Emerging Approaches for Fluorescence-Based Newborn Screening of Mucopolysaccharidoses.

Interest in newborn screening for mucopolysaccharidoses (MPS) is growing, due in part to ongoing efforts to develop new therapies for these disorders and new screening assays to identify increased risk for the individual MPSs on the basis of deficiency in the cognate enzyme. Existing tests for MPSs utilize either fluorescence or mass spectrometry detection methods to measure biomarkers of disease (e.g., enzyme function or glycosaminoglycans) using either urine or dried blood spot (DBS) samples. There are currently two approaches to fluorescence-based enzyme function assays from DBS: (1) manual reaction mixing, incubation, and termination followed by detection on a microtiter plate reader; and (2) miniaturized automation of these same assay steps using digital microfluidics technology. This article describes the origins of laboratory assays for enzyme activity measurement, the maturation and clinical application of fluorescent enzyme assays for MPS newborn screening, and considerations for future expansion of the technology.

Fluorimetric assay with a novel substrate for quantification of galactocerebrosidase activity in dried blood spot specimens.

BACKGROUND: Decreased galactocerebrosidase (GALC) enzyme activity is causative for Krabbe disease, a lysosomal storage disorder with devastating neurodegenerative consequences. Quantitative fluorimetric assays for GALC activity in isolated blood and skin cells have been described; however, no such assay has been described using dried blood spot (DBS) specimens. METHODS: GALC enzyme activity was measured quantitatively using fluorescence from a novel glycosidic substrate: carboxy derived from 6-hexadecanoylamino-4-methylumbelliferone. GALC activity was demonstrated on newborn DBS specimens, known Krabbe disease patient specimens, proficiency testing and quality control samples. RESULTS: We present data on characterization of the novel substrate and assay, including pH optimization and enzyme kinetics using a fluorimetric profile. Single and multi-day precision analyses revealed tight analytical measurements with %CV ranging from 5.2% to 14.1%. GALC enzyme activity was linear over the range of 0.31 - 12.04 µmol/l/h with a limit of detection of 0.066 µmol/l/h. Our results with this assay show a clear discrimination between GALC activities in samples from Krabbe disease patients versus presumed normal newborn samples. CONCLUSIONS: A fluorimetric assay for GALC enzyme activity measurement on dried blood spot specimens is feasible. Improvements to the assay including novel substrate design, increased substrate concentration and removal of sodium chloride maximize the specificity of the assay and minimize interference from ß-galactosidase.

The role of antigen specificity in the binding of murine monoclonal anti-DNA antibodies to microparticles from apoptotic cells.

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and markers of underlying immune system disturbances. These antibodies bind to both single-stranded and double-stranded DNA, mediating pathogenesis by forming immune complexes. As shown recently, DNA in blood exists in both free and particulate forms, with DNA representing an important component of microparticles. Microparticles are membrane-bound vesicles containing nuclear molecules, released by membrane blebbing during cell death and activation. A panel of monoclonal NZB/NZW F1 anti-DNA antibodies was tested for binding to microparticles generated from apoptotic THP-1 and Jurkat cells. These studies showed that only certain anti-DNA antibodies in the panel, specific for double-stranded DNA, bound to microparticles. Binding to particles was reduced by soluble DNA or DNase treatment. Together, these results indicate that particle binding is a feature of only certain anti-DNA antibodies, reflecting immunochemical properties of the antibodies and the nature of the exposed DNA antigens.

Development of a fluorometric microtiter plate based enzyme assay for MPS IVA (Morquio type A) using dried blood spots.

Mucopolysaccharidosis type IVA or Morquio type-A disease is a hereditary lysosomal storage disorder caused by deficient activity of the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). The disease is caused by lysosomal accumulation of unprocessed glycosaminoglycans (GAGs) that manifests with severe to mild skeletal and cardiopulmonary abnormalities. We have developed a modified microtiter plate-based enzyme activity assay using dried blood spots and a fluorescent substrate for measuring specific GALNS activity to identify patients with MPS IVA.

Development of a fluorometric microtiter plate-based enzyme assay for arylsulfatase B (MPS VI) using dried blood spots.

