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North Adelaide, Australia

Hodge G.,SA Pathology | Scott J.,SA Pathology | Osborn M.,SA Pathology | To L.B.,SA Pathology | And 3 more authors.
Cytokine | Year: 2011

Background: Paediatric oncology patients with febrile neutropenia are usually hospitalised and treated with empirical broad-spectrum antibiotic therapy to counter the risk of infection. However, there is currently no method available to rapidly identify bacteremia in these patients. T-helper-type-1 (Th1) cytokines are required for effective immune response to many pathogenic organisms and T regulatory cells are known suppressors of Th1 cells. We hypothesized that characterization of reduced intracellular Th1 cytokines and increased T regulatory cells (Tregs) may prove useful in identifying infection in childhood oncology patients with febrile neutropenia. Methods: Intracellular Th 1 cytokines and Tregs were enumerated in peripheral blood from a group of childhood oncology patients with febrile neutropenia using multiparameter flow cytometry. Results: There was a significant increase in the percentage of CD25+ CD127- CD8- CD3+ Tregs and a significant decrease in Th1 intracellular cytokines IFNγ, IL-2 and TNFα in the blood of culture positive patients compared with culture negative patients. Conclusions: Enumeration of Tregs and intracellular Th1 cytokines may provide a sensitive, specific test for determining infection in childhood oncology patients before blood culture results become available. © 2010.

Davenport M.H.,R Samuel Mclaughlin Foundation Exercise And Pregnancy Laboratory | Goswami R.,Neurovascular Research Laboratory | Kevin Shoemaker J.,Neurovascular Research Laboratory | Mottola M.F.,R Samuel Mclaughlin Foundation Exercise And Pregnancy Laboratory | And 2 more authors.
American Journal of Physiology - Regulatory Integrative and Comparative Physiology | Year: 2012

Endothelial dysfunction is commonly observed in women with a previous diagnosis of gestational diabetes mellitus (GDM). Whether arterial stiffness is also related to pregnancy and/or postpartum glucose intolerance has not been determined. We examined the influence of GDM during pregnancy and hyperglycemia in the postpartum period on arterial function. Thirty postpartum women were stratified into one of three groups: 1) normoglycemic pregnancy, normoglycemic postpartum (NORM), 2) GDM during pregnancy, normoglycemic postpartum (GDM-N); and 3) GDM during pregnancy, hyperglycemic postpartum (GDM-H). Ten never-pregnant controls were also recruited (Control). All measures were made at 2 mo postpartum or in the early follicular phase in Control women. Arterial stiffness was assessed by pulse wave velocity (PWV) and brachial and carotid artery distensibility. Endothelial function was determined by flowmediated dilation (FMD). PWV was not different between the four groups. Distensibility of the brachial and carotid arteries was lower in GDM-N women (brachial: 1.1 X 10 -3 mmHg -1 ± 3.6 × 10 -4; carotid: 2.0 X 10 -3 ± 3.3 × 10 -4) and GDM-H (brachial: 1.4 × 10 -3 mmHg-1 ± 4.1 × 10 -4; carotid: 1.8 × 10 -3 mmHg -1 ± 5.0 × 10 -4) compared with NORM women (brachial: 3.4 × 10 -3 mmHg -1 ± 7.0 × 10 -4; carotid: 3.9 × 10 -3 ± 7.4 × 10 -4). However, only brachial artery distensibility returned to Control levels by 2 mo postpartum in the NORM women. FMD was lower in previously GDM women (GDM-N: 4.1% ± 2.3; GDM-H: 4.4% ± 0.9) compared with NORM women (10.8% ± 1.3; P < 0.01). These findings suggest that the vascular function of women in the early postpartum period is influenced by GDM during pregnancy and the persistence of clinical and/or subclinical hyperglycemia after delivery. © 2012 the American Physiological Society.

