Cardiac magnetic field map topology quantified by Kullback-Leibler entropy identifies patients with clinically suspected myocarditis.

Background: Myocarditis is a condition that can have severe adverse outcomes and lead to sudden cardiac death if remaining undetected. This study tested the capability of cardiac magnetic field mapping to detect patients with clinically suspected myocarditis. This could open up the way for rapid, non-invasive, and cost-effective screening of suspected cases before a gold standard assessment via endomyocardial biopsy. Methods: Historical cardiac magnetic field maps (n = 97) and data from a state-of-the-art magnetocardiography device (n = 30) were analyzed using the Kullback-Leibler entropy (KLE) for dimensionality reduction and topological quantification. Linear discriminant analysis was used to discern between patients with ongoing myocarditis and healthy controls. Results: The STT segment of a magnetocardiogram, i.e., the section between the end of the S wave and the end of the T wave, was best suited to discern both groups. Using a 250-ms excerpt from the onset of the STT segment gave a reliable classification between the myocarditis and control group for both historic data (sensitivity: 0.83, specificity: 0.85, accuracy: 0.84) and recent data (sensitivity: 0.69, specificity: 0.88, accuracy: 0.80) using the KLE to quantify the topology of the cardiac magnetic field map. Conclusion: The implementation based on KLE can reliably distinguish between clinically suspected myocarditis patients and healthy controls. We implemented an automatized feature selection based on LDA to replace the observer-dependent manual thresholding in previous studies.

Functional regeneration of tendons using scaffolds with physical anisotropy engineered via microarchitectural manipulation.

Structural and hierarchical anisotropy underlies the structure-function relationship of most living tissues. Attempts to exploit the interplay between cells and their immediate environment have rarely featured macroscale, three-dimensional constructs required for clinical applications. Furthermore, compromises to biomechanical robustness during fabrication often limit the scaffold's relevance in translational medicine. We report a polymeric three-dimensional scaffold with tendon-like mechanical properties and controlled anisotropic microstructures. The scaffold was composed of two distinct portions, which enabled high porosity while retaining tendon-like mechanical properties. When tenocytes were cultured in vitro on the scaffold, phenotypic markers of tenogenesis such as type-I collagen, decorin, and tenascin were significantly expressed over nonanisotropic controls. Moreover, highly aligned intracellular cytoskeletal network and high nuclear alignment efficiencies were observed, suggesting that microstructural anisotropy might play the epigenetic role of mechanotransduction. When implanted in an in vivo micropig model, a neotissue that formed over the scaffold resembled native tendon tissue in composition and structure.

Effects of active HCV replication on neurologic status in HIV RNA virally suppressed patients.

BACKGROUND: Hepatitis C virus (HCV) is a frequent copathogen with HIV. Both viruses appear to replicate in the brain and both are implicated in neurocognitive and peripheral neuropathy syndromes. Interaction of the viruses is likely to be complicated and better understanding of the contributions of each virus will be necessary to make evidence-based therapeutic decisions. METHODS: This study was designed to determine if active HCV infection, identified by quantitative HCV RNA determination, is associated with increased neurocognitive deficits or excess development of distal sensory peripheral neuropathy in HIV coinfected patients with stable HIV viral suppression. The AIDS Clinical Trials Group Longitudinal Linked Randomized Trials (ALLRT) study was the source of subjects with known HIV treatment status, neurocognitive and neuropathy evaluations, and HCV status. Subjects were selected based on HCV antibody status (249 positive; 310 negative). RESULTS: HCV RNA viral loads were detectable in 172 participants with controlled HIV infection and available neurologic evaluations in the ALLRT. These participants were compared with 345 participants with undetectable HCV viral load and the same inclusion criteria from the same cohort. Neurocognitive performance measured by Trail-Making A or B and digit symbol testing was not dissimilar between the 2 groups. In addition, there was no significant association between active HCV replication and distal sensory neuropathy. CONCLUSION: Clinically significant neurocognitive dysfunction and peripheral neuropathy were not exacerbated by active hepatitis C virus infection in the setting of optimally treated HIV infection.