Mucopolysaccharidosis type VI or Maroteaux-Lamy syndrome is an autosomal recessive lysosomal storage disorder caused by deficiency of arylsulfatase B (ARS-B) enzyme activity. It results in mild to severe multi-organ system failure from accumulation of undigested glycosaminoglycans (GAGs); dermatan sulfate and chondroitin-4-sulfate. We have developed a single-step enzyme assay using a fluorescent substrate and dried blood spots to measure ARS-B activity to identify disease patients. This assay is robust, reproducible, specific and convenient to perform.

The role of microparticles in the generation of immune complexes in murine lupus.

Systemic lupus erythematosus is a systemic inflammatory disease characterized by antibodies to nuclear molecules in association with immune complex deposition. As shown previously, microparticles (MPs), which are small membrane-bound vesicles released from dying and activated cells, contain nucleic acids and can form immune complexes found in patient blood. To assess the role of MPs in murine lupus, we used flow cytometry to measure the presence of MPs with bound IgG in the blood of MRL-lpr/lpr and NZB/W mice. These studies showed much higher numbers of MPs with bound IgG in the blood of MRL lpr/lpr compared to NZB/W mice. Furthermore, these studies showed that antibodies from MRL-lpr/lpr mice bound better to MPs from apoptotic cells than those from NZB/W mice. Together, these studies indicate important differences in the serological features of the two strains as reflected by the capacity of antibodies to bind to MPs.

Microparticles as mediators and biomarkers of rheumatic disease.

Microparticles (MPs) are small membrane-bound vesicles that arise from activated and dying cells and enter the blood to display pro-inflammatory and pro-thrombotic activities. MPs are 0.1-1.0 µm in size and incorporate nuclear, cytoplasmic and membrane molecules as they detach from cells. This process can occur with cell activation as well as cell death, with particles likely corresponding to blebs that form on the cell surface during apoptosis. To measure particle expression, flow cytometry allows determination of particle numbers based on size as well as surface markers that denote the cell of origin; platelet MPs are usually the most abundant type in blood. As shown in in vitro and in vivo systems, MPs can promote inflammation and thrombosis resulting from their content of cytokines like IL-1 and pro-coagulant molecules like tissue factor. Certain particle types can be anti-inflammatory, however, suggesting a range of immunomodulatory activities depending on the cell of origin. Studies on patients with a wide range of rheumatic disease show increased MP numbers in blood, with platelet and endothelial particles associated with vascular manifestations; increased numbers of particles also occur in the joint fluid where they may drive cytokine production and activate synoviocytes. In autoimmune diseases such as SLE and RA, MPs may also contribute to disease pathogenesis by the formation of immune complexes. MPs thus represent novel subcellular structures that can impact on the pathogenesis of rheumatic disease and serve as biomarkers of underlying cellular disturbances.

Microparticles as antigenic targets of antibodies to DNA and nucleosomes in systemic lupus erythematosus.

Systemic lupus erythematosus is a prototypic autoimmune disease characterized by antibodies to DNA and other nuclear molecules. While these antibodies can form immune complexes, the mechanisms generating the bound nuclear antigens are not known. These studies investigated whether microparticles can form complexes with anti-DNA and other anti-nucleosomal antibodies. Microparticles are small membrane-bound vesicles released from dead and dying cells; these particles contain a variety of cellular components, including DNA. To assess antigenicity, microparticles generated in vitro from apoptotic cell lines were tested using murine monoclonal anti-DNA and anti-nucleosomal antibodies as well as plasma from lupus patients. Antibody binding was assessed by flow cytometry. As these studies showed, some but not all of the monoclonal antibodies bound to microparticles prepared from apoptotic HL-60, THP-1 and Jurkat cells. For HL-60 cells, both staurosporine and UV radiation led to the production of antigenically active particles. For the anti-DNA antibody with high particle binding, prior treatment of DNase reduced activity. With plasma from patients with SLE, antibody binding to microparticles was present although a clear relationship with anti-DNA antibody levels was not observed. To determine whether lupus plasma contains immune complexes with particle properties, particle preparations were tested for bound IgG by flow cytometry. These studies indicated that lupus plasma contains particles with IgG binding, with numbers correlated with anti-DNA levels. Together, these findings indicate that microparticles display DNA and nucleosomal molecules in an antigenic form and could represent a source of immune complexes in SLE.

HMGB1 and microparticles as mediators of the immune response to cell death.

In a wide variety of diseases, cell death represents both an outcome and an important step in pathogenesis. This duality occurs because cell death leads to the extracellular release of molecules and structures that can potently induce the innate immune system. These mediators include the alarmins which are endogenous cellular constituents that exit activated or dying cells to stimulate toll-like receptors (TLRs) as well as non-TLR receptors. Of alarmins, the nonhistone protein HMGB1 is the prototype. Like DNA and RNA, HMGB1 can translocate from cells as they die. The activity of HMGB1 may reflect its interaction with other molecules such as LPS, DNA, and cytokines. In addition to alarmins, dead and dying cells can release subcellular organelles called microparticles that contain cytoplasmic and nuclear constituents, including DNA and RNA. These particles can impact on many cell types to induce inflammation. The release of HMGB1 and microparticles shows important similarities, occurring with cell death as well as stimulation of certain but not all TLRs. Furthermore, nitric oxide can induce the release of both. These observations suggest that the products of dead cells can serve as important mediators to drive immune responses and promote inflammation and autoreactivity.

Application of antimicrobial polypeptide host defenses to aquaculture: Exploitation of downregulation and upregulation responses.

Antimicrobial polypeptides (AMPPs), consisting of peptides and small proteins with antimicrobial activity, are an integral component of innate immunity. Their often potent properties and widespread prevalence in fish suggests that designing means of manipulating their levels has considerable potential for maintaining or improving fish health. There is evidence that a number of chronic stresses lead to significant downregulation of AMPPs and thus their monitoring could be a highly sensitive measure of health status and risk of an infectious disease outbreak. Conversely, upregulation of AMPP expression could be used to enhance disease resistance in stressful environments, as well as improve the efficacy of traditional antimicrobial drugs. However, further work is required in linking levels of a number of AMPPs to physiological function since, while a number of studies have documented the down- or upregulation of AMPPs via gene expression, relatively few studies have quantitatively examined changes in protein expression. In addition, not all AMPPs appear to be expressed at microbicidal levels in vivo, suggesting that at least some may have functions other than being directly protective. Nonetheless, in fish, there is evidence that some constitutively expressed AMPPs, such as piscidins and histone-like proteins, are expressed at microbicidal levels and that they decline with stress. Furthermore, certain AMPPs derived from hemoglobin-ß are upregulated to microbicidal levels after experimental challenge. The likely widespread distribution of these three AMPP groups in fish provides the opportunity to design strategies to greatly improve the health of cultured fish populations.

Microparticles as a source of extracellular DNA.

Microparticles are small membrane-bound vesicles that display pro-inflammatory and pro-thrombotic activities important in the pathogenesis of a wide variety of diseases. These particles are released from activated and dying cells and incorporate nuclear and cytoplasmic molecules for extracellular export. Of these molecules, DNA is a central autoantigen in systemic lupus erythematosus (SLE). As studies in our laboratory show, DNA occurs prominently in microparticles, translocating into these structures during apoptotic cell death. This DNA is antigenically active and can bind to lupus anti-DNA autoantibodies. These findings suggest that microparticles are an important source of extracellular DNA to serve as an autoantigen and autoadjuvant in SLE.

The blood nucleome in the pathogenesis of SLE.

Systemic lupus erythematosus (SLE) is prototypic autoimmune disease characterized by the production of autoantibodies to DNA among other nuclear molecules. These antibodies can form immune complexes that promote pathogenesis by stimulating cytokine production and depositing in the kidney to instigate nephritis. The antigens that form these complexes arise from the blood nucleome, a pool of circulating macromolecules comprised of DNA, RNA and nuclear proteins released from cells. Cell death is a major source of these molecules, releasing DNA in a process that can be modeled in mice by the administration of cells killed ex vivo. In the mouse model, the appearance of blood DNA requires macrophages and differs between males and females. This finding raises the possibility that augmented levels of extracellular DNA and other nuclear antigens can contribute to the increased frequency of SLE in females. Extracellular DNA can occur in both a soluble and particulate form, with microparticles generated in vitro displaying antigenically active DNA. Together, these findings suggest that cell death is an important event in lupus pathogenesis and can provide a supply of blood DNA essential for immune complex formation.

