In vivo measurement of dose distribution in patients' lymphocytes: helical tomotherapy versus step-and-shoot IMRT in prostate cancer

In radiotherapy, in vivo measurement of dose distribution within patients'' lymphocytes can be performed by detecting gamma-H2AX foci in lymphocyte nuclei. This method can help in determining the whole-body dose. Options for risk estimations for toxicities in normal tissue and for the incidence of secondary malignancy are still under debate. In this investigation, helical tomotherapy (TOMO) is compared with step-and-shoot IMRT (SSIMRT) of the prostate gland by measuring the dose distribution within patients'' lymphocytes. In this prospective study, blood was taken from 20 patients before and 10 min after their first irradiation fraction for each technique. The isolated leukocytes were fixed 2 h after radiation. DNA double-stranded breaks in lymphocyte nuclei were stained immunocytochemically using anti-gamma-H2AX antibodies. Gamma-H2AX foci distribution in lymphocytes was determined for each patient. Using a calibration line, dose distributions in patients'' lymphocytes were determined by studying the gamma-H2AX foci distribution, and these data were used to generate a cumulative dose–lymphocyte histogram (DLH). Measured in vivo (DLH), significantly fewer lymphocytes indicated low-dose exposure (<40% of the applied dose) during TOMO compared with SSIMRT. The dose exposure range, between 45 and 100%, was equal with both radiation techniques. The mean number of gamma-H2AX foci per lymphocyte was significantly lower in the TOMO group compared with the SSIMRT group. In radiotherapy of the prostate gland, TOMO generates a smaller fraction of patients'' lymphocytes with low-dose exposure relative to the whole body compared with SSIMRT. Differences in the constructional buildup of the different linear accelerator systems, e.g. the flattening filter, may be the cause thereof. The influence of these methods on the incidence of secondary malignancy should be investigated in further studies.     

Tissue-type plasminogen activator (t-PA) is stored in Weibel-Palade bodies in human endothelial cells both in vitro and in vivo

Vascular endothelial cells are thought to be the main source of plasma tissue-type plasminogen activator (t-PA) and von Willebrand factor (VWF). Previous studies have suggested that both t-PA and VWF are acutely released in response to the same stimuli, both in cultured endothelial cells and in vivo. However, the subcellular storage compartment in endothelial cells has not been definitively established. We tested the hypothesis that t-PA is localized in Weibel-Palade (WP) bodies, the specialized endothelial storage granules for VWF. In cultured human umbilical vein endothelial cells (HUVECs), t-PA was expressed in a minority of cells and found in WP bodies by immunofluorescence. After up-regulation of t-PA synthesis either by vascular endothelial growth factor (VEGF) and retinoic acid or by sodium butyrate, there was a large increase in t-PA-positive cells. t-PA was exclusively located to WP bodies, an observation confirmed by immunoelectron microscopy. Incubation with histamine, forskolin, and epinephrine induced the rapid, coordinate release of both t-PA and VWF, consistent with a single storage compartment. In native human skeletal muscle, t-PA was expressed in endothelial cells from arterioles and venules, along with VWF. The 2 proteins were found to be colocalized in WP bodies by immunoelectron microscopy. These data indicate that t-PA and VWF are colocalized in WP bodies, both in HUVECs and in vivo. Release of both t-PA and VWF from the same storage pool likely accounts for the coordinate increase in the plasma level of the 2 proteins in response to numerous stimuli, such as physical activity, beta-adrenergic agents, and 1-deamino-8d-arginine vasopressin (DDAVP) among others.     

Studies on the regulation of myocardial blood flow in man II. Effects of acute arterial hypoxia