Morphology and metabolism of Ba-alginate-encapsulated hepatocytes with galactosylated chitosan and poly(vinyl alcohol) as extracellular matrices.

Lactobionic acid, bearing a beta-galactose group, was coupled with chitosan to provide synthetic extracellular matrices together with poly(vinyl alcohol) (PVA). The hepatocytes encapsulated in Ba-alginate capsules with galactosylated chitosan (GC) and PVA as extracellular matrices showed aggregation morphologies as the incubation time increased. Ba-alginate-encapsulated hepatocytes with GC exhibited a higher metabolic function in albumin secretion compared to those entrapped in Ba-alginate beads and monolayer-cultured on a collagen-immobilized polystyrene dish. The ammonia removal ability of monolayer-cultured hepatocytes decreased with increasing culture time and disappeared completely after three days. In contrast, the ammonia removal ability of encapsulated and entrapped hepatocytes increased with increasing incubation time in the first seven and five days, respectively. Thereafter, the entrapped hepatocytes lost ammonia removal ability quickly while the encapsulated hepatocytes kept a relatively high ammonia removal ability up to 13 days. The trace amount of GC in the core matrices enabled encapsulated cells to enhance their ammonia removal and albumin secretion ability. The results obtained with 3-(3,4-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) also showed that the capsules incorporated with GC can provide a better microenvironment for cell aggregation along with nutrition and metabolite transfer. Due to the nature of the liquid core, the encapsulated hepatocytes showed very good mobility. This facilitated cell-cell interaction and cell-matrix interaction.

Autosomal telomere exchange results in the rapid amplification and dispersion of Csf2ra genes in wild-derived mice.

Common laboratory strains such as C57BL/6J carry a single Csf2ra gene that maps to the distal end of Chromosome (Chr) 19. Here we report that several species of wild mice contain multiple Csf2ra genes. Using interspecific backcross mapping and in situ hybridization, we demonstrate that one of these species, Mus spretus, carries four Csf2ra genes dispersed among the distal tips of Chrs 4, 10, 13, and 19. Our data further suggest that these additional Csf2ra genes are not generated by retrotransposition, but rather by nonhomologous subtelomeric exchanges that could be mediated in part by ribosomal genes located at the subtelomeric regions of Chrs 4, 13, and 19. Although we do not know whether these additional Csf2ra genes are functionally active, our studies suggest that subtelomeric exchange provides a potent means for rapid gene amplification in the mouse.

Comparative cellular catabolism and retention of astatine-, bismuth-, and lead-radiolabeled internalizing monoclonal antibody.

UNLABELLED: Monoclonal antibodies (mAbs) labeled with alpha-emitting radionuclides such as (211)At, (212)Bi, (213)Bi, and (212)Pb (which decays by beta-emission to its alpha-emitting daughter, (212)Bi) are being evaluated for their potential applications for cancer therapy. The fate of these radionuclides after cells are targeted with mAbs is important in terms of dosimetry and tumor detection. METHODS: In this study, we attached various radionuclides that result in alpha-emissions to T101, a rapidly internalizing anti-CD5 mAb. We then evaluated the catabolism and cellular retention and compared them with those of (125)I- and (111)In-labeled T101. T101 was labeled with (211)At, (125)I, (205,6)Bi, (111)In, and (203)Pb. CD5 antigen-positive cells, peripheral blood mononuclear cells (PBMNC), and MOLT-4 leukemia cells were used. The labeled T101 was incubated with the cells for 1 h at 4 degrees C for surface labeling. Unbound activity was removed and 1 mL medium added. The cells were then incubated at 37 degrees C for 0, 1, 2, 4, 8, and 24 h. The activity on the cell surface that internalized and the activity on the cell surface remaining in the supernatant were determined. The protein in the supernatant was further precipitated by methanol for determining protein-bound and non-protein-bound radioactivity. Sites of internal cellular localization of radioactivity were determined by Percoll gradient centrifugation. RESULTS: All radiolabeled antibodies bound to the cells were internalized rapidly. After internalization, (205,6)Bi, (203)Pb, and (111)In radiolabels were retained in the cell, with little decrease of cell-associated radioactivity. However, (211)At and (125)I were released from cells rapidly ((211)At < (125)I) and most of the radioactivity in the supernatant was in a non-protein-bound form. Intracellular distribution of radioactivity revealed a transit of the radiolabel from the cell surface to the lysosome. The catabolism patterns of MOLT-4 cells and PBMNC were similar. CONCLUSION: (211)At catabolism and release from cells were somewhat similar to that of (125)I, whereas (205,6)Bi and (203)Pb showed prolonged cell retention similar to that of (111)In. These catabolism differences may be important in the selection of alpha-radionuclides for radioimmunotherapy.

