The diagnosis and management of Gaucher disease in pediatric patients: Where do we go from here?

Gaucher disease (GD) is an autosomal recessive inherited lysosomal storage disease that often presents in early childhood and is associated with damage to multiple organ systems. Many challenges associated with GD diagnosis and management arise from the considerable heterogeneity of disease presentations and natural history. Phenotypic classification has traditionally been based on the absence (in type 1 GD) or presence (in types 2 and 3 GD) of neurological involvement of varying severity. However, patient management and prediction of prognosis may be best served by a dynamic, evolving definition of individual phenotype rather than by a rigid system of classification. Patients may experience considerable delays in diagnosis, which can potentially be reduced by effective screening programs; however, program implementation can involve ethical and practical challenges. Variation in the clinical course of GD and an uncertain prognosis also complicate decisions concerning treatment initiation, with differing stakeholder perspectives around efficacy and acceptable cost/benefit ratio. We review the challenges faced by physicians in the diagnosis and management of GD in pediatric patients. We also consider future directions and goals, including acceleration of accurate diagnosis, improvements in the understanding of disease heterogeneity (natural history, response to treatment, and prognosis), the need for new treatments to address unmet needs for all forms of GD, and refinement of the tools for monitoring disease progression and treatment efficacy, such as specific biomarkers.

Assessment of iron nanoparticle distribution in mouse models using ultrashort-echo-time MRI.

Microscopic magnetic field inhomogeneities caused by iron deposition or tissue-air interfaces may result in rapid decay of transverse magnetization in MRI. The aim of this study is to detect and quantify the distribution of iron-based nanoparticles in mouse models by applying ultrashort-echo-time (UTE) sequences in tissues exhibiting extremely fast transverse relaxation. In 24 C57BL/6 mice (two controls), suspensions containing either non-oxidic Fe or AuFeOx nanoparticles were injected into the tail vein at two doses (200 µg and 600 µg per mouse). Mice underwent MRI using a UTE sequence at 4.7 T field strength with five different echo times between 100 µs and 5000 µs. Transverse relaxation times T2 * were computed for the lung, liver, and spleen by mono-exponential fitting. In UTE imaging, the MRI signal could reliably be detected even in liver parenchyma exhibiting the highest deposition of nanoparticles. In animals treated with Fe nanoparticles (600 µg per mouse), the relaxation time substantially decreased in the liver (3418 ± 1534 µs (control) versus 228 ± 67 µs), the spleen (2170 ± 728 µs versus 299 ± 97 µs), and the lungs (663 ± 101 µs versus 413 ± 99 µs). The change in transverse relaxation was dependent on the number and composition of the nanoparticles. By pixel-wise curve fitting, T2 * maps were calculated showing nanoparticle distribution. In conclusion, UTE sequences may be used to assess and quantify nanoparticle distribution in tissues exhibiting ultrafast signal decay in MRI.

Biochemical Studies in Fibroblasts to Interpret Variants of Unknown Significance in the ABCD1 Gene.

Due to newborn screening for X-linked adrenoleukodystrophy (ALD), and the use of exome sequencing in clinical practice, the detection of variants of unknown significance (VUS) in the ABCD1 gene is increasing. In these cases, functional tests in fibroblasts may help to classify a variant as (likely) benign or pathogenic. We sought to establish reference ranges for these tests in ALD patients and control subjects with the aim of helping to determine the pathogenicity of VUS in ABCD1. Fibroblasts from 36 male patients with confirmed ALD, 26 healthy control subjects and 17 individuals without a family history of ALD, all with an uncertain clinical diagnosis and a VUS identified in ABCD1, were included. We performed a combination of tests: (i) a test for very-long-chain fatty acids (VLCFA) levels, (ii) a D3-C22:0 loading test to study the VLCFA metabolism and (iii) immunoblotting for ALD protein. All ALD patient fibroblasts had elevated VLCFA levels and a reduced peroxisomal ß-oxidation capacity (as measured by the D3-C16:0/D3-C22:0 ratio in the D3-C22:0 loading test) compared to the control subjects. Of the VUS cases, the VLCFA metabolism was not significantly impaired (most test results were within the reference range) in 6/17, the VLCFA metabolism was significantly impaired (most test results were within/near the ALD range) in 9/17 and a definite conclusion could not be drawn in 2/17 of the cases. Biochemical studies in fibroblasts provided clearly defined reference and disease ranges for the VLCFA metabolism. In 15/17 (88%) VUS we were able to classify the variant as being likely benign or pathogenic. This is of great clinical importance as new variants will be detected.