To determine whether adjustment of myocardial blood flow (MBF), myocardial oxygen consumption (MVO₂) and myocardial substrate uptake (MSU) to acute arterial hypoxia is influenced by training effects on the heart, 7 trained and 7 untrained healthy individuals were investigated. MBF (argon method), MVO₂ and MSU of glucose, lactate and free fatty acids were measured at rest during normoxia and two different stages of acute arterial hypoxia: a) 12.82 vol% O₂; b) 8.74 vol% O₂. Measurements were carried out during hemodynamic and respiratory steady state conditions. Myocardial flow and metabolism of athletes were significantly (p<0.01) lower compared to untrained subjects. In the trained cohort, MBF increased from 65 ± 19 to 73 ± 16 (a) and 98 ± 23 (b) ml/min·100 g. MVO₂ remained at normoxic control level of 8.00 ± 2.27 ml/min·100g. In the untrained group, MBF increased from 77 ± 15 to 84 ± 20 (a) and 108 ± 18 (b) ml/min · 100g. Again, there was no significant deviation in MVO₂ from the normoxic level of 10.11 ± 1.90 ml/min·100g. Decrease in arterial oxygen content was overcompensated by an increase in coronary conductance resulting in a significantly improved efficiency of myocardial perfusion during severe hypoxia. MSU of glucose, lactate and free fatty acids as well as calculated ATP production did not change significantly during hypoxia. It is concluded that training effects on the heart do not influence regulation of MBF, MVO₂ and MSU during moderate or severe acute arterial hypoxia. Reaction of coronary smooth muscle tone to a decrease in oxygen partial pressure is independent from training effects. However, both acute arterial hypoxia and physical training exert synergetic effects on the heart by reducing myocardial oxygen consumption per heart beat. Thus, it is assumed that adaptive properties of myocardial blood flow and metaboüsm to severe hypoxia are more pronounced in trained than in untrained individuals.     

Berichtigung

Ohne Zusammenfassung     

Microparticles as mediators of cellular cross-talk in inflammatory disease

Microparticles are a heterogeneous population of membrane-coated vesicles which can be released from virtually all cell types during activation or apoptosis. Release occurs from the cell surface in an exogenous budding process involving local rearrangement of the cytoskeleton. Given their origin, these particles can be identified by staining for cell surface markers and annexin V. As shown in in vitro studies, microparticles may represent a novel subcellular element for intercellular communication in inflammation. Thus, microparticles can transfer chemokine receptors and arachidonic acid between cells, activate complement, promote leukocyte rolling and stimulate the release of pro-inflammatory mediators. Under certain conditions, however, microparticles may also exert anti-inflammatory properties by inducing immune cell apoptosis and the production of anti-inflammatory mediators. Microparticles may play an important role in the pathogenesis of rheumatologic diseases as evidenced by their elevation in diseases such as systemic sclerosis (SSc), systemic vasculitis and antiphospholipid antibody syndrome and correlation with clinical events. A role in inflammatory arthritis is suggested by the finding that leukocyte-derived microparticles induce the production of matrix metalloproteinases and cytokines by synovial fibroblasts. Together, these findings point to novel signaling pathways of cellular cross-talk that may operate along the spectrum of soluble cytokines and mediators of direct cell–cell contact.     

Slam Haplotype 2 Promotes NKT But Suppresses Vγ4+ T-Cell Activation in Coxsackievirus B3 Infection Leading to Increased Liver Damage But Reduced Myocarditis