Improvement of water treatment performance by using polyamine flocculants.

In this study, polyamine flocculants were synthesized and applied to Nak-dong river raw water in Korea to examine their efficiency in reducing turbidity, total organic carbon (TOC) and UV254. Synthesized polyamines were effective as flocculants for water treatment and the addition of organic polymer caused a reduction of 50-80% of the consumption of polyaluminium chloride (PAC). The effects of polyamine on the removal of turbidity, TOC and UV254 were investigated via both jar and pilot tests. The adsorption and separation mechanisms for the removal of turbidity and TOC by using the polymer flocculants were also observed.

Cloning of the murine thymic stromal lymphopoietin (TSLP) receptor: Formation of a functional heteromeric complex requires interleukin 7 receptor.

The cellular receptor for murine thymic stromal lymphopoietin (TSLP) was detected in a variety of murine, but not human myelomonocytic cell lines by radioligand binding. cDNA clones encoding the receptor were isolated from a murine T helper cell cDNA library. TSLP receptor (TSLPR) is a member of the hematopoietin receptor family. Transfection of TSLPR cDNA resulted in only low affinity binding. Cotransfection of the interleukin 7 (IL-7)Ralpha chain cDNA resulted in conversion to high affinity binding. TSLP did not activate cells from IL-7Ralpha(-/)- mice, but did activate cells from gammac(-/)- mice. Thus, the functional TSLPR requires the IL-7Ralpha chain, but not the gammac chain for signaling.

Impact of recent welfare and immigration reforms on use of Medicaid for prenatal care by immigrants in California.

This study investigates the impact of the recent welfare and immigration changes on the use of Medicaid by low-income pregnant immigrant women in California. The study presents findings from interviews with government officials, safety-net prenatal care providers, and immigrant advocates who serve low-income pregnant Asian and Latina immigrants at the national, state, or local levels. These informants spoke of policy actions that affect immigrants' abilities to use Medicaid for coverage of prenatal care. These actions include (1) the sharing of information between the California Department of Health Services and the federal Immigration and Naturalization Service, (2) the slow and confusing implementation of the reforms, and (3) the intimidating Medicaid eligibility process. The findings demonstrate how the policies changed the immigrant women's relationship with safety-net prenatal care providers, and sparked intense actions on the part of their advocates to sustain the women's access to perinatal care.

Soluble cellular adhesion molecules in proliferative vitreoretinopathy and proliferative diabetic retinopathy.