Dynamic Measurement of Tumor Vascular Permeability and Perfusion using a Hybrid System for Simultaneous Magnetic Resonance and Fluorescence Imaging.

PURPOSE: Assessing tumor vascular features including permeability and perfusion is essential for diagnostic and therapeutic purposes. The aim of this study was to compare fluorescence and magnetic resonance imaging (MRI)-based vascular readouts in subcutaneously implanted tumors in mice by simultaneous dynamic measurement of tracer uptake using a hybrid fluorescence molecular tomography (FMT)/MRI system. PROCEDURE: Vascular permeability was measured using a mixture of extravascular imaging agents, GdDOTA and the dye Cy5.5, and perfusion using a mixture of intravascular agents, Endorem and a fluorescent probe (Angiosense). Dynamic fluorescence reflectance imaging (dFRI) was integrated into the hybrid system for high temporal resolution. RESULTS: Excellent correspondence between uptake curves of Cy5.5/GdDOTA and Endorem/Angiosense has been found with correlation coefficients R > 0.98. The two modalities revealed good agreement regarding permeability coefficients and centers-of-gravity of the imaging agent distribution. CONCLUSION: The FMT/dFRI protocol presented is able to accurately map physiological processes and poses an attractive alternative to MRI for characterizing tumor neoangiogenesis.

In vivo imaging of mouse tumors by a lipidated cathepsin S substrate.

The synthesis and evaluation of two cathepsin S-specific probes is described. For long-term retention of the probe at the target site and a high signal-to-noise ratio, we introduced a lipidation approach via the simple attachment of palmitoic acid to the reporter. After cathepsin S-specific cleavage in cultured cells and in a grafted tumor mouse model, fluorescence increased owing to dequenching and we observed an intracellular accumulation of the fluorescence in the target tissue. The lipidated probe provided a prolonged and strongly fluorescent signal in tumors when compared to the very similar non-lipidated probe, demonstrating that non-invasive tumor identification is feasable. The homing principle by probe lipidation might also work for selective administration of cytotoxic compounds to specifically reduce tumor mass.

Longitudinal evaluation of hepatic lipid deposition and composition in ob/ob and ob/+ control mice.

Obesity is associated with insulin resistance (IR) and hepatosteatosis. Understanding the link between IR and hepatosteatosis could be relevant to chronic clinical outcomes. The objective of this study was to quantitatively assess lipid deposition (fractional lipid mass, fLM) and composition (fraction of polyunsaturated lipids, fPUL and mean chain length, MCL) in livers of ob/ob mice, a genetic model of obesity and mild diabetes, and ob/+ heterozygous control animals in a noninvasive manner using (1) H-MRS at 9.4T. For accurate quantification, intensity values were corrected for differences in T2 values while T1 effects were considered minimal due to the long TR values used. Values of fLM, fPUL and MCL were derived from T2 -corrected signal intensities of lipids and water resonance. Hepatic lipid signals were compared with fasted plasma insulin, glucose and lipid levels. Statistically significant correlations between fPUL and fasting plasma insulin/glucose levels were found in adolescent ob/ob mice. A similar correlation was found between fLM and fasting plasma insulin levels; however, the correlation between fLM and fasting plasma glucose levels was less obvious in adolescent ob/ob mice. These correlations were lost in adult ob/ob mice. The study showed that in adolescent ob/ob mice, there was an obvious link between lipid deposition/composition in the liver and plasma insulin/glucose levels. This correlation was lost in adult animals, probably due to the limited lipid storage capacity of the liver.

Prenatally engineered autologous amniotic fluid stem cell-based heart valves in the fetal circulation.

