairings with ⩾1 DSA against crossmatched donors there was a negative CDC and flow crossmatch. These results show excellent correlation between matching using virtual crossmatch and actual crossmatch results. Using the 2000MFI cut-off the number of potentially unacceptable CDC and flow crossmatch positive pairings identified by virtual crossmatching is low, but some potential crossmatch negative pairings are missed., (Copyright © 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

In this study, a flow-cytometric cell cycle analysis method to assess instantaneous growth rate of whole larvae of the Australian barramundi Lates calcarifer was developed and validated. High-resolution DNA measurements of either fresh, frozen or RNAlater-preserved larvae (gap0-gap1, G(0) -G(1), coefficient of variation (c.v.) < 3, 4 and 5%, respectively) enabled the deconvolution of the DNA histogram and assignment of the proportion of nuclei into cell cycle compartments G(0) -G(1), S (DNA synthesis) and G(2) -M (Gap2-Mitosis). This technique can be also used for individual fish tissues such as brain, liver, fin and muscle. For the first time, the combined proportion of replicating nuclei (into S and G(2) -M phases) of whole fish larvae and absolute growth rate in length (mm day(-1)) has been correlated in commercial aquaculture conditions. Fast growing L. calcarifer larvae had an overall hyperplasia advantage as indicated by a greater proportion of cells in the S+G(2) -M phase compared with slow growing larvae, which might explain the increasing differences in size during culture. In a fasting trial, larvae ceased growth while maintaining the constant initial rates of cell division throughout a 6 day period. For a highly fed fast growing control group, cell division rates significantly increased after day 4. Flow-cytometric cell cycle analysis of whole fish larvae may provide fish biologists and aquaculturists with a better understanding of how cell division rates influence early growth in natural and artificial environments., (© 2012 The Authors. Journal of Fish Biology © 2012 The Fisheries Society of the British Isles.)

Background: Environmental changes, including declining microbial exposure, have been linked with the rising incidence of allergic and autoimmune diseases in 'western' populations. This potentially occurs by altering early development of immuno-regulatory pathways including T regulatory cells (T(reg)). There is now increasing evidence that such conditioning begins in utero., Methods: We compared neonatal T(reg) from children born under typical western conditions (Australia, AUS) with those of neonates born under more traditional conditions of high microbial burden (Papua New Guinea, PNG)., Results: The frequency of neonatal T(reg), defined as CD4(+) Foxp3(+) CD127(-) CD25(+/high) was found to be higher in the cord blood of AUS compared to PNG newborns. However, cord T(reg) suppressive function in a small subset of children was qualitatively similar between PNG and AUS newborns in both a T(reg) depletion assay and a T(reg) supplementation assay., Conclusions: These findings do not support the hypothesis that living in a 'western' versus more traditional environment leads to poor induction or suppressive function of neonatal T(reg). However, environmentally-induced immuno-regulation may potentially occur via alternative mechanisms in PNG newborns that should now be investigated further., (© 2011 John Wiley & Sons A/S.)

Rationale: Approximately 100 million doses of bacille Calmette-Guérin (BCG) vaccine are given each year to protect against tuberculosis (TB). More than 20 genetically distinct BCG vaccine strains are in use worldwide. Previous studies suggest that BCG vaccine strain influences the immune response and protection against TB. Current data on which BCG vaccine strain induces the optimal immune response in humans are insufficient., Objectives: To compare the immune response to three different BCG vaccine strains given to infants at birth., Methods: Newborn infants in a tertiary women's hospital were immunized at birth with one of three BCG vaccine strains. A stratified randomization according to the mother's region of birth was used., Measurements and Main Results: The presence of mycobacterial-specific polyfunctional CD4 T cells measured by flow cytometry 10 weeks after immunization. Of the 209 infants immunized, data from 164 infants were included in the final analysis (BCG-Denmark, n = 54; BCG-Japan, n = 54; BCG-Russia, n = 57). The proportion of polyfunctional CD4 T cells was significantly higher in infants immunized with BCG-Denmark (0.013%) or BCG-Japan (0.016%) than with BCG-Russia (0.007%) (P = 0.018 and P = 0.003, respectively). Infants immunized with BCG-Japan had higher concentrations of secreted Th1 cytokines; infants immunized with BCG-Denmark had higher proportions of CD107-expressing cytotoxic CD4 T cells., Conclusions: There are significant differences in the immune response induced by different BCG vaccine strains in newborn infants. Immunization with BCG-Denmark or BCG-Japan induced higher frequencies of mycobacterial-specific polyfunctional and cytotoxic T cells and higher concentrations of Th1 cytokines. These findings have potentially important implications for global antituberculosis immunization policies and future tuberculosis vaccine trials.