PURPOSE: To measure vitreous levels of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cellular adhesion molecule-1 (sVCAM-1) in the eyes of patients with retinal detachment (RD) due to proliferative diabetic retinopathy (PDR) or proliferative vitreoretinopathy (PVR) and to determine whether the levels of these mediators correlated with clinical parameters of disease. METHODS: Undiluted vitreous specimens were collected from 50 eyes of 48 patients undergoing vitrectomy for traction RD due to PDR (21 specimens) and recurrent RD due to PVR (19 specimens). Control vitreous specimens were obtained from patients undergoing macular hole repair (10 specimens). The levels of sICAM-1 and sVCAM-1 were measured in each sample by specific enzyme-linked immunoadsorbent assays. RESULTS: Vitreous levels of sICAM-1 were significantly increased in vitreous specimens from both PVR (median +/- SD; 12.0 +/- 76.3 ng/ml; P < 0.01) and PDR (8.4 +/- 24.0 ng/ml; P < 0.01) when compared to vitreous from eyes with macular holes (0. 3 +/- 4.2 ng/ml). Vitreous levels of sVCAM-1 were significantly increased in both PVR (36.5 +/- 255.2 ng/ml; P < 0.001) and PDR (26. 2 +/- 93.5 ng/ml; P < 0.01) when compared to control vitreous (17.7 +/- 7.8 ng/ml). The vitreous levels of sICAM-1 were higher in cases of PDR which developed recurrent proliferative disease (P < 0.01) and recurrent RD (P = 0.01), whereas the levels of sICAM-1 in PVR and sVCAM-1 in PDR and PVR did not significantly correlate with these clinical parameters. CONCLUSIONS: Soluble forms of ICAM-1 and VCAM-1 are increased in the vitreous cavity of patients with RD due to PDR or PVR, reflecting the inflammatory nature of these conditions and suggesting a possible role for these mediators in the pathogenesis of proliferative retinal disease. The vitreous levels of these sCAMs at the time of surgery may serve as a marker of inflammation, but their specific levels do not predict the likelihood of recurrent proliferation or surgical anatomic success in most cases of PVR and PDR.

Molecular characterization of the human interleukin (IL)-17 receptor.

Human interleukin 17 (hIL-17) is a T-cell derived cytokine that exhibits 63% amino acid sequence identity to mouse IL-17 (mIL-17) and 57% identity to a viral protein encoded by the herpesvirus saimiri (HSV) gene 13 (HVS13). The IL-17 family of proteins binds to a unique mouse receptor (mIL-17R). Using nucleic acid hybridization techniques, a cDNA encoding a human homologue of the mIL-17R (hIL-17R) was isolated from a human T cell library. The predicted amino acid sequence of the hIL-17R is 69% identical to the mIL-17R, shares no homology with previously identified cytokine receptor families, and exhibits a broad tissue distribution. The hIL-17R gene was localized to chromosome 22. Monoclonal antibodies (mAbs) generated against the hIL-17R were able to block the IL-17-induced production of cytokine from human foreskin fibroblast (HFF) cells. Binding studies suggest that recombinant hIL-17 binds to the hIL-17R with low affinity.

Dual oncostatin M (OSM) receptors. Cloning and characterization of an alternative signaling subunit conferring OSM-specific receptor activation.

Oncostatin M (OSM) is a cytokine whose structural and functional features are similar to other members of the interleukin (IL)-6 family of cytokines (IL-6, IL-11, leukemia inhibitory factor (LIF), granulocyte colonystimulating factor, ciliary neurotrophic factor, and cardiotrophin-1), many of which utilize gp130 as a common receptor subunit. A biologically active OSM receptor has been previously described that consists of a heterodimer of leukemia inhibitory factor receptor (LIFR) and gp130. This LIFR.gp130 complex is also a functional receptor for LIF. We have cloned and characterized an alternative subunit (OSMRbeta) for an OSM receptor complex (a heterodimer of gp130 and OSMRbeta) that is activated by OSM but not by LIF. The signaling capability of specific receptor subunit combinations was analyzed by independent assays measuring cell proliferation or induction of acute phase protein synthesis. Our results demonstrate that both LIF and OSM cause tyrosine phosphorylation and activation of the gp130.LIFR combination, but the gp130.OSMRbeta complex is activated by OSM only. OSM-induced cellular responses, initiated through low affinity binding to gp130, are mediated by two heterodimeric receptor complexes that utilize alternative signal transducing subunits that confer different cytokine specificities to the receptor complex.

Cell-cell adhesion mediated by binding of membrane-anchored ligand LERK-2 to the EPH-related receptor human embryonal kinase 2 promotes tyrosine kinase activity.

Human embryonal kinase 2 (HEK2) is a protein-tyrosine kinase that is a member of the EPH family of receptors. Transcripts for HEK2 have a wide tissue distribution. Recently, a still growing family of ligands, which we have named LERKs, for ligands of the eph-related kinases, has been isolated. In order to analyze functional effects between the LERKs and the HEK2 receptor, we expressed HEK2 cDNA in an interleukin-3-dependent progenitor cell line 32D that grows as single cells in culture. Within the group of LERKs, LERK-2 and -5 were shown to bind to HEK2. Membrane-bound and soluble forms of LERK-2 were demonstrated to signal through HEK2 as judged by receptor phosphorylation. Coincubation of HEK2 and LERK-2 expressing cells induced cell-cell adhesion and formation of cell aggregates. This interaction could be inhibited by preincubation of HEK2 expressing cells with soluble LERK-2. Coexpression of HEK2 and LERK-2 in 32D cells showed reduced kinase activity and autophosphorylation of HEK2 compared with the juxtacrine stimulation, which seems to be due to a reduced sensitivity of the receptor.

Characterization of interleukin-15 (IL-15) and the IL-15 receptor complex.

IL-15 interacts with a heterotrimeric receptor that consists of the beta and gamma subunits of the IL-2 receptor (IL-2R) as well as a specific, high-affinity IL-15-binding subunit, which is designated IL-15R alpha. Since both the beta and the gamma subunits of the IL-2R are required for signaling by either IL-2 or IL-15, it is not surprising that these cytokines share many activities in vitro. However, the differential expression of these cytokines and the alpha chains of their receptors within various tissues and cell types suggests that IL-2 and IL-15 may perform at least partially distinct physiological functions. The production of IL-15 by macrophages, and possibly other cell types, in response to environmental stimuli and infectious agents suggests that IL-15 may play a role in protective immune responses, allograft rejection, and the pathogenesis of autoimmune diseases.

Isolation of LERK-5: a ligand of the eph-related receptor tyrosine kinases.

Hek and elk are members of the eph-related family of receptor tyrosine kinases. Recently we isolated four cDNAs encoding membrane-bound ligands to hek and elk [Beckman et al. (1994) EMBO J. 13, 3757-3762; Kozlosky et al. (1995) Oncogene 10, 299-306]. Because of the promiscuous nature of their binding, we have termed these proteins ligands of the eph-related kinases or LERKs. A search of GenBank revealed an expressed sequence tag (EST) with homology to the LERKs. Using this EST as a probe, we have isolated human and murine cDNAs that encode a protein which we call LERK-5. The human and murine cDNAs encode proteins of 333 and 336 amino acids, respectively, with a 97% amino acid identity; LERK-5 has an amino acid identity of 27-59% with the other reported LERKs. LERK-5 is a ligand for both elk and hek and induces receptor phosphorylation. It is expressed in adult lung and kidney and the fetal tissues heart, lung, kidney, and brain. In addition, Southern blot analysis of DNA from interspecific backcross mice indicated that LERK-5 (Eplg5) maps to the proximal region of mouse chromosome 8.

Identification and cloning of a novel IL-15 binding protein that is structurally related to the alpha chain of the IL-2 receptor.

Interleukin-15 (IL-15) is a novel cytokine of the four-helix bundle family which shares many biological activities with IL-2, probably due to its interaction with the IL-2 receptor beta and gamma (IL-2R beta and gamma c) chains. We report here the characterization and molecular cloning of a distinct murine IL-15R alpha chain. IL-15R alpha alone displays an affinity of binding for IL-15 equivalent to that of the heterotrimeric IL-2R for IL-2. A biologically functional heteromeric IL-15 receptor complex capable of mediating IL-15 responses was generated through reconstruction experiments in a murine myeloid cell line. IL-15R alpha is structurally similar to IL-2R alpha; together they define a new cytokine receptor family. The distribution of IL-15 and IL-15R alpha mRNA suggests that IL-15 may have biological activities distinct from IL-2.

IL-15, a novel T cell growth factor that shares activities and receptor components with IL-2.

Interleukin 15 is a newly discovered cytokine that shares biological activities with IL-2 and, like IL-2, is a member of the four-helix bundle cytokine family. We have shown that IL-15 shares components of the receptor for IL-2: the alpha chain of the IL-2R is not required, but both the beta and gamma chains are needed for IL-15 mediated bioactivities. A defect in IL-15 signaling may therefore contribute to the phenotype of X-linked severe combined immunodeficiency in humans, resulting from mutations in the common gamma chain. Differential ability of cells to bind and respond to IL-2 and IL-15 suggested the existence of an additional IL-15 specific receptor component. We identified an IL-15 specific binding protein (IL-15R alpha) on a murine T cell and isolated the corresponding cDNA. The IL-15R alpha is not a member of the hematopoietin receptor superfamily, but is structurally related to the alpha chain of the IL-2R. Differences in the expression pattern of IL-15 and its receptor compared to the IL-2 system suggest unique in vivo roles for IL-15.

Conserved amino acids in the human granulocyte-macrophage colony-stimulating factor receptor-binding subunit essential for tyrosine phosphorylation and proliferation.

A superfamily of growth factor and cytokine receptors has recently been identified, which is characterized by four spatially conserved cysteine residues and a tryptophan-serine motif (WSXWS) in the extracellular domain and proline-rich cytoplasmic domain. The high-affinity human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, hGM-CSFR, consists of two subunits, alpha (hGM-CSFR alpha), which is required for ligand binding, and beta (hGM-CSFR beta), which is required for signal transduction. Both the alpha and beta subunits are members of the cytokine receptor superfamily. In this study, we analyzed mutations in the conserved amino acids of the alpha subunit to determine their function in signal transduction, as assayed by tyrosine phosphorylation and proliferation. Disruption of either of the conserved disulfide bonds in the extracellular domain abolishes low-affinity binding but not binding to a preformed heterodimeric complex with the beta-chain. Cells expressing receptors with mutations in cysteines 2 or 3 grew as well as cells expressing wild-type receptors in human GM-CSF (hGM-CSF) and phosphorylated the same proteins on tyrosine residues, although the level of phosphorylation may be attenuated; cysteine 3 appears to be required for generation of the true high-affinity binding site. The WSXWS motif and the cytoplasmic domain are required for function of the human GM-CSF receptor, as stable cell lines expressing receptors with these mutations were unable to proliferate continuously in hGM-CSF. Surprisingly, no function for the conserved proline-rich region of the cytoplasmic domain could be ascertained from these studies; cells expressing these receptors were indistinguishable from wild-type in both binding and functional assays.

Interleukin 15 and its receptor.

Interleukin 15 (IL-15) is a member of the four-helix bundle cytokine family that shares many in vitro biological activities with IL-2. Previous work demonstrated that IL-15 utilizes the beta and gamma chains of the IL-2 receptor (IL-2R), and that these are essential for IL-15-mediated signal transduction. However, several lines of evidence indicated the existence of an additional, IL-15-specific receptor component. An IL-15 binding chain was identified on a murine T cell clone, and direct expression cloning was used to isolate the corresponding cDNA. The predicted structure of this protein shows sequence similarity to the IL-2R alpha chain. Transfection of this cDNA into a murine, IL-3-dependent myeloid cell line, 32D-01, conferred IL-15 binding and, together with transfection of the IL-2R beta chain, rendered the cells responsive to IL-15 stimulation. This experiment confirmed that the IL-15 binding chain is part of the IL-15 receptor, and it is designated as the IL-15R alpha subunit. The expression pattern of the IL-15R alpha mRNA is distinct from that of IL-2R alpha mRNA. Recombinant expression of a soluble form of IL-15R alpha demonstrated that it is a potent inhibitor of IL-15 biological activity.

Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice.

Interleukin 7 (IL-7) stimulates the proliferation of B cell progenitors, thymocytes, and mature T cells through an interaction with a high affinity receptor (IL-7R) belonging to the hematopoietin receptor superfamily. We have further addressed the role of IL-7 and its receptor during B and T cell development by generating mice genetically deficient in IL-7R. Mutant mice display a profound reduction in thymic and peripheral lymphoid cellularity. Analyses of lymphoid progenitor populations in IL-7R-deficient mice define precisely those developmental stages affected by the mutation and reveal a critical role for IL-7R during early lymphoid development. Significantly, these studies indicate that the phase of thymocyte expansion occurring before the onset of T cell receptor gene rearrangement is critically dependent upon, and mediated by the high affinity receptor for IL-7.