The release of microparticles by Jurkat leukemia T cells treated with staurosporine and related kinase inhibitors to induce apoptosis.

Microparticles (MPs) are small membrane-bound vesicles released from cells undergoing activation or cell death. These particles display potent biological activities that can impact on physiologic and pathologic processes. Previous studies with the Jurkat T leukemia cell line demonstrated that staurosporine (STS) induces the release of MPs as cells undergo apoptosis. To investigate further this process, we tested the effects of STS, its analogue, 7-hydroxystaurosporine (UCN-01), and other protein kinase C (PKC) and cyclin-dependent kinase (CDK) inhibitors. FACS analysis was used to assess MP release. Results of these studies indicate that STS and UCN-01 induce MP release by Jurkat cells; in contrast, other PKC and CDK inhibitors failed to induce comparable release, suggesting that release does not result from simple inhibition of either kinase alone. Time course experiments indicated that STS-induced particle release occurred as early as 2 h after treatment, with the early release MPs displaying low levels of binding of annexin V and propidium iodide (PI). Early-release MPs, however, matured in culture to an annexin V- and PI-positive phenotype. Together, these results indicate that STS and UCN-01 induce MPs that are phenotypically distinct and reflect specific patterns of kinase inhibition during apoptosis.

Use of SYTO 13, a fluorescent dye binding nucleic acids, for the detection of microparticles in in vitro systems.

Microparticles (MPs) are small membrane-bound vesicles that are released from activated or dying cells by a blebbing process. These particles contain nuclear and cytoplasmic components and represent unique biomarkers for disease. The small size of particles, however, limits detection using flow cytometry with either light scatter or staining for surface markers. Because MPs contain DNA and RNA, we have explored the use of SYTO 13, a member of the class of SYTO dyes, for particle detection. SYTO 13 is cell permeable and has a high fluorescent yield when bound to DNA or RNA. In this study, we compared detection of MPs using either light scatter or SYTO 13 staining, testing the hypothesis that, with fluorescence detection with SYTO 13, problems of "noise" with light scatter are reduced and the range of MP sizes detected is increased. In these experiments, particles were obtained from lymphoid cell lines treated in vitro to undergo apoptosis. As these results showed, STYO 13 allowed the detection of 1.5-2.9 times as many particles as did light scatter. The increased sensitivity was observed with three different cell lines and was independent of inducing stimulus. Treatment of fixed and permeabilized MPs with DNase and RNase showed that SYTO 13 binding resulted from interaction with both DNA and RNA. Together, these findings indicate that the nucleic acid content of MPs provides the basis for their detection in in vitro systems and suggests the utility of fluorescent dyes like SYTO 13 for more sensitive quantitative assays.

Antimicrobial peptides derived from hemoglobin are expressed in epithelium of channel catfish (Ictalurus punctatus, Rafinesque).

The beta-chain of the respiratory protein hemoglobin (Hbbeta), has recently been identified in novel sites, including mammalian macrophages and alveolar epithelium, as well as in gill microsomes of fish. However, the functional significance of extra-erythrocytically expressed hemoglobin has been unclear. Here we show inducible expression and upregulation of antimicrobial peptides (AMPs) homologous to Hbbeta in the gill epithelium of channel catfish (Ictalurus punctatus) in response to parasitic (Ichthyophthirius multifiliis, ich) infection. One peptide (HbbetaP-1), while having activity against some fish bacterial pathogens (e.g., Aeromonas hydrophila), had especially potent antiparasitic activity that was specifically lethal (lytic) to the feeding (trophont) stage of ich and also appeared to accelerate the differentiation of trophonts. However, it had no apparent effect on either the disseminative (theront) or reproductive (tomont) stages, nor was it lytic to channel catfish erythrocytes. Fish experimentally challenged with ich confirmed that the HbbetaP-1 sequence was both transcribed and translated in skin and gill epithelium, the target tissues for ich. The Hb AMP concentration expressed in vivo appeared to be well within the antiparasitic concentrations measured in vitro. Our findings suggest that hemoglobin-derived AMPs might play a significant role in the non-specific immune response.