The effect of three human recombinant hematopoietic growth factors (granulocyte-macrophage colony-stimulating factor, granulocyte colony- stimulating factor, and interleukin-3) on phagocyte oxidative activity

Hematopoietic growth factors not only modulate blood progenitor cell activity but also alter the function of mature phagocytes. Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; 1 ng/mL for 60 min) did not stimulate luminol-enhanced chemiluminescence of polymorphonuclear leukocytes (PMNs) in suspension but primed PMN for as much as a 15-fold increase in chemiluminescence in response to f-met- leu-phe (fMLP). Mixed mononuclear leukocytes (monocytes [approximately 20%] and lymphocytes [approximately 80%]; MNL) chemiluminescence was very low even after rhGM-CSF priming, but MNLs added to the PMNs (PMN- MNL) resulted in near doubling of rhGM-CSF-primed PMN fMLP-stimulated chemiluminescence. The enhancing factor(s) from MNLs were inherent rather than induced by the GM-CSF, and purified lymphocytes increased GM-CSF-primed PMN chemiluminescence equal to mixed MNLs. We could not detect cell-free "enhancing factor(s)," but cell-to-cell contact further enhanced rhGM-CSF-primed fMLP-stimulated PMN-MNL oxidative activity by 40%. Polyclonal rabbit anti-tumor necrosis factor (TNF) (but not preimmune serum) decreased both fMLP-stimulated rhGM-CSF- primed PMNs and PMN-MNL chemiluminescence, suggesting that TNF on the PMN surface is enhancing GM-CSF-primed chemiluminescence. GM-CSF priming markedly increased PMN superoxide release (sevenfold), but PMN superoxide release was not further enhanced by the presence of MNLs. Recombinant human granulocyte colony-stimulating factor (rhG-CSF) and interleukin-3 (rhIL-3) displayed much smaller effects on pure PMNs and mixed PMN-MNL chemiluminescence and superoxide release than rhGM-CSF. rhGM-CSF primes PMNs for increased oxidative activity more than rhG-CSF and rhIL-3. Maximal oxidative activity was observed when mixed PMN-MNL were primed with GM-CSF in a cell pellet-promoting cell-to-cell contact. This enhanced activity can be attributed, in part, to both inherent enhancing factor(s) on lymphocytes and PMN-associated TNF induced by GM-CSF.     

Effects of a high polyunsaturated fat diet and vitamin E supplementation on high-density lipoprotein oxidation in humans.

Oxidative modification of high-density lipoproteins (HDL) impairs several biologic functions critical to its role in reverse cholesterol transport. We therefore investigated the effect of dietary polyunsaturated fat and vitamin E on the kinetics of HDL oxidation. Ten subjects were fed sequentially: a baseline diet in which the major fat source was olive oil; a high polyunsaturated fat diet in which the major fat source was safflower oil; and the safflower oil diet plus 800 I.U. vitamin E per day. Plasma lipoprotein levels, vitamin E content, fatty acid composition, and oxidation lag time and rate were determined after 3 weeks on each diet. The polyunsaturated fat diet increased the mean HDL(2) lag time from 45.8+/-12.5 to 83.3+/-11.6 min with no change in oxidation rate. Addition of vitamin E further increased the HDL(2) lag time to 115.6+/-4.4 min and decreased the HDL(2) oxidation rate 10-fold. Neither the polyunsaturated diet alone nor the diet with vitamin E supplementation had any effect on HDL(3) oxidation. We conclude that under conditions of controlled dietary fat intake, a high polyunsaturated fat intake does not increase the oxidation susceptibility of HDL subfractions, and that in this setting, vitamin E supplementation reduces the oxidation susceptibility of HDL(2). These data suggest that antioxidants could influence HDL function in vivo.     

