Healthcare for everyone, but how? Two policy experts debate problems and solutions. Interview by Glen C. Griffin.
Explore the source record for details and available documents.
Health Care Reform↗
Biomedical subjects
P Ferrara
Publications and source records attributed to P Ferrara.
Interleukin 13: novel role in direct regulation of proliferation and differentiation of primitive hematopoietic progenitor cells.
The recently cloned interleukin 13 (IL-13) shares most investigated biological activities on B lymphocytes and monocytes with IL-4. In this study we investigated for the first time the potential role of IL-13 in the regulation of the growth of hematopoietic progenitor cells. IL-13 enhanced stem cell factor (SCF)-induced proliferation of Lin-Sca-1+ bone marrow progenitor cells more potently than IL-4. The effect of IL-13 was purely synergistic, since IL-13 alone stimulated no colony formation. Single cell experiments suggested that the synergistic effect of IL-13 on Lin-Sca-1+ progenitors was directly mediated. In contrast, IL-13 had no synergistic activity on SCF-induced proliferation of the more mature Lin-Sca-1- progenitor cells. Thus, the cloning frequency in response to SCF + IL-13 was at least 20-fold higher in the Lin-Sca-1+ than the Lin-Sca-1- progenitor cell population. Furthermore, IL-13 but not IL-4 synergistically enhanced colony formