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Results for “cell deconvolution”

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PubMed2006

A 'bottom-up' approach for endo-PK/PD analysis.

A 'bottom-up' PK/PD analysis approach employing system analysis principles of convolution/deconvolution and special nonparametric estimation procedures is presented to resolve the complex 'endo-PK/PD' of the endogenous form of recombinant drugs using erythropoietin (EPO) as an example. A novel cellular deconvolution algorithm is presented that facilitates the identification of the functional relationship between the variables involved in EPO's complex PK/PD. Five sheep each underwent two phlebotomies spaced 4-6 weeks apart when their hemoglobin levels were reduced from 12 g/dl to 3-4 g/dl. EPO levels and reticulocyte counts were frequently sampled. The data were analysed using end-constrained cubic splines. The rate of reticulocyte production was determined using the novel deconvolution methodology. The erythroid progenitor cells activation rate by EPO was estimated from the reticulocyte production rate using a lag-time parameter which determines the delay in the reticulocyte appearance in the blood relative to the activation of erythroid progenitors. Hysteresis minimization combined with cellular deconvolution was employed to determine the population PK/PD transduction function relating the progenitor activation rate to EPO concentrations in a nonparametric manner without assuming a specific structure. The proposed approach provides a rational informative starting point for developing parametric PK/PD models to resolve the complex endo-PK/PD of recombinant drugs.

Anemia↗

PubMed2006

Clear imaging of Förster resonance energy transfer (FRET) signals of Ras activation by a time-lapse three-dimensional deconvolution system.

A procedure for a time series of three-dimensional Förster resonance energy transfer observation of a cell has been established. It employs quantitative deconvolution and three-dimensional views of intensity-modulated displays. The development was done with Raichu, a synthetic Ras protein capable of producing Förster resonance energy transfer upon its activation, for which two-dimensional imaging has been established. This method gave much clearer images of Förster resonance energy transfer than does the usual method without deconvolution, even though the signals were relatively weak. The results suggest that this procedure is compatible with weak fluorescent light, which is prone to photobleaching.

Animals↗

PubMed2001

Membrane association of glutathione S-transferase mGSTA4-4, an enzyme that metabolizes lipid peroxidation products.

Lipid peroxidation products have signaling functions and at higher concentrations are toxic and may trigger cell death. The compounds are metabolized predominantly by glutathione S-transferases exemplified by mGSTA4-4, an enzyme highly efficient in glutathione conjugation of 4-hydroxyalkenals, and possessing glutathione peroxidase activity toward phospholipid hydroperoxides. mGSTA4-4 belongs to the predominant group of "canonical" glutathione S-transferases that are soluble and generally localized in the cytoplasm. The intracellular localization of mGSTA4-4 was examined in hepatocytes of normal mouse liver and in transfected HepG2 cells by fluorescence microscopy and digital deconvolution. mGSTA4-4 was found to be predominantly localized at or near the plasma membrane in transfected HepG2 cells, as well as in hepatocytes endogenously expressing the protein. In vitro, mGSTA4-4 associated with liposomes, and this interaction was potentiated when the liposomes contained negatively charged phospholipids. Mutating lysine 115 to glutamic acid resulted in a loss of the plasma membrane targeting of mGSTA4-4 as well as in a significant reduction of its binding to liposomes in vitro. These data suggest preferential targeting of mGSTA4-4 to the plasma membrane that may contain the major substrate(s) for this enzyme. Lysine 115 is critically important for the membrane association of mGSTA4-4, most likely by entering into an electrostatic interaction with negatively charged phospholipid headgroups.

Animals↗

PubMed2006

The internalization and degradation of human copper transporter 1 following cisplatin exposure.

The human copper transporter 1 (hCTR1), the major transporter responsible for copper influx, mediates one component of the cellular accumulation of cisplatin (DDP). Both copper and DDP cause rapid down-regulation of hCTR1 expression in human ovarian carcinoma cells. In this study, we investigated the mechanism of this effect using digital deconvolution microscopy and Western blot analysis of cells stained with antibodies directed at both ends of the protein. Treatment of 2008 cells with DDP in combination with inhibitors of various endosomal pathways (amiloride, cytochalasin D, nystatin, and methyl-beta-cyclodextrin) showed that hCTR1 degradation was blocked by amiloride and cytochalasin D, indicating that hCTR1 was internalized primarily by macropinocytosis. Expression of transdominant-negative forms of dynamin I and Rac showed that loss of hCTR1 was not dependent on pathways regulated by either of these proteins. DDP-induced loss of hCTR1 was blocked by the proteasome inhibitors lactacystin, proteasome inhibitor 1, and MG132. This study confirms that DDP triggers the rapid loss of hCTR1 from ovarian carcinoma cells at clinically relevant concentrations. The results indicate that DDP-induced loss of hCTR1 involves internalization from the plasma membrane by macropinocytosis followed by proteasomal degradation. Because hCTR1 is a major determinant of early DDP uptake, prevention of its degradation offers a potential approach to enhancing tumor sensitivity.

Amiloride↗

PubMed2026

Paired analysis of primary adenoid cystic carcinoma and derived cell lines reveals a mesenchymal and stem-like shift associated with therapy resistance.

Adenoid cystic carcinoma (ACC) is a salivary gland malignancy characterized by slow but persistent growth, frequent local recurrence, and late metastatic progression. Patients with unresectable, recurrent, or metastatic disease have limited therapeutic options. Efforts to identify effective therapeutic targets have been hindered by the limited availability of well-characterized ACC models. In this study, we established 11 ACC cell lines and performed RNA sequencing of nine cell lines and their matched primary tumors to evaluate the preservation and evolution of molecular and lineage-associated characteristics during cell line establishment. Comparative transcriptomic analysis revealed reduced epithelial and luminal differentiation programs in the cell lines, accompanied by enrichment of myoepithelial, EMT-, and cancer stem cell-associated transcriptional programs. Digital deconvolution and single-sample gene set enrichment analysis supported enrichment of hybrid EMT/stem-like states during in vitro propagation, while comparison with publicly available primary-recurrent ACC data demonstrated partial preservation of recurrence-associated plasticity and invasion programs. Protein-level validation of representative epithelial, myoepithelial, EMT, and stemness markers supported the major transcriptomic changes. In addition, a cell line with a higher stemness signature showed reduced sensitivity to cisplatin. Together, these findings indicate that ACC cell line establishment is associated with transcriptional reprogramming and enrichment of plastic, EMT/stem-like states while retaining selected ACC lineage characteristics. These models provide experimentally tractable platforms for investigating ACC progression, therapeutic response, and mechanisms of treatment resistance.

Adenoid cystic carcinoma↗

PubMed2007

Carboxymethyl beta-glucan binds to corneal epithelial cells and increases cell adhesion to laminin and resistance to oxidative stress.

PURPOSE: Polysaccharides are frequently used as viscoelastic agents to improve pharmacokinetics of ophthalmic preparations. Recently, polysaccharides from yeast cell walls such as beta-glucans have emerged as bioactive molecules endowed with immunomodulatory and cytoprotective properties. In this study, we investigated the effects of carboxymethyl beta-glucan (CMG), a water-soluble derivative of yeast beta-glucan, on cultured rabbit corneal epithelial cells. METHODS: We developed a fluorescein-labeled CMG to visualize its binding to corneal cells by means of digital microscopy and image deconvolution. The effects of CMG on adhesion and survival of corneal epithelial cells exposed to noxious stimuli were also studied. RESULTS: CMG binds defined regions scattered throughout the body of corneal cells, suggesting binding specificity. Tridimensional reconstruction of fluorescence shows that binding is localized mainly at the plasma and nuclear membranes. Interestingly, CMG binding is highly represented at the level of focal adhesion of cells spreading onto laminin. Accordingly, CMG promotes adhesion of corneal epithelial cells to laminin without affecting their proliferation rate. CMG also protects cells from oxidative stress-dependent cell death, being ineffective in preventing ultraviolet B cytotoxicity. CONCLUSIONS: Data show that CMG dynamically binds to corneal epithelial cells, promoting cell adhesion and resistance to oxidative stress.

Animals↗

PubMed2005

Activation of matrix metalloproteinase-9 via neuronal nitric oxide synthase contributes to NMDA-induced retinal ganglion cell death.

PURPOSE: Understanding the mechanism of neuronal cell death in retinal diseases like glaucoma is important for devising new treatments. One factor involves excitatory amino acid stimulation of N-methyl-D-aspartate (NMDA)-type glutamate receptors, excessive Ca2+ influx, and formation of nitric oxide (NO) via neuronal NO synthase (nNOS). Another factor is the abnormal activation of matrix metalloproteinases (MMPs), in particular MMP-9, which triggers an extracellular signaling cascade leading to apoptosis. This study was designed to investigate the mechanism of excitotoxic retinal ganglion cell (RGC) death in vivo and its relationship to MMP activation. METHODS: NMDA and glycine were injected into the vitreous of the eye in rats and in nNOS-deficient mice (nNOS-/-) versus control. Gelatinolytic activity of MMP-9 and MMP-2 by zymography and cellular localization by immunohistochemistry were examined, and the effect of MMP inhibition on NMDA-induced RGC death was tested. RESULTS: NMDA was found to upregulate the proform of MMP-9 in the retina and to increase MMP-9 gelatinolytic activity. Retrograde labeling with aminostilbamidine to identify RGCs confirmed that MMP activity occurred only in these retinal neurons and not in glial or other retinal cell types after excitotoxic insult. Deconvolution fluorescence microscopy revealed that MMP activity colocalized with immunoreactive S-nitrosylated protein. NMDA-induced MMP activation was diminished in the retina of nNOS-/- mice, implying that S-nitrosylation of MMP had indeed occurred. In addition, the broad-spectrum MMP inhibitor GM6001 protected RGCs after intravitreal NMDA injection. CONCLUSIONS: These findings suggest that an extracellular proteolytic pathway in the retina contributes to RGC death via NO-activated MMP-9.

Animals↗

PubMedDate not supplied

Basal and gonadotropin-releasing hormone-releasable serum follicle-stimulating hormone charge isoform distribution and in vitro biological-to-immunological ratio in male puberty.

Follicle-stimulating hormone is synthesized and secreted as a mixture of heterogeneous isoforms that differ from each other in carbohydrate structure, biological potency, and plasma half-life. The relative abundance of the FSH isoforms will depend on the endocrine status of the donor at the time of sample collection. In the present study, we attempted to define the impact of the changing endocrine milieu characteristic of male puberty on the charge heterogeneity and plasma half-life of the serum FSH isoforms released under endogenous and exogenous GnRH drives, and examined whether such a varying hormone milieu modifies the capability of the circulating hormone to trigger intracellular signal transduction at the human FSH receptor level. Forty healthy male subjects at Tanner stages (Ts) 1 to 5 were sampled at 10 min intervals for 10 h. Serum from successive samples collected across 2-4 h intervals containing FSH released under basal, low-dose (10 microg), and high-dose (90 microg) exogenous GnRH-stimulated conditions was subjected to preparative chromatofocusing and tested for bioactivity employing a homologous cell in vitro bioassay system. Deconvolution analysis was applied to estimate the apparent endogenous FSH plasma half-life in samples obtained after administration of low-dose exogenous GnRH. Under all conditions studied, serum FSH charge isoforms were distributed along a pH range of 7.0 to less than 3.0. Comparisons across the different Tanner stages revealed a significant and selective increase in the ratio of FSH isoforms with elution pH values <4.50 relative to those with values >/=4.50 at Ts-2. At Ts-3, this ratio returned to that present at Ts-1, to decline thereafter during the ensuing pubertal stages. Serum bioactive FSH concentrations progressively increased (from 3.72 +/- 1.3 to 16.2 +/- 5.3 IU/L) throughout puberty, and in all conditions bioactive FSH concentrations exceeded those detected by a specific radioimmunoassay. The biological to immunological (B:I) FSH ratio at baseline was significantly (p < 0.05) lower at Ts-1 and Ts-2 (1.33 +/- 0.30 and 1.62 +/- 0.34, respectively) than at more advanced stages of pubertal development (2.28 +/- 0.20, 2.96 +/- 0.38, and 2.77 +/- 0.63 at Ts-3-, 4-, and -5, respectively) Similar differences were detected in samples containing FSH molecules released after low- and high-dose GnRH administration. The apparent endogenous FSH half-life of the deconvolved GnRH-induced FSH pulses was similar in the five study groups. These results demonstrate that the transition from infancy to sexual maturity in men is accompanied by qualitative changes in the circulating FSH isoform mixture. Although the changes in FSH glycosylation occurring throughout puberty are not of sufficient magnitude to alter the survival of the gonadotropin in circulation, they allow preferential secretion of bioactive FSH. The enrichment of the circulating mix of FSH isoforms with highly bioactive variants throughout spontaneous puberty may potentially favor the development of spermatogenesis and acquisition of reproductive competence.

Adolescent↗

PubMed1998

Altered instantaneous and calcium-modulated oscillatory PTH secretion patterns in patients with secondary hyperparathyroidism.

The relative contributions of increased parathyroid cell mass and altered control mechanisms of parathyroid hormone (PTH) secretion in secondary hyperparathyroidism are still controversial. In this study, endogenous pulsatile PTH secretion was analyzed by the multiparameter deconvolution technique to differentiate alterations in cell mass-dependent (PTH burst mass) and regulation-dependent (frequency, synchrony, calcium responsiveness) PTH release in uremic patients. PTH concentration versus time profiles were obtained in 13 uremic and 16 healthy adults under baseline conditions and during acute hypo- and hypercalcemia. Plasma PTH half-life was increased in patients compared with control subjects (4.7+/-1.9 versus 2.6+/-0.1 min, P < 0.005). The baseline PTH secretion rate was elevated eightfold in the patients as a result of an increased PTH mass secreted per burst (17.1+/-4.7 versus 2.0+/-0.4 pM, P = 0.0001), higher burst frequency (8.0+/-0.3 versus 6.8+/-0.3 h(-1), P < 0.01), and a higher tonic secretion rate (343+/-99 versus 30+/-4 pM/h, P = 0.0001). Acute hypocalcemia elicited an immediate, frequency- and amplitude-mediated selective increase in the pulsatile secretory component, which was fractionally weaker in patients (+595%) than control subjects (+1755%, P < 0.001). The acceleration and the amplification of PTH bursts were 35 and 60% lower in the patient group. Acute hypercalcemia suppressed total PTH secretion by 79% in control subjects but only by 63% in patients (P < 0.002). PTH burst frequency was reduced during hypercalcemia by 30% in control subjects, but remained unchanged in patients. In conclusion, uremic hyperparathyroidism is mediated by a marked increase in glandular secretion, but also by reduced PTH elimination. The increased spontaneous PTH burst frequency and the blunted responsiveness to changes in Ca2+ indicate partial uncoupling of hyperplastic parathyroid glands from the physiologic regulatory mechanisms that direct pulsatile PTH release.

Adolescent↗

PubMed2002

Mechanisms of hyperinsulinaemia in Child's disease grade B liver cirrhosis investigated in free living conditions.

AIMS: Human liver cirrhosis is commonly associated with increased fasting and glucose induced insulin concentrations. However, whether the hyperinsulinaemia is a consequence of increased pancreatic insulin secretion, decreased hepatic insulin removal, or impaired feedback regulation of insulin secretion is still doubtful. To investigate these issues, insulin secretion-during 24 hours of standardised living conditions-insulin sensitivity, and hepatic insulin extraction were assessed in cirrhotic patients compared with matched healthy subjects. PATIENTS: Nine Child's disease grade B cirrhotic patients and seven healthy volunteers, participated in the study. The subjects were studied on two separate days, one for the assessment of insulin secretion during a standardised 24 hour life period (calorimetric chamber), and one for the determination of insulin sensitivity. METHODS: Insulin secretion rates were reconstructed from plasma C peptide concentrations by deconvolution, and indices of beta cell function were derived using a mathematical model describing the functional dependence of insulin secretion on plasma glucose concentrations. Insulin sensitivity was determined using the euglycaemic hyperinsulinaemic clamp technique. RESULTS: Cirrhotic patients showed a marked hypersecretory response, both in absolute terms (mean (SEM) 295 (53) versus 138 (11) nmol/m(2), p<0.02), and in relation to glucose (175 (26) versus 57 (5) pmol/min/m(2), p<0.02). In particular, the beta cell dose-response function was shifted upward compared with controls. The sensitivity of insulin secretion to the rate of glucose change was also increased. Insulin sensitivity, markedly reduced in cirrhosis (157 (10) versus 296 (30) ml/min/m(2), p<0.002), was strongly inversely correlated (r=0.89, p<0.002) in these patients with insulin secretion at 5 mM glucose. Insulin clearance and hepatic insulin extraction were not reduced. A frank hypermetabolism with increased lipid oxidation was found in this series. CONCLUSIONS: This study suggests that hyperinsulinaemia, at least in Child's disease grade B cirrhotic patients, is the consequence of increased beta cell sensitivity to glucose, while hepatic insulin extraction does not seem to play a significant part.

Biomarkers↗

PubMed2003

Upon thyrotropin binding the thyrotropin receptor is internalized and localized to endosome.

To study the fate of TSH receptor (TSHR) on TSH binding, we constructed a chimeric cDNA that encodes a yellow fluorescent protein (YFP) fused to the carboxyl terminus of human TSHR. The protein expression in transfected cells was confirmed using flow cytometry. The functionality of the chimeric protein was determined by its ability to transduce signal leading to activation of cAMP in a TSH dose-dependent manner. The levels of cAMP produced by these cells were comparable with the levels seen in cells transfected with unfused TSHR without the YFP. Using deconvolution microscopy, we observed that the receptor is largely expressed on the cell surface, but on addition of TSH, some of the receptors were rapidly internalized. This conclusion was supported by several independent observations involving different cells expressing either native or recombinant TSHR. On TSH treatment, we observed internalization of human TSHR-YFP and human TSHR, expressed on 293 and CHO cells, respectively. This was further substantiated when we observed colocalization of rhodamine-labeled TSH with TSHR-YFP within the cell and by the uptake of radiolabeled TSH. Furthermore, shortly after ligand binding, there was a profound change in the morphology of the cells and some of the receptors accumulated in the perinuclear region of the cell. The TSHR-YFP was colocalized with RhoB-cyan fluorescent protein, indicating that it accumulated within the endosomes. These results indicate that the receptor internalization might in part be responsible for TSHR desensitization on TSH binding.

Animals↗

PubMed2006

Spectral unmixing of multicolored bioluminescence emitted from heterogeneous biological sources.

A wide variety of bioluminescent luciferase proteins are available for use in transcriptional or biochemical reporter assays. However, spectral overlap normally prevents them from being monitored simultaneously. To address this problem, a Java plug-in for ImageJ was written to deconvolute bioluminescent images composed of signals from multiple luciferases. The methodology was validated by testing the program with both simulated and real luciferase images. Bioluminescent images were acquired using a CCD camera equipped with optical filters, and the images were deconvoluted using the ImageJ plug-in. HeLa cells were transfected with either click beetle red luciferase (CBR), click beetle green luciferase (CBG99), or Renilla luciferase (Rluc), and mixed lysates were imaged in varying proportions in a 96-well plate to biochemically validate the methodology. After spectral deconvolution, the predicted, pure luciferase signals could be recovered with maximal cross-talk errors of +/-1.5%. In addition, live cells expressing CBR, CBG99, and Rluc were simultaneously imaged and deconvoluted in 96-well plates to demonstrate the feasibility of applying this methodology to high-throughput applications. Finally, multicolor transcriptional and posttranslational modification reporters were simultaneously imaged and shown to deconvolute normalized IkappaBeta kinase activity in longitudinal assays. Thus, our software provided a rapid, simple, and accurate method for simultaneously measuring multiple bioluminescent reporters in living cells.

Cell-Free System↗

PubMed2005

Measurements of insulin responses as predictive markers of pancreatic beta-cell mass in normal and beta-cell-reduced lean and obese Göttingen minipigs in vivo.

At present, the best available estimators of beta-cell mass in humans are those based on measurement of insulin levels or appearance rates in the circulation. In several animal models, these estimators have been validated against beta-cell mass in lean animals. However, as many diabetic humans are obese, a correlation between in vivo tests and beta-cell mass must be evaluated over a range of body weights to include different levels of insulin sensitivity. For this purpose, obese (n = 10) and lean (n = 25) Göttingen minipigs were studied. Beta-cell mass had been reduced (n = 16 lean, n = 5 obese) with a combination of nicotinamide (67 mg/kg) and streptozotocin (125 mg/kg), acute insulin response (AIR) to intravenous glucose and/or arginine was tested, pulsatile insulin secretion was evaluated by deconvolution (n = 30), and beta-cell mass was determined histologically. AIR to 0.3 (r(2) = 0.4502, P < 0.0001) or 0.6 g/kg glucose (r(2) = 0.6806, P < 0.0001), 67 mg/kg arginine (r(2) = 0.5730, P < 0.001), and maximum insulin concentration (r(2) = 0.7726, P < 0.0001) were all correlated to beta-cell mass when evaluated across study groups, and regression lines were not different between lean and obese groups except for AIR to 0.3 g/kg glucose. Baseline pulse mass was not significantly correlated to beta-cell mass across the study groups (r(2) = 0.1036, NS), whereas entrained pulse mass did show a correlation across groups (r(2) = 0.4049, P < 0.001). This study supports the use of in vivo tests of insulin responses to evaluate beta-cell mass over a range of body weights in the minipig. Extensive stimulation of insulin secretion by a combination of glucose and arginine seems to give the best correlation to beta-cell mass.

Animals↗

PubMed2003

Osmotic stress alters the intracellular distribution of non-erythroidal spectrin (fodrin) in bovine aortic endothelial cells.

Cell swelling is known to result in unfolding of membrane invaginations and restructuring of F-actin. The effect of cell swelling on the intracellular distributions of other cytoskeletal proteins that constitute the submembrane cortical cytoskeleton is virtually unknown. This study focuses on the effects of cell swelling on non-erythroidal spectrin (fodrin, also known as spectrin II), a predominant component of the membrane cytoskeleton. The intracellular distribution of spectrin in vascular endothelial cells was studied by optical sectioning using a 3-D deconvolution microscopy system. Our results show that once bovine aortic endothelial cells (BAECs) reach confluency, the non-erythroidal spectrin is localized in the submembrane regions of the cells. Analysis of the intensity profiles of the non-erythroidal spectrin under isotonic and hypotonic conditions show that: (a) the width of the submembrane spectrin staining increases gradually with time within the first 5 minutes after the osmotic shock; (b) significant recovery is observed after 10 minutes even if the cells are maintained in hypotonic medium, and (c) spectrin distribution is altered by disrupting F-actin with latrunculin A but not by stabilizing F-actin with jasplakinolide. We suggest that cell swelling results in partial translocation of the submembrane spectrin to the cytosol and that it may play a major role in initiation of swelling-induced cellular events.

Adaptation, Physiological↗

PubMed2002

Findings on T cell specificity revealed by synthetic combinatorial libraries.

Combinatorial libraries and in particular positional scanning synthetic combinatorial libraries (PS-SCL) allow the study of T cell specificity. This is a systematic and unbiased approach that does not require any previous knowledge about the clones to be studied, neither their specificity nor they major histocompatibility complex (MHC) restriction. Two different types of T cell clone ligands can be identified: (1) peptides that do not necessarily correspond to proteins described in the databases, and (2) peptides that are fragments of natural proteins. In this paper, relevant examples of the application of PS-SCL and the deconvolution strategies followed to identify T cell epitopes for clones of known and unknown specificity will be reviewed. Also, important issues like the immunogenicity of such T cell ligands will be discussed.

Animals↗

PubMed2006

A natural antibody to oxidized cardiolipin binds to oxidized low-density lipoprotein, apoptotic cells, and atherosclerotic lesions.

OBJECTIVE: Cardiolipin (CL) is found in membranes of bacteria, in the inner membrane of mitochondria and in plasma low-density lipoprotein (LDL). Anticardiolipin antibodies (aCL) are associated with disease states, and we have suggested that many aCL bind to oxidized CL (oxCL) but not native CL. To determine the immunogenicity and origins of oxCL in vivo, we cloned a natural antibody to oxCL. METHODS AND RESULTS: A monoclonal IgM antibody to oxCL (LRO1) was cloned from a nonimmunized LDLR-/- mouse. The V(H) sequence originated from the V(H)Gam3.8 germline with one nucleotide difference, and the Vkappa was 100% identical to Vkappa19-20 germline gene, making LRO1 a natural antibody. LRO1 bound specifically to oxCL and oxidized-LDL, but not to native CL or native LDL. LRO1 epitopes were demonstrated in apoptotic, but not in viable, Jurkat cells by flow cytometry, immunofluorescence and deconvolution microscopy. Human and rabbit atherosclerotic lesions contained LRO1 epitopes. Human LDL (n=113) showed LRO1 immunoreactivity, which correlated with aCL IgG titers (r=0.32, P=0.0004). CONCLUSIONS: These data demonstrate that some aCL antibodies are highly conserved natural antibodies binding to oxCL in oxLDL, apoptotic cells, and atherosclerotic lesions. This suggests that oxCL is one of the pathogen-associated molecular patterns of innate immunity and gives insight into the pathogenic events of diseases with increased titers of aCL antibodies.

Animals↗

PubMed2005

Appreciating the heterogeneity in autoimmune disease: multiparameter assessment of intracellular signaling mechanisms.

Autoimmune disease pathologies are multifactorial with complex interactive networks of cells and chemical messengers that initiate cascades of aberrant cellular activity. Rheumatoid arthritis (RA) is a chronic inflammatory disease that is characterized by systemic inflammation, destruction of the joints, and production of autoantibodies recognizing dozens of putative autoantigens. The presence of autoreactive T cells in individuals leads to pathological autoimmunity by activating additional cellular constituents to mediate inflammation and joint destruction. The etiology of RA is unknown, and knowledge is lacking of the molecular mechanisms underlying the production and subsequent regulation of autoreactive T cells and predicting patient responses to treatments. Biochemical investigations into mechanisms of the disease have relied on animal models that are helpful in dissecting elements of the disease but that are not necessarily reflective of human RA development. The study of multiple activated signaling pathways in complex populations of cells, such as peripheral blood, at the single-cell level has not previously been possible. This article describes how intracellular phosphoepitope staining methodology in conjunction with surface-cell immunophenotyping can be used to deconvolute cellular subsets and allow functional characterization of patient-derived material. Multiparameter flow cytometric analysis allows for small subpopulations-representing different cellular subsets and differentiation or activation states-to be discerned and simultaneously assessed for intracellular biochemical activities. This article also describes how single-cell signal network analysis can be used to stratify patients and may be useful for understanding mechanisms of disease progression, treatment resistance, and development of diagnostic indicators.

Autoimmune Diseases↗

PubMed2006

Hinge peptide combinatorial libraries for inhilbitors of botulinum neurotoxins and saxitoxin: deconvolution strategy.

Abstract Combinatorial library screening offers a rapid process for identifying potential therapies to toxins. Hinge peptide libraries, which rely on conformational diversity rather than traditional molecular diversity, reduce the need for huge numbers of syntheses and screening steps and greatly expedite the discovery process of active molecules. Hinge peptide libraries having the structures: Acetyl-X1-X2-hinge-X3-X4-NH2 (capped) and X1-hinge-X2-X3 (uncapped), where X1 through X4 are near-equimolar mixtures of twelve L-amino acids and hinge = 4-aminobutyric acid, were screened for inhibitory activity in bioassays for botulinum neurotoxins A and B (BoNT/A, BoNT/B) and saxitoxin. The zinc protease activity of the reduced light chains of BoNT/A and /B was assayed by measuring the cleavage of synthetic substrates. Saxitoxin activity was measured by the restoration of the viability of neuroblastoma cells treated with ouabain and veratridine. Deconvolution of libraries was accomplished by fixing one position at a time beginning with the C-terminus. Primary library subsets in which position 4 was fixed showed moderate levels of inhibition for BoNT/A. Secondary library subsets showed stronger inhibition in the bioassays. In each of the bioassays, inhibitory potency was stronger when the second position to be fixed was on the opposite side of the hinge, rather than on the same side with respect to the C-terminus, suggesting that the hinge facilitates the interaction of side chains. Inhibitors for all three of the toxins studied were discovered within library subsets, although not necessarily in primary subsets. These studies demonstrate that (1) the best strategy for deconvoluting hinge peptide libraries is by fixing residues alternately on each side of the hinge moiety, and (2) it is essential to investigate secondary subsets even when primary subsets are inactive. The present findings support the concept that the increased flexibility imposed by the inclusion of a central hinge residue in small peptides increases the opportunity for side chain interactions, providing a distinct advantage for hinge peptide libraries over conventional peptide libraries. Hinge peptide libraries are a rich source of novel ligands for modulation of biomechanisms. The library subsets uncovered in this study may possess peptides that will lead to effective therapies to neurotoxin poisoning.

Biological Assay↗