Prenatal heart valve interventions aiming at the early and systematic correction of congenital cardiac malformations represent a promising treatment option in maternal-fetal care. However, definite fetal valve replacements require growing implants adaptive to fetal and postnatal development. The presented study investigates the fetal implantation of prenatally engineered living autologous cell-based heart valves. Autologous amniotic fluid cells (AFCs) were isolated from pregnant sheep between 122 and 128 days of gestation via transuterine sonographic sampling. Stented trileaflet heart valves were fabricated from biodegradable PGA-P4HB composite matrices (n = 9) and seeded with AFCs in vitro. Within the same intervention, tissue engineered heart valves (TEHVs) and unseeded controls were implanted orthotopically into the pulmonary position using an in-utero closed-heart hybrid approach. The transapical valve deployments were successful in all animals with acute survival of 77.8% of fetuses. TEHV in-vivo functionality was assessed using echocardiography as well as angiography. Fetuses were harvested up to 1 week after implantation representing a birth-relevant gestational age. TEHVs showed in vivo functionality with intact valvular integrity and absence of thrombus formation. The presented approach may serve as an experimental basis for future human prenatal cardiac interventions using fully biodegradable autologous cell-based living materials.

Hybrid small animal imaging system combining magnetic resonance imaging with fluorescence tomography using single photon avalanche diode detectors.

The high sensitivity of fluorescence imaging enables the detection of molecular processes in living organisms. However, diffuse light propagation in tissue prevents accurate recovery of tomographic information on fluorophore distribution for structures embedded deeper than 0.5 mm. Combining optical with magnetic resonance imaging (MRI) provides an accurate anatomical reference for fluorescence imaging data and thereby enables the correlation of molecular with high quality structural/functional information. We describe an integrated system for small animal imaging incorporating a noncontact fluorescence molecular tomography (FMT) system into an MRI detector. By adopting a free laser beam design geometrical constraints imposed by the use of optical fibers could be avoided allowing for flexible fluorescence excitation schemes. Photon detection based on a single-photon avalanche diode array enabled simultaneous FMT/MRI measurements without interference between modalities. In vitro characterization revealed good spatial accuracy of FMT data and accurate quantification of dye concentrations. Feasibility of FMT/MRI was demonstrated in vivo by simultaneous assessment of protease activity and tumor morphology in murine colon cancer xenografts.

N-acetyl-D-glucosaminylphosphatidylinositol de-N-acetylase from Entamoeba histolytica: metal alters catalytic rates but not substrate affinity.

PIG-L/GPI12 proteins are endoplasmic reticulum-resident membrane proteins involved in the second step of glycosylphosphatidylinositol anchor biosynthesis in eukaryotes. We show that the Entamoeba histolytica PIG-L protein is optimally active in the acidic pH range. The enzyme has an intrinsic low level of de-N-acetylase activity in the absence of metal and is significantly stimulated by divalent cations. Metal binding induces a large conformational change in the protein that appears to improve catalytic rates while not altering the affinity of the enzyme for its substrate.

Improving the accuracy of a solid spherical source radius and depth estimation using the diffusion equation in fluorescence reflectance mode.

BACKGROUND: Non-invasive planar fluorescence reflectance imaging (FRI) is used for accessing physiological and molecular processes in biological tissue. This method is efficiently used to detect superficial fluorescent inclusions. FRI is based on recording the spatial radiance distribution (SRD) at the surface of a sample. SRD provides information for measuring structural parameters of a fluorescent source (such as radius and depth). The aim of this article is to estimate the depth and radius of the source distribution from SRD, measured at the sample surface. For this reason, a theoretical expression for the SRD at the surface of a turbid sample arising from a spherical light source embedded in the sample, was derived using a steady-state solution of the diffusion equation with an appropriate boundary condition. METHODS: The SRD was approximated by solving the diffusion equation in an infinite homogeneous medium with solid spherical sources in cylindrical geometry. Theoretical predications were verified by experiments with fluorescent sources of radius 2-6 mm embedded at depths of 2-4 mm in a tissue-like phantom. RESULTS: The experimental data were compared with the theoretical values which shows that the root mean square (RMS) error in depth measurement for nominal depth values d = 2, 2.5, 3, 3.5, 4 mm amounted to 17%, 5%, 2%, 1% and 5% respectively. Therefore, the average error in depth estimation was < or = 4% for depths larger than the photon mean free path. CONCLUSIONS: An algorithm is proposed that allows estimation of the location and radius of a spherical source in a homogeneous tissue-like phantom by accounting for anisotropic light scattering effect using FRI modality. Surface SRD measurement enabled accurate estimates of fluorescent depth and radius in FRI modality, and can be used as an element of a more general tomography reconstruction algorithm.