How I investigate acute myeloid leukemia

Acute myeloid leukemia (AML) is a neoplasm of immature myeloid cells and is associated with a wide variety of clinical presentations, morphological features, immunophenotypes, and genetic findings. Recent advances in identification of cytogenetic abnormalities and mutations have provided novel insights into the pathogenesis of AML. Based on the above‐mentioned parameters, the World Health Organization (WHO) classified AML into 25 subtypes, including 2 provisional entities, which differ in prognosis and treatment. In addition, certain mutations are associated with germline predisposition and increase the risk of inherited AML, which warrants family screening. Therefore, precise diagnosis and classification of AML are the most important steps in patient management. Both these steps require incorporation of history, clinical presentation, and laboratory results with studies performed by a pathologist. Pathologist‐initiated studies include morphologic evaluation on the bone marrow aspirate and/or core biopsy, immunophenotyping by flow cytometry and/or immunohistochemistry, cytogenetic analysis by karyotyping and/or fluorescence in situ hybridization, and molecular testing using gene panels and/or next‐generation sequencing. A similar approach is employed during follow‐up of patients after beginning treatment. Here, we describe in detail the various aspects of the workup, including purpose, limitations, and practice guidelines for the different studies. The process of choosing appropriate materials for the different studies is also addressed. We also provide an algorithm for the workup and risk stratification of AML based on guidelines recommended by the WHO, College of American Pathologists, National Comprehensive Cancer Network, American Society of Clinical Oncology, European Society of Medical Oncology, and the European LeukemiaNet.     

Mutation of Plekha7 attenuates salt-sensitive hypertension in the rat

PLEKHA7 (pleckstrin homology domain containing family A member 7) has been found in multiple studies as a candidate gene for human hypertension, yet functional data supporting this association are lacking. We investigated the contribution of this gene to the pathogenesis of salt-sensitive hypertension by mutating Plekha7 in the Dahl salt-sensitive (SS/JrHsdMcwi) rat using zinc-finger nuclease technology. After four weeks on an 8% NaCl diet, homozygous mutant rats had lower mean arterial (149 ± 9 mmHg vs. 178 ± 7 mmHg; P < 0.05) and systolic (180 ± 7 mmHg vs. 213 ± 8 mmHg; P < 0.05) blood pressure compared with WT littermates. Albumin and protein excretion rates were also significantly lower in mutant rats, demonstrating a renoprotective effect of the mutation. Total peripheral resistance and perivascular fibrosis in the heart and kidney were significantly reduced in Plekha7 mutant animals, suggesting a potential role of the vasculature in the attenuation of hypertension. Indeed, both flow-mediated dilation and endothelium-dependent vasodilation in response to acetylcholine were improved in isolated mesenteric resistance arteries of Plekha7 mutant rats compared with WT. These vascular improvements were correlated with changes in intracellular calcium handling, resulting in increased nitric oxide bioavailability in mutant vessels. Collectively, these data provide the first functional evidence that Plekha7 may contribute to blood pressure regulation and cardiovascular function through its effects on the vasculature.Hypertension is a complex disease that is characterized by increased blood pressure, renal damage, and vascular dysfunction which collectively increase risk of atherosclerosis, stroke, heart disease, and renal failure in one-quarter of all adults worldwide (1–3). Because there is strong evidence of heritability in hypertension (2, 4, 5), considerable effort has been put toward identifying novel candidate genes and their molecular mechanisms. Genome-wide association studies (GWAS) have identified many potential hypertension loci, which shed light on the genetic complexity of this disease (5–8) but have provided little mechanistic insight. As such, validation and elucidation of the functional roles and disease mechanisms for these gene candidates are the next important challenges (4).Because hypertension is a complex disease (i.e., multiple variants of small effect sizes contributing to disease risk), we hypothesized candidate gene targeting on a genetically sensitized background would reveal functional role(s) of genetic disease modifiers. The Dahl salt-sensitive (SS) rat is an inbred genetic model of salt-sensitive hypertension that displays hypertension-induced renal damage, cardiac hypertrophy and vascular dysfunction (9–11). These phenotypes are induced by exposing SS rats to a high-salt diet, which results in rapid induction of hypertensive phenotypes that closely resemble salt-induced hypertension seen in humans (12–15). Knockout of specific genes in this disease model using zinc-finger nuclease (ZFN) technology have revealed the importance of key mechanisms contributing to hypertension risk, such as the protection from salt-induced hypertension and renal injury by selective ablation of adaptive immune cells in the SS-Rag1^em1Mcwi and SS-Cd247^em1Mcwi knockout rats (16, 17) and reduced hypertension and renal injury in the SS-Ncf2^em1Mcwi (p67phox) null model exhibiting reduced medullary oxidative stress (18). Additionally, we have recently demonstrated multiple genes at a single hypertension GWAS-nominated locus (Agtrap-Plod1 locus) can have additive or subtractive effects on blood pressure and renal function when mutated in the SS rat (19). These previous studies highlight the utility of this model system for testing the roles of GWAS candidate human disease genes by disrupting their specific rat orthologs using ZFN technology (20).A single-nucleotide polymorphism (SNP) (rs381815, minor allele frequency 0.26) in intron 1 of the pleckstrin homology domain containing family A member 7 (PLEKHA7) gene, was identified by five independent GWAS to be associated with elevated systolic blood pressure and hypertension in multiple populations (5, 6, 8, 21, 22). The associated locus contains only the PLEKHA7 gene (5); however, the genetic mechanism(s) underlying this locus have not yet been functionally characterized. PLEKHA7 is highly expressed in the kidney and heart, where it may be involved in formation and maintenance of the apical junction complex of epithelial cells (23). However, limited data on PLEKHA7 function are available to extrapolate its potential role(s) in the pathogenesis of hypertension. Here we used ZFN mutagenesis to obtain the first evidence to our knowledge in any model system that Plekha7 has a functional role in several hypertension-associated phenotypes in the rat. We found that mutation of Plekha7 in the SS rat attenuated salt-induced hypertension, reduced renal damage, and improved cardiac function. We also show that Plekha7 modulates calcium handling and nitric oxide (NO) bioavailability, both of which are required for normal vascular health. Collectively, these studies provide significant mechanistic insight to the role of Plekha7 in salt-sensitive hypertension.     

Postsynaptic distribution of IRSp53 in spiny excitatory and inhibitory neurons

The 53 kDa insulin receptor substrate protein (IRSp53) is highly enriched in the brain. Despite evidence that links mutations of IRSp53 with autism and other neuropsychiatric problems, the functional significance of this protein remains unclear. We used light and electron microscopic immunohistochemistry to demonstrate that IRSp53 is expressed throughout the adult rat brain. Labeling concentrated selectively in dendritic spines, where it was associated with the postsynaptic density (PSD). Surprisingly, its organization within the PSD of spiny excitatory neurons of neocortex and hippocampus differed from that within spiny inhibitory neurons of neostriatum and cerebellar cortex. The present data support previous suggestions that IRSp53 is involved in postsynaptic signaling, while hinting that its signaling role may differ in different types of neurons. J. Comp. Neurol. 522:2164–2178, 2014. © 2013 Wiley Periodicals, Inc.     

Common variation at 6p21.31 (BAK1) influences the risk of chronic lymphocytic leukemia

We performed a meta-analysis of 3 genome-wide association studies to identify additional common variants influencing chronic lymphocytic leukemia (CLL) risk. The discovery phase was composed of genome-wide association study data from 1121 cases and 3745 controls. Replication analysis was performed in 861 cases and 2033 controls. We identified a novel CLL risk locus at 6p21.33 (rs210142; intronic to the BAK1 gene, BCL2 antagonist killer 1; P = 9.47 × 10(-16)). A strong relationship between risk genotype and reduced BAK1 expression was shown in lymphoblastoid cell lines. This finding provides additional support for polygenic inheritance to CLL and provides further insight into the biologic basis of disease development.     

In vivo degradation performance of micro-arc-oxidized magnesium implants: A micro-CT study in rats

Biodegradable Mg alloys are of great interest for osteosynthetic applications because they do not require surgical removal after they have served their purpose. In this study, fast-degrading ZX50 Mg-based implants were surface-treated by micro-arc oxidation (MAO), to alter the initial degradation, and implanted along with untreated ZX50 controls in the femoral legs of 20 male Sprague–Dawley rats. Their degradation was monitored by microfocus computed tomography (μCT) over a total observation period of 24 weeks, and histological analysis was performed after 4, 12 and 24 weeks. While the MAO-treated samples showed almost no corrosion in the first week, they revealed an accelerated degradation rate after the third week, even faster than that of the untreated ZX50 implants. This increase in degradation rate can be explained by an increase in the surface-area-to-volume ratio of MAO-treated implants, which degrade inhomogeneously via localized corrosion attacks. The histological analyses show that the initially improved corrosion resistance of the MAO implants has a positive effect on bone and tissue response: The reduced hydrogen evolution (due to reduced corrosion) makes possible increased osteoblast apposition from the very beginning, thus generating a stable bone–implant interface. As such, MAO treatment appears to be very interesting for osteosynthetic implant applications, as it delays implant degradation immediately after implantation, enhances fracture stabilization, minimizes the burden on the postoperatively irritated surrounding tissue and generates good bone–implant connections, followed by accelerated degradation in the later stage of bone healing.