Preferential invasion by Plasmodium merozoites and the self-regulation of parasite burden

The preferential invasion of particular red blood cell (RBC) age classes may offer a mechanism by which certain species of Plasmodia regulate their population growth. Asexual reproduction of the parasite within RBCs exponentially increases the number of circulating parasites; limiting this explosion in parasite density may be key to providing sufficient time for the parasite to reproduce, and for the host to develop a specific immune response. It is critical that the role of preferential invasion in infection is properly understood to model the within-host dynamics of different Plasmodia species. We develop a simulation model to show that limiting the range of RBC age classes available for invasion is a credible mechanism for restricting parasite density, one which is equally as important as the maximum parasite replication rate and the duration of the erythrocytic cycle. Different species of Plasmodia that regularly infect humans exhibit different preferences for RBC invasion, with all species except P. falciparum appearing to exhibit a combination of characteristics which are able to selfregulate parasite density.

Tissue viability imaging of skin microcirculation following exposure to whole body cryotherapy (-110°C) and cold water immersion (8°C)

Cryotherapy is currently used in various clinical, rehabilitative, and sporting settings. However, very little is known regarding the impact of cooling on the microcirculatory response. Objectives: The present study sought to examine the influence of two commonly employed modalities of cryotherapy, whole body cryotherapy (WBC; -110°C) and cold water immersion(CWI; 8±1°C), on skin microcirculation in the mid- thigh region. Methods: The skin area examined was a 3 × 3 cm located between the most anterior aspect of the inguinal fold and the patella. Following 10 minutes of rest, 5 healthy, active males were exposed to either WBC for 3 minutes or CWI for 5 minutes in a randomised order. Volunteers lay supine for five minutes after treatment, in order to monitor the variation of red blood cell (RBC) concentration in the region of interest for a duration of 40 minutes. Microcirculation response was assessed using a non-invasive, portable instrument known as a Tissue Viability imaging system. After a minimum of seven days, the protocol was repeated. Subjective assessment of the volunteer’s thermal comfort and thermal sensation was also recorded. Results: RBC was altered following exposure to both WBC and CWI but appeared to stabilise approximately 35 minutes after treatments. Both WBC and CWI affected thermal sensation (p < 0.05); however no betweengroup differences in thermal comfort or sensation were recorded (p > 0.05). Conclusions: As both WBC and CWI altered RBC, further study is necessary to examine the mechanism for this alteration during whole body cooling.

Sequence variants in three loci influence monocyte counts and erythrocyte volume

Blood cells participate in vital physiological processes, and their numbers are tightly regulated so that homeostasis is maintained. Disruption of key regulatory mechanisms underlies many blood-related Mendelian diseases but also contributes to more common disorders, including atherosclerosis. We searched for quantitative trait loci (QTL) for hematology traits through a whole-genome association study, because these could provide new insights into both hemopoeitic and disease mechanisms. We tested 1.8 million variants for association with 13 hematology traits measured in 6015 individuals from the Australian and Dutch populations. These traits included hemoglobin composition, platelet counts, and red blood cell and white blood cell indices. We identified three regions of strong association that, to our knowledge, have not been previously reported in the literature. The first was located in an intergenic region of chromosome 9q31 near LPAR1, explaining 1.5% of the variation in monocyte counts (best SNP rs7023923, p=8.9x10(-14)). The second locus was located on chromosome 6p21 and associated with mean cell erythrocyte volume (rs12661667, p=1.2x10(-9), 0.7% variance explained) in a region that spanned five genes, including CCND3, a member of the D-cyclin gene family that is involved in hematopoietic stem cell expansion. The third region was also associated with erythrocyte volume and was located in an intergenic region on chromosome 6q24 (rs592423, p=5.3x10(-9), 0.6% variance explained). All three loci replicated in an independent panel of 1543 individuals (p values=0.001, 9.9x10(-5), and 7x10(-5), respectively). The identification of these QTL provides new opportunities for furthering our understanding of the mechanisms regulating hemopoietic cell fate.

Human papillomavirus DNA detected in peripheral blood samples from healthy Australian male blood donors

Recent studies have shown that human papillomavirus (HPV) DNA can be found in circulating blood, including peripheral blood mononuclear cells (PBMCs), sera, plasma, and arterial cord blood. In light of these findings, DNA extracted from PBMCs from healthy blood donors were examined in order to determine how common HPV DNA is in blood of healthy individuals. Blood samples were collected from 180 healthy male blood donors (18-76 years old) through the Australian Red Cross Blood Services. Genomic DNA was extracted and specimens were tested for HPV DNA by PCR using a broad range primer pair. Positive samples were HPV-type determined by cloning and sequencing. HPV DNA was found in 8.3% (15/180) of the blood donors. A wide variety of different HPV types were isolated from the PBMCs; belonging to the cutaneous beta and gamma papillomavirus genera and mucosal alpha papillomaviruses. High-risk HPV types that are linked to cancer development were detected in 1.7% (3/180) of the PBMCs. Blood was also collected from a healthy HPV-positive 44-year-old male on four different occasions in order to determine which blood cell fractions harbor HPV. PBMCs treated with trypsin were negative for HPV, while non-trypsinized PBMCs were HPV-positive. This suggests that the HPV in blood is attached to the outside of blood cells via a protein-containing moiety. HPV was also isolated in the B cells, dendritic cells, NK cells, and neutrophils. To conclude, HPV present in PBMCs could represent a reservoir of virus and a potential new route of transmission.

Neutrophil oxidative burst is primed by supernatant from stored red cells : implications for Transfusion-Related Acute Lung Injury (TRALI)

Aim/Background: Transfusion-related acute lung injury (TRALI) is a potentially fatal adverse transfusion reaction. It is hypothesised to occur via a two-insult mechanism: the recipient’s underlying co-morbidity in addition to the transfusion of blood products activate neutrophils in the lung resulting in damaged endothelium and capillary leakage. Neutrophil activation may occur by antibody or non-antibody related mechanisms, with the length of storage of cellular blood products implicated in the latter. This study investigated non-antibody mediated priming and/or activation of neutrophil oxidative burst. Methods: A cytochrome C reduction assay was used to assess priming and activation of neutrophil oxidative burst by pooled supernatant (SN) from day 1 (D1; n=75) and day 42 (D42; n=113) packed red blood cells (PRBC). Pooled PRBC-SN were assessed in parallel with PAF (priming), fMLP (activating), PAF + fMLP (priming + activating) and buffer only (negative) controls. Cytochrome C reduction was measured over 30min at 37oC (inclusive of 10min priming). Neutrophil activation by PRBC-SN was assessed cf. buffer only and neutrophil priming by PRBC-SN was assessed by co-incubation with fMLP cf. fMLP alone. One-way ANOVA; Newman-Keuls post-test; p<0.05; n=10 independent assays. Results: Neither D1- nor D42- PRBC-SN alone activated neutrophil oxidative burst. In addition, D1-PRBC-SN did not prime fMLP-activated neutrophil oxidative burst. D42-PRBC-SN did, however, prime neutrophils for subsequent activation of oxidative burst by fMLP, the magnitude of response being similar to PAF (a known neutrophil priming agonist). Conclusion: These findings are consistent with the two-insult mechanism of TRALI. Factors released into the SN during PRBC storage contributed to neutrophil priming synergistically with other neutrophil stimulating agonists. This implicates PRBC storage duration as a key factor contributing to non-immune neutrophil activation in the development of TRALI in patients with pre-disposing inflammatory conditions.

Stored blood products augment inflammation in a two-event model of Transfusion Related Acute Lung Injury (TRALI)

Aim/Background TRALI is hypothesised to develop via a two-event mechanism involving both the patieint's underlying morbidity and blood product factors. The storage of cellular products has been implicated in cases of non-antibody mediated TRALI, however the pathophysiological mechanisms are undefined. We investigated blood product storage-related modulation of inflmmatory cells and medicators involved in TRALI. Methods In an in vitro mode, fresh human whole blood was mixed with culture media (control) or LPS as a 1st event and "transfused" with 10% (v/v) pooled supernatant (SN) from Day 1 (d1, n=75) or Day 42 (D42, n=113) packed red blood cells (PRBCs) as a 2nd event. Following 6hrs, culture SN was used to assess the overall inflammatory response (cytometric bead array) and a duplicate assay containing protein transport inhibitor was used to assess neutrophil- and monocyte-specific inflmamatory responses using multi-colour flow cytometry. Panels: IL-6, IL-8, IL-10, IL-12, IL-1, TNF, MCP-1, IP-10, MIP-1. One-way ANOVA 95% CI. Results In the absence of LPS, exposure to D1 or D42 PRBC-SN reduced monocyte expression of IL-6, IL-8 and Il-10. D42 PRBC-SN also reduced monocyte IP-10, and the overall IL-8 production was increased. In the presence of LPS, D1-PRBC SN only modified overall IP-10 levels which were reduced. However, cf LPS alone, the combination of LPS and D42 PRBC-SN resulted in increased neutrophil and monocyte productionof IL-1 and IL-8 as well as reduced monocyte TNF production. Additionally, LPS and D42 PRBC-SN resulted in overall inflmmatory changes: elevated IL-8, reduced TNF and IP-10. Conclusion In the absence of LPS, exposure to both fresh and stored PRBC-SN predominantly resulted in an anti-imflammatory response. However, in the presence of LPS, stored but not fresh PRBC-SN augmented a pro-inflammatory profile. These data support the two-event mechanism of TRALI pathogenesis, and highlights blood product storage duration as a factor contributing to the development of inflmamatory responses in non-antibody mediated TRALI.