Alternative M2 activation of Kupffer cells by PPARdelta ameliorates obesity-induced insulin resistance.

Macrophage infiltration and activation in metabolic tissues underlie obesity-induced insulin resistance and type 2 diabetes. While inflammatory activation of resident hepatic macrophages potentiates insulin resistance, the functions of alternatively activated Kupffer cells in metabolic disease remain unknown. Here we show that in response to the Th2 cytokine interleukin-4 (IL-4), peroxisome proliferator-activated receptor delta (PPARdelta) directs expression of the alternative phenotype in Kupffer cells and adipose tissue macrophages of lean mice. However, adoptive transfer of PPARdelta(-/-) (Ppard(-/-)) bone marrow into wild-type mice diminishes alternative activation of hepatic macrophages, causing hepatic dysfunction and systemic insulin resistance. Suppression of hepatic oxidative metabolism is recapitulated by treatment of primary hepatocytes with conditioned medium from PPARdelta(-/-) macrophages, indicating direct involvement of Kupffer cells in liver lipid metabolism. Taken together, these data suggest an unexpected beneficial role for alternatively activated Kupffer cells in metabolic syndrome and type 2 diabetes.

Bioinformatics and functional analysis of an Entamoeba histolytica mannosyltransferase necessary for parasite complement resistance and hepatical infection.

The glycosylphosphatidylinositol (GPI) moiety is one of the ways by which many cell surface proteins, such as Gal/GalNAc lectin and proteophosphoglycans (PPGs) attach to the surface of Entamoeba histolytica, the agent of human amoebiasis. It is believed that these GPI-anchored molecules are involved in parasite adhesion to cells, mucus and the extracellular matrix. We identified an E. histolytica homolog of PIG-M, which is a mannosyltransferase required for synthesis of GPI. The sequence and structural analysis led to the conclusion that EhPIG-M1 is composed of one signal peptide and 11 transmembrane domains with two large intra luminal loops, one of which contains the DXD motif, involved in the enzymatic catalysis and conserved in most glycosyltransferases. Expressing a fragment of the EhPIG-M1 encoding gene in antisense orientation generated parasite lines diminished in EhPIG-M1 levels; these lines displayed reduced GPI production, were highly sensitive to complement and were dramatically inhibited for amoebic abscess formation. The data suggest a role for GPI surface anchored molecules in the survival of E. histolytica during pathogenesis.

Macrophage-specific PPARgamma controls alternative activation and improves insulin resistance.

Obesity and insulin resistance, the cardinal features of metabolic syndrome, are closely associated with a state of low-grade inflammation. In adipose tissue chronic overnutrition leads to macrophage infiltration, resulting in local inflammation that potentiates insulin resistance. For instance, transgenic expression of Mcp1 (also known as chemokine ligand 2, Ccl2) in adipose tissue increases macrophage infiltration, inflammation and insulin resistance. Conversely, disruption of Mcp1 or its receptor Ccr2 impairs migration of macrophages into adipose tissue, thereby lowering adipose tissue inflammation and improving insulin sensitivity. These findings together suggest a correlation between macrophage content in adipose tissue and insulin resistance. However, resident macrophages in tissues display tremendous heterogeneity in their activities and functions, primarily reflecting their local metabolic and immune microenvironment. While Mcp1 directs recruitment of pro-inflammatory classically activated macrophages to sites of tissue damage, resident macrophages, such as those present in the adipose tissue of lean mice, display the alternatively activated phenotype. Despite their higher capacity to repair tissue, the precise role of alternatively activated macrophages in obesity-induced insulin resistance remains unknown. Using mice with macrophage-specific deletion of the peroxisome proliferator activated receptor-gamma (PPARgamma), we show here that PPARgamma is required for maturation of alternatively activated macrophages. Disruption of PPARgamma in myeloid cells impairs alternative macrophage activation, and predisposes these animals to development of diet-induced obesity, insulin resistance, and glucose intolerance. Furthermore, gene expression profiling revealed that downregulation of oxidative phosphorylation gene expression in skeletal muscle and liver leads to decreased insulin sensitivity in these tissues. Together, our findings suggest that resident alternatively activated macrophages have a beneficial role in regulating nutrient homeostasis and suggest that macrophage polarization towards the alternative state might be a useful strategy for treating type 2 diabetes.

Quantitative expansion of ES cell-derived myeloid progenitors capable of differentiating into macrophages.

Macrophages participate in physiologic and pathologic processes through elaboration of distinct activation programs. Studies with macrophage cell systems have revealed much concerning the importance of this pleiotropic cell; however, these studies are inherently limited by three factors: heterogeneity of the target cell population, poor capacity to elaborate various activation programs, and lack of a genetically tractable model system for loss- and gain-of-function studies. Although definitive, hematopoietic lineages can be isolated from embryonic stem (ES) cells, these isolation procedures are inefficient and time-consuming and require elaborate cell-sorting protocols. We therefore examined whether myeloid precursors, capable of differentiating into macrophages, could be conditionally expanded in vitro. Here, we report methods for selective isolation and immortalization of ES cell-derived myeloid precursors by estrogen-regulated HoxA9 protein. Using this new macrophage differentiation system, an unlimited number of custom-designed macrophages with defined functional characteristics can be generated from any targeted ES cell. In combination with knockout or small interfering RNA knockdown technologies, this macrophage differentiation system provides a powerful tool for high throughput analysis of regulatory mechanisms controlling macrophage activation in health and disease.

Oxidative metabolism and PGC-1beta attenuate macrophage-mediated inflammation.

Complex interplay between T helper (Th) cells and macrophages contributes to the formation and progression of atherosclerotic plaques. While Th1 cytokines promote inflammatory activation of lesion macrophages, Th2 cytokines attenuate macrophage-mediated inflammation and enhance their repair functions. In spite of its biologic importance, the biochemical and molecular basis of how Th2 cytokines promote maturation of anti-inflammatory macrophages is not understood. We show here that in response to interleukin-4 (IL-4), signal transducer and activator of transcription 6 (STAT6) and PPARgamma-coactivator-1beta (PGC-1beta) induce macrophage programs for fatty acid oxidation and mitochondrial biogenesis. Transgenic expression of PGC-1beta primes macrophages for alternative activation and strongly inhibits proinflammatory cytokine production, whereas inhibition of oxidative metabolism or RNAi-mediated knockdown of PGC-1beta attenuates this immune response. These data elucidate a molecular pathway that directly links mitochondrial oxidative metabolism to the anti-inflammatory program of macrophage activation, suggesting a potential role for metabolic therapies in treating atherogenic inflammation.

Reduction of cell surface glycosylphosphatidylinositol conjugates in Entamoeba histolytica by antisense blocking of E. histolytica GlcNAc-phosphatidylinositol deacetylase expression: effect on cell proliferation, endocytosis, and adhesion to target cells.

Glycosylphosphatidylinositol (GPI)-anchored molecules such as cell surface Gal/GalNAc lectin and proteophosphoglycans of the protozoan parasite Entamoeba histolytica are thought to be involved in pathogenesis. Here, we report the identification of genes that may be involved in the GPI biosynthetic pathway of E. histolytica by use of bioinformatic tools applied to the recently published genome sequence. Of the genes identified, one of the early genes, GlcNAc-phosphatidylinositol deacetylase (PIG-L), was partially characterized. Cell lines deficient in E. histolytica PIG-L (EhPL-AS) or overproducing it (EhPL-S) were generated by expressing the gene in the antisense or sense orientation, respectively, in a tetracycline-inducible system. The overexpressing cells showed higher EhPIG-L activity and increased production of GlcN-PI. Conversely, cells expressing the antisense RNA displayed reduced GlcN-PI production. The total number of GPI-containing molecules was also reduced in these cells, as demonstrated by Alexa 488 fluorescently labeled proaerolysin labeling. The distribution of GPI-linked PPG and Gal/GalNAc lectin was altered in the tetracycline-induced EhPL-AS cell lines. Further, the antisense-blocked cells showed 36% suppression of cell growth, 50 to 60% inhibition of fluid phase endocytosis, and about 50% inhibition of adhesion to target cells. Therefore, our data suggest the importance of GPI anchors in regulating some of the events in amoebic pathogenesis. They also demonstrated the use of antisense RNA-mediated inhibition of GPI biosynthetic enzymes as an approach to decrease the amount of GPI conjugates in E. histolytica.