Rawat V.P.S.,University of Ulm | Arseni N.,Helmholtz Center Munich | Arseni N.,Ludwig Maximilians University of Munich | Ahmed F.,Helmholtz Center Munich | And 15 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2010

Recent data indicate that a variety of regulatory molecules active in embryonic development may also play a role in the regulation of early hematopoiesis. Here we report that the human Vent-like homeobox gene VENTX, a putative homolog of the Xenopus xvent2 gene, is a unique regulatory hematopoietic gene that is aberrantly expressed in CD34+ leukemic stem-cell candidates in human acute myeloid leukemia (AML). Quantitative RT-PCR documented expression of the gene in lineage positive hematopoietic subpopulations, with the highest expression in CD33+ myeloid cells. Notably, expression levels of VENTX were negligible in normal CD34 +/CD38- or CD34+ human progenitor cells. In contrast to this, leukemic CD34+/CD38- cells from AML patients with translocation t(8,21) and normal karyotype displayed aberrantly high expression of VENTX. Gene expression and pathway analysis demonstrated that in normal CD34+ cells enforced expression of VENTX initiates genes associated with myeloid development and down-regulates genes involved in early lymphoid development. Functional analyses confirmed that aberrant expression of VENTX in normal CD34+ human progenitor cells perturbs normal hematopoietic development, promoting generation of myeloid cells and impairing generation of lymphoid cells in vitro and in vivo. Stable knockdown of VENTX expression inhibited the proliferation of human AML cell lines. Taken together, these data extend our insights into the function of embryonic mesodermal factors in human postnatal hematopoiesis and indicate a role for VENTX in normal and malignant myelopoiesis.

Badurdeen S.,Child Health Research Institute | Hodge G.,SA Pathology at Womens and Childrens Hospital | Osborn M.,SA Pathology at Womens and Childrens Hospital | Scott J.,SA Pathology at Womens and Childrens Hospital | And 4 more authors.
Journal of Pediatric Hematology/Oncology | Year: 2012

Neutropenic patients with bacteraemia need prolonged intravenous antibiotic treatment. Using cytometric bead array technology, we show in children with febrile neutropenia that bacteraemia is associated with an elevation of at least 1 of 3 plasma cytokines plus C-reactive protein. The combination of interleukin (IL)-8, IL-6, IL-10, and C-reactive protein values above operator-defined cutoff levels identified 15 of 16 episodes of bacteraemia, making this a potentially useful technique in identifying high-risk patients who should not be discharged early from hospital. Furthermore, low risk of bacteraemia may be predicted by a combination of below threshold cytokines and negative clinical examination. Copyright © 2011 by Lippincott Williams & Wilkins.

News Article
Site: www.biosciencetechnology.com

In his role as a pediatrician, Manish Butte, M.D., Ph.D., will often push and prod a patient’s abdomen, feeling for abnormalities — a swollen spleen, a hardened lymph node or an unusual lump in the intestines or liver. There are still some things that can only be gleaned by touch, and Butte believes this notion applies to individual cells as well. Yet researchers’ ability to probe and measure the features of living cells has been almost nonexistent. Recently, a team of Stanford scientists and engineers set out to right that imbalance with a new technique for rapidly mapping cells. They succeeded by engineering a major advancement in a technology known as atomic force microscopy, or AFM, which itself was invented at Stanford in 1986. A paper describing the work was published online Nov. 11 in ACS Nano. Butte, an assistant professor of pediatric immunology, is the senior author. Lead authorship is shared by Andrew Wang, Ph.D., a former postdoctoral scholar in Butte’s lab, and Karthik Vijayrhagavan, Ph.D., who was a graduate student and member of the microphotonics lab led by Olav Solgaard, Ph.D., a professor of electrical engineering. “What a cell feels like — its mechanical properties that affect how it makes contact with other cells and tissues — is much more important than what it looks like, but the technology just wasn’t there to allow us to examine it,” Butte said. “There is a lot to be learned from studying the mechanics of a cell and its structures just beneath the surface.” The way Butte and his colleagues use AFM to measure the mechanical properties of cells is akin to the way a builder taps her knuckles along a drywall, listening for the change in pitch that will tell her a wooden stud is on the other side. When an AFM probe taps the surface of a cell, it vibrates, and the pattern of these vibrations, like the sound waves reflecting from the stud, gives mechanical information about the structures of the cell being touched. However, existing AFM probes are relatively large and, as a result, insensitive to high frequencies, which communicate much of the key information about a cell’s innards. The Stanford team’s device couples a very small probe with a traditional one. This assembly allows the device to sense faster oscillations than conventional devices and, accordingly, to take more detailed and much faster measurements. “The main difference between this and previous atomic force microscopes is that we are able to measure the impact of the probe on the cell very fast and get specific readings, whereas typical AFMs simply provide an average. This allows us to accurately measure some very soft materials for the first time,” said Solgaard, who also is a co-author of the paper. Current probes measure cellular stiffness by tapping against the cell around one or two times per second — the fastest that the large probes can make measurements. The small probe, however, can make detailed measurements easily at five to 10,000 taps per second because of its sensitivity. He likened the leap in sensitivity to the difference between driving a Cadillac Escalade down the road and pushing a Hot Wheel toy car along the same surface: “The small Hot Wheel will feel every little bump so much more than the large Cadillac.” AFMs measure movement of the probe by bouncing a laser off its tip. As the tip moves up and down, the laser is reflected. The Stanford invention couples the small probe with the large one by means of a fork-shaped structure called an interferometric grating. The grating produces a diffraction pattern based on the movements of the small probe, and allows the AFM to conveniently capture its measurements. “Our tip actually produces a second signal, and that is what allows us to get much greater detail. From an engineering standpoint, it’s an extremely simple, beautiful solution,” Solgaard said, referring to the diffracted signals from the grating. Best of all, the team’s device can be directly attached to existing AFMs, potentially saving millions of dollars on new equipment that could otherwise be spent on research. A new AFM can run as much as $500,000, according to Solgaard. The objective is the cellular equivalent of Butte pressing a child’s abdomen. “We want to study cell stiffness to understand what is beneath the surface and how cells are structured,” Wang said. As a demonstration, the team measured a section of a red blood cell, making approximately 4 million total measurements in about 10 minutes — all without damaging the delicate cellular exterior. “The same measurements would have taken more than a month to complete using conventional atomic force microscopes,” said Vijayraghavan. The technology is so fast that the team was able to create a series of time-lapse images of a living cell, each taken just seven minutes apart, a previously unimaginable pace. The practical applications of the device range from basic scientific understanding of cellular structure to immunology and oncology. Scientific understanding of the mechanical forces at play in cells is so lacking that the field — now being called mechanobiology — is really in its infancy, according to Butte. The mechanical forces in the body can come from tissues, which range in stiffness from softest brain matter to stiffest bones, from gravity, and even from the pushing and pulling movements of other cells. Cancer cells make their environment mechanically rigid by secreting chemicals that stiffen up the extracellular matrix. Cancer cells likewise interpret the mechanical forces of a tissue to make decisions about growth and metastasis. Surprising feedback loops like this also appear to occur for stem cells in the bone marrow and during embryonic development. How immune cells interpret mechanical forces is still totally unknown. “The lowest-hanging fruit is cancer. Cancers are often stiffer than normal, healthy tissues and we can use that knowledge to diagnose disease. But first, you have to have good data, which our device provides,” Wang said. He has already used an early form of the new Stanford probe in pilot work on breast cancer specimens taken from mastectomies. For his part, Butte plans to use fast AFM to study the immune system. He hopes to explore why otherwise disease-fighting T cells often remain dormant once inside a tumor. He theorizes that the mechanical stiffness of the tumorous tissue may be preventing T cells from freely making contact with cancer cells and from triggering their cancer-fighting functions. In essence, the tumor may be too crowded for the T cells to work. On the other end of the stiffness gamut, he believes that the soft mechanical properties of chronically inflamed or infected tissues provoke the immune system into over-activity, like autoimmunity. It is a theory no one has yet explored due to technical barriers, which the fast AFM could overcome. Butte’s lab has begun a broad effort to link mechanical forces with immune responses at the molecular, cellular and tissue scales. “There is so much we don’t know about the mechanical properties of various cell types and diseased tissues. Almost nothing, in fact,” Butte said. “The first step is to probe. Now, we can do that.” The work was funded by the National Institutes of Health, the National Science Foundation, the Stanford Center for Probing the Nanoscale, the Stanford Child Health Research Institute and Stanford Bio-X.

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