Age of blood and recipient factors determine the severity of transfusion-related acute lung injury (TRALI)

Introduction Critical care patients frequently receive blood transfusions. Some reports show an association between aged or stored blood and increased morbidity and mortality, including the development of transfusion-related acute lung injury (TRALI). However, the existence of conflicting data endorses the need for research to either reject this association, or to confirm it and elucidate the underlying mechanisms. Methods Twenty-eight sheep were randomised into two groups, receiving saline or lipopolysaccharide (LPS). Sheep were further randomised to also receive transfusion of pooled and heat-inactivated supernatant from fresh (Day 1) or stored (Day 42) non-leucoreduced human packed red blood cells (PRBC) or an infusion of saline. TRALI was defined by hypoxaemia during or within two hours of transfusion and histological evidence of pulmonary oedema. Regression modelling compared physiology between groups, and to a previous study, using stored platelet concentrates (PLT). Samples of the transfused blood products also underwent cytokine array and biochemical analyses, and their neutrophil priming ability was measured in vitro. Results TRALI did not develop in sheep that first received saline-infusion. In contrast, 80% of sheep that first received LPS-infusion developed TRALI following transfusion with "stored PRBC." The decreased mean arterial pressure and cardiac output as well as increased central venous pressure and body temperature were more severe for TRALI induced by "stored PRBC" than by "stored PLT." Storage-related accumulation of several factors was demonstrated in both "stored PRBC" and "stored PLT", and was associated with increased in vitro neutrophil priming. Concentrations of several factors were higher in the "stored PRBC" than in the "stored PLT," however, there was no difference to neutrophil priming in vitro. Conclusions In this in vivo ovine model, both recipient and blood product factors contributed to the development of TRALI. Sick (LPS infused) sheep rather than healthy (saline infused) sheep predominantly developed TRALI when transfused with supernatant from stored but not fresh PRBC. "Stored PRBC" induced a more severe injury than "stored PLT" and had a different storage lesion profile, suggesting that these outcomes may be associated with storage lesion factors unique to each blood product type. Therefore, the transfusion of fresh rather than stored PRBC may minimise the risk of TRALI.

Velocity jump processes with proliferation

Cell invasion involves a population of cells that migrate along a substrate and proliferate to a carrying capacity density. These two processes, combined, lead to invasion fronts that move into unoccupied tissues. Traditional modelling approaches based on reaction–diffusion equations cannot incorporate individual–level observations of cell velocity, as information propagates with infinite velocity according to these parabolic models. In contrast, velocity jump processes allow us to explicitly incorporate individual–level observations of cell velocity, thus providing an alternative framework for modelling cell invasion. Here, we introduce proliferation into a standard velocity–jump process and show that the standard model does not support invasion fronts. Instead, we find that crowding effects must be explicitly incorporated into a proliferative velocity–jump process before invasion fronts can be observed. Our observations are supported by numerical and analytical solutions of a novel coupled system of partial differential equations, including travelling wave solutions, and associated random walk simulations.

Bioreactor for blood product production

The feasibility of ex vivo blood production is limited by both biological and engineering challenges. From an engineering perspective, these challenges include the significant volumes required to generate even a single unit of a blood product, as well as the correspondingly high protein consumption required for such large volume cultures. Membrane bioreactors, such as hollow fiber bioreactors (HFBRs), enable cell densities approximately 100-fold greater than traditional culture systems and therefore may enable a significant reduction in culture working volumes. As cultured cells, and larger molecules, are retained within a fraction of the system volume, via a semipermeable membrane it may be possible to reduce protein consumption by limiting supplementation to only this fraction. Typically, HFBRs are complex perfusion systems having total volumes incompatible with bench scale screening and optimization of stem cell-based cultures. In this article we describe the use of a simplified HFBR system to assess the feasibility of this technology to produce blood products from umbilical cord blood-derived CD34+ hematopoietic stem progenitor cells (HSPCs). Unlike conventional HFBR systems used for protein manufacture, where cells are cultured in the extracapillary space, we have cultured cells in the intracapillary space, which is likely more compatible with the large-scale production of blood cell suspension cultures. Using this platform we direct HSPCs down the myeloid lineage, while targeting a 100-fold increase in cell density and the use of protein-free bulk medium. Our results demonstrate the potential of this system to deliver high cell densities, even in the absence of protein supplementation of the bulk medium.

Stochastic dynamics of interacting haematopoietic stem cell niche lineages

Since we still know very little about stem cells in their natural environment, it is useful to explore their dynamics through modelling and simulation, as well as experimentally. Most models of stem cell systems are based on deterministic differential equations that ignore the natural heterogeneity of stem cell populations. This is not appropriate at the level of individual cells and niches, when randomness is more likely to affect dynamics. In this paper, we introduce a fast stochastic method for simulating a metapopulation of stem cell niche lineages, that is, many sub-populations that together form a heterogeneous metapopulation, over time. By selecting the common limiting timestep, our method ensures that the entire metapopulation is simulated synchronously. This is important, as it allows us to introduce interactions between separate niche lineages, which would otherwise be impossible. We expand our method to enable the coupling of many lineages into niche groups, where differentiated cells are pooled within each niche group. Using this method, we explore the dynamics of the haematopoietic system from a demand control system perspective. We find that coupling together niche lineages allows the organism to regulate blood cell numbers as closely as possible to the homeostatic optimum. Furthermore, coupled lineages respond better than uncoupled ones to random perturbations, here the loss of some myeloid cells. This could imply that it is advantageous for an organism to connect together its niche lineages into groups. Our results suggest that a potential fruitful empirical direction will be to understand how stem cell descendants communicate with the niche and how cancer may arise as a result of a failure of such communication.

Plasma ATP concentration and venous oxygen content in the forearm during dynamic handgrip exercise

Background: It has been proposed that adenosine triphosphate (ATP) released from red blood cells (RBCs) may contribute to the tight coupling between blood flow and oxygen demand in contracting skeletal muscle. To determine whether ATP may contribute to the vasodilatory response to exercise in the forearm, we measured arterialised and venous plasma ATP concentration and venous oxygen content in 10 healthy young males at rest, and at 30 and 180 seconds during dynamic handgrip exercise at 45% of maximum voluntary contraction (MVC). Results: Venous plasma ATP concentration was elevated above rest after 30 seconds of exercise (P < 0.05), and remained at this higher level 180 seconds into exercise (P < 0.05 versus rest). The increase in ATP was mirrored by a decrease in venous oxygen content. While there was no significant relationship between ATP concentration and venous oxygen content at 30 seconds of exercise, they were moderately and inversely correlated at 180 seconds of exercise (r = -0.651, P = 0.021). Arterial ATP concentration remained unchanged throughout exercise, resulting in an increase in the venous-arterial ATP difference. Conclusions: Collectively these results indicate that ATP in the plasma originated from the muscle microcirculation, and are consistent with the notion that deoxygenation of the blood perfusing the muscle acts as a stimulus for ATP release. That ATP concentration was elevated just 30 seconds after the onset of exercise also suggests that ATP may be a contributing factor to the blood flow response in the transition from rest to steady state exercise.

Active regulation of the epidermal calcium profile

A distinct calcium profile is strongly implicated in regulating the multi-layered structure of the epidermis. However, the mechanisms that govern the regulation of this calcium profile are currently unclear. It clearly depends on the relatively impermeable barrier of the stratum corneum (passive regulation) but may also depend on calcium exchanges between keratinocytes and extracellular fluid (active regulation). Using a mathematical model that treats the viable sublayers of unwounded human and murine epidermis as porous media and assumes that their calcium profiles are passively regulated, we demonstrate that these profiles are also actively regulated. To obtain this result, we found that diffusion governs extracellular calcium motion in the viable epidermis and hence intracellular calcium is the main source of the epidermal calcium profile. Then, by comparison with experimental calcium profiles and combination with a hypothesised cell velocity distribution in the viable epidermis, we found that the net influx of calcium ions into keratinocytes from extracellular fluid may be constant and positive throughout the stratum basale and stratum spinosum, and that there is a net outflux of these ions in the stratum granulosum. Hence the calcium exchange between keratinocytes and extracellular fluid differs distinctly between the stratum granulosum and the underlying sublayers, and these differences actively regulate the epidermal calcium profile. Our results also indicate that plasma membrane dysfunction may be an early event during keratinocyte disintegration in the stratum granulosum.