There are two major haplotypes of signal lymphocytic activation molecule (Slam) in inbred mouse strains, with the Slam haplotype 1 expressed in C57Bl/6 mice and the Slam haplotype 2 expressed in most other commonly used inbred strains, including 129 mice. Because signaling through Slam family receptors can affect innate immunity [natural killer T cell (NKT) and γ-δ T-cell receptor], and innate immunity can determine susceptibility to coxsackievirus B3 (CVB3) infection, the present study evaluated the response of C57Bl/6 and congenic B6.129c1 mice (expressing the 129-derived Slam locus) to CVB3. CVB3-infected C57Bl/6 male mice developed increased myocarditis but reduced hepatic injury compared with infected B6.129c1 mice. C57Bl/6 mice also had increased γδ⁺ and CD8⁺interferon-γ⁺ cells but decreased numbers of NKT (T-cell receptor β chain + mCD1d tetramer⁺) and CD4⁺FoxP3⁺ cells compared with B6.129c1 mice. C57Bl/6 mice were infected with CVB3 and treated with either α-galactosylceramide, an NKT cell-specific ligand, or vehicle (dimethyl sulfoxide/PBS). Mice treated with α-galactosylceramide showed significantly reduced myocarditis. Liver injuries, as determined by alanine aminotransferase levels in plasma, were increased significantly, confirming that NKT cells are protective for myocarditis but pathogenic in the liver.Myocarditis is an inflammation of the cardiac muscle that follows microbial infections.¹ Among viruses, enteroviruses including coxsackie B viruses are common etiologic agents.^2,3 Although infectious agents act as a trigger for myocarditis, there is considerable debate as to the actual mechanism(s) of myocardial injury. Viruses directly cause cellular dysfunction either through induced cell death, shut down of cell RNA and protein synthesis, or viral protease cleavage of contractile proteins.^4,5 In addition, cytokines such as IL-1β, IL-6, and tumor necrosis factor α, which are elicited from resident cells in the heart subsequent to infection, can suppress contractility, leading to cardiac dysfunction.⁶ Finally, host immune responses to infection may kill myocytes, leading to cardiac stress. Host response can be directed specifically toward virally infected cardiocytes or infection can trigger autoimmunity to cardiac antigens (autoimmunity), which destroys both infected and uninfected myocytes.⁷Host innate immune responses occur rapidly, subsequent to viral infections, and usually have broad specificity, unlike the classic adaptive immune response, which requires a week or more for development of a measurable response in the naive individual but is highly specific to the inducing pathogen. The innate immune response both helps to control microbe load before generation of the adaptive immune response and has a major impact on the phenotype and intensity of the adaptive response. Two types of T cells representing innate immunity are natural killer T cells (NKT) and T cells expressing the γ-δ T-cell receptor (γδ⁺). A study by Wu et al⁸ showed that in vivo administration of α-galactosylceramide, a ligand that specifically activates NKT cells, protects mice from coxsackievirus B3 (CVB3)-induced myocarditis. Prior studies have shown that signaling through Slam family receptors has a major impact on NKT cell development,^9–11 and that different Slam haplotypes can have distinct effects on NKT cell response and function.^9,12 There are two major Slam haplotypes, Slam haplotype 1 and Slam haplotype 2, that distinguish commonly used inbred mouse strains.^13,14 Slam haplotype 1 is present in C57Bl/6, and Slam haplotype 2 is present in most other commonly used mouse strains including 129S1/SvImJ and BALB/c mice. The congenic B6.129c1 mouse expresses the genetic region of chromosome 1 containing the 129-derived Slam haplotype 2 locus on the C57Bl/6 background and was used previously to show Slam haplotype control of liver NKT cell numbers and NKT cell cytokine production.¹² In addition, Slam haplotypes previously were shown to regulate macrophage tumor necrosis factor production in response to lipopolysaccharide.¹² Although less well studied, Slam family–receptor signaling also has been shown to affect γδ⁺ T-cell development. Studies using human peripheral blood mononuclear cells stimulated in vitro with antibody to CD3 and either IL-2, anti-CD150 (SLAM), or IL-15 showed that all three stimulation protocols resulted in γδ⁺ T-cell survival. However, co-culture with anti-CD3 and anti-CD150 resulted in selective proliferation of CD8⁺CD56⁺γδ⁺ T cells expressing the Vδ1 chain, and cells co-cultured with anti-CD3 and IL-15 resulted in preferential generation of CD8⁻CD56⁻γδ⁺ cells expressing the Vδ2 chain.¹⁵ Therefore, SLAM signaling can impact the generation of a subpopulation of the total γδ⁺ cell population in humans. Prior studies from the Huber laboratory have shown that a subpopulation of γδ⁺ cells is crucial to myocarditis susceptibility subsequent to CVB3 infection¹⁶ and that the relevant γδ⁺ cell expresses both CD8 and the Vγ4 chain.^16,17 This raised the question of whether Slam haplotypes modulated selected γδ⁺ cell subsets in the mouse, as it does in humans, and whether the Slam haplotype specifically could affect activation of the CD8⁺Vγ4⁺ T cell, which is known to be pathogenic in CVB3-induced myocarditis.CVB3 infection of mice results in multiple organ infection, including pancreas, liver, and heart with accompanying tissue injury in all tissues. There are well-established differences in disease susceptibility between BALB/c and C57Bl/6 mice, strains expressing the two distinct Slam haplotypes. C57Bl/6 mice are highly susceptible to type 2 autoimmune hepatitis and develop extensive hepatic inflammation, whereas BALB/c mice are resistant to this disease and show no inflammation.¹⁸ In contrast, BALB/c mice are more susceptible to myocarditis^19–22 compared with the more resistant C57Bl/6 strain. However, there are many genetic differences between BALB/c and C57Bl/6 mice, which may influence disease development or development and activation of specific innate effectors such as NKT and γδ T cells. The goal of the current study was to determine whether Slam haplotype affected NKT and Vγ4⁺ T-cell responses subsequent to CVB3 infection using C57Bl/6 congenic mice in which the Slam locus alone differed between the mouse strains, and whether haplotype-dependent NKT/Vγ4⁺ cell response had a distinct effect in different organs infected with the virus in the absence of the many other genetic differences between BALB/c and C57Bl/6 mice.