There are limited data concerning the lethal and sublethal effects of environmental pollutants on Australian freshwater fish and consequently many of the Australian water quality guidelines are based on data from exotic fish species. This study used a flow cytometric assay to assess the effect of in vitro exposures to commonly used pesticides, on the phagocytic function and cellular composition of head kidney cells from four Australian native fish, i.e. crimson-spotted rainbowfish (Melanotaenia fluviatilis), silver perch (Bidyanus bidyanus), golden perch (Macquaria ambigua) and Murray cod (Maccullochella peelii). Head kidney immune cells were isolated from the four native fish and incubated (1 x 10(6) cells/mL) under previously optimised conditions to measure the phagocytosis of fluorescent-latex beads. These cell cultures were exposed to three classes of pesticides, i.e. the organotins, tributyltin and dibutyltin, the organochlorine endosulfan and the organophosphate chlorpyrifos. The in vitro organotin exposures were highly immunotoxic to head kidney cells from the Australian fish, although there were some differences in immunotoxic responses between species. At the highest concentration (i.e. 10 mg/L), endosulfan exposure resulted in the modulation of phagocytic responses in all species except for silver perch. Chlorpyrifos displayed little immunotoxicity, although there was a dose-dependent reduction in Murray cod lymphocytes. These studies describe the first investigation of the phagocytic response of Australian freshwater fish immunocytes in the presence of environmental pollutants, and will help to determine appropriate ecotoxicity testing for Australian freshwater environments.

Background and Objectives: The aim of the study was to evaluate a lyophilized anti-D immunoglobulin preparation to serve as a global standard for potency assays of anti-D immunoglobulin products., Materials and Methods: The candidate global standard, 01/572, was calibrated against the World Health Organization (WHO) International Reference Preparation (IRP) for anti-D immunoglobulin, human (68/419), along with two reserve candidate reference preparations, in an international collaborative study involving 25 laboratories in 15 countries. The United States Food and Drug Administration (US-FDA) Center for Biologics Evaluation and Research (CBER) Standard for anti-D immunoglobulin, Lot 3, was included for comparison. Most laboratories (20/25) performed AutoAnalyser methodology, competitive enzyme-linked immunoassay (EIA) and/or flow cytometry., Results: The overall mean potency of the candidate global standard, 01/572, was 284.5 international units (IU)/ampoule, with an interlaboratory variability, expressed as a percentage geometric coefficient of variation (% gcv), of 9.7. The mean potency of the US Standard was 859.4 IU/ml with an interlaboratory variability of 9.5% gcv, excluding an outlier. The mean potencies of the reserve preparations per ampoule/vial were 110.6 IU and 106.7 IU when calibrated against the IRP, and 112.2 IU and 106.6 IU when calibrated against 01/572, respectively, with interlaboratory % gcv values of 9.6-18.3 (excluding outliers)., Conclusions: Preparation 01/572 proved more suitable for use as a global standard than the reserve candidate preparations and was established, with an assigned potency of 285 IU/ampoule, by the WHO as the 2nd International Standard for anti-D immunoglobulin; by FDA-CBER as the Standard for anti-D immunoglobulin, Lot 4; and by the European Directorate for the Quality of Medicines (EDQM) as the 1st Biological Reference Preparation for anti-D immunoglobulin.

This study aimed to determine the prognostic significance of DNA ploidy and S-phase fraction (SPF) measurements in our laboratory for patients with node-negative breast cancer. Frozen tumors from axillary node-negative breast cancer patients (n = 50) treated at Westmead Hospital, NSW, between 1988 and 1991 were analysed by flow cytometry. The median duration of follow-up for all patients was 8.4 years. Forty-six specimens provided evaluable DNA histograms with 43% (n = 20) diploid and 56% (n = 26) aneuploid tumors identified. Comparisons of DNA ploidy status and SPF were made with traditional prognostic variables, which included age, menopausal status, tumor size, histologic grade and hormone receptor status. Our results showed that there was no significant difference in disease-free or overall survival between patients with diploid and aneuploid tumors. Histologic grade 3 tumors were more likely to be aneuploid and had higher SPF than grade 1 or 2 tumors. Patients with grade 3 tumors and a high SPF were four times more likely to relapse than the rest of the population. These results indicate that DNA flow cytometric analysis in our laboratory provides additional prognostic data that could be utilised alongside traditional clinical and histopathologic indicators for predicting outcome for patients.

The DNA content and proliferative activity of paraffin-embedded lymph node tissue from 111 patients with poor histology non-Hodgkin's lymphoma were measured by flow cytometry. These patients had been entered into a prospective randomized trial which, to date, has shown no survival difference by treatment arm. Forty-four (40%) samples showed evidence of aneuploidy with three samples having more than one aneuploid population. The aneuploid populations had a bimodal distribution with one group having a DNA index between 1.1-1.3 and the other 1.8-2.2. The incidence of aneuploidy did not correlate with age, stage or survival. In 56 diploid samples the S phase values below 10% had a significantly better survival than those with S phase values above 10% (P less than 0.011). For patients with diffuse large cell histology the corresponding discriminatory S phase value was 19% (P less than 0.009).