A screen for modifiers of epigenetic reprogramming

Epigenetic modifiers are the proteins involved in establishing and maintaining the epigenome of an organism. They are particularly important for development. Changes in epigenetic modifiers have been shown be lethal, or cause diseases. Our laboratory has developed an ENU mutagenesis screen to produce mouse mutants displaying altered epigenetic gene silencing. The screen relies on a GFP transgene that is expressed in red blood cells in a variegated manner. In the orginal transgenic FVB mice expression occurs in approximately 55% of red blood cells. During the course of my Masters, I characterised four different Mommes (Modifiers of murine metastable epiallele), MommeD32, MommeD33, MommeD35 and MommeD36. For each Momme, I identified the underlying mutation, and observed the corresponding phenotype. In MommeD32 the causative mutation is in Dnmt1, (DNA methyltransferase 1). This gene was previously identified in the screen, as MommeD2, and the new allele, MommeD32 has a change in the BAH domain of the protein. MommeD33 is the result of a change at the transgene itself. MommeD35 carries a mutation in Suv39h1 (suppressor of variegation 3-9 homolog 1). This gene has not previously been identified in the screen, but it is a known epigenetic modifier. MommeD36 had the same ENU treated sire as MommeD32, and I found that it has the same mutation as MommeD32. These mutant strains provide valuable tools that can be used to further our knowledge of epigenetic reprogramming. An example being the cancer study done with MommeD9 which has a mutation in Trim28. By crossing MommeD9+/- mutant mice with Trp53+/- mice, it can be seen if Trim28 has an effect on the rate of tumour genesis. However no clear effect of Trim28 haploinsufficiency can be observed in Trp53+/- mice.

Implications of lipid biology for the pathogenesis of schizophrenia

Objective: Preclinical and clinical data suggest that lipid biology is integral to brain development and neurodegeneration. Both aspects are proposed as being important in the pathogenesis of schizophrenia. The purpose of this paper is to examine the implications of lipid biology, in particular the role of essential fatty acids (EFA), for schizophrenia. Methods: Medline databases were searched from 1966 to 2001 followed by the crosschecking of references. Results: Most studies investigating lipids in schizophrenia described reduced EFA, altered glycerophospholipids and an increased activity of a calcium-independent phospholipase A2 in blood cells and in post-mortem brain tissue. Additionally, in vivo brain phosphorus-31 Magnetic Resonance Spectroscopy (31P-MRS) demonstrated lower phosphomonoesters (implying reduced membrane precursors) in first- and multi-episode patients. In contrast, phosphodiesters were elevated mainly in first-episode patients (implying increased membrane breakdown products), whereas inconclusive results were found in chronic patients. EFA supplementation trials in chronic patient populations with residual symptoms have demonstrated conflicting results. More consistent results were observed in the early and symptomatic stages of illness, especially if EFA with a high proportion of eicosapentaenoic acid was used. Conclusion: Peripheral blood cell, brain necropsy and 31P-MRS analysis reveal a disturbed lipid biology, suggesting generalized membrane alterations in schizophrenia. 31P-MRS data suggest increased membrane turnover at illness onset and persisting membrane abnormalities in established schizophrenia. Cellular processes regulating membrane lipid metabolism are potential new targets for antipsychotic drugs and might explain the mechanism of action of treatments such as eicosapentaenoic acid.

A cost-outcome description of the septic work-up for bacterial infection in neonates in a tertiary care hospital

Analysis of the septic work-up of 194 neonates at Women's College Hospital, Toronto, showed that the only antepartum condition predicting neonatal sepsis was the mother being on antibiotics. The only postnatal condition predicting sepsis was a maternal postpartum white blood cell count over 11,000. The average cost for tests for a septic work-up in these 194 mother-neonate pairs was $71.48 (Canadian dollars), and the average cost of tests to find a septic case was $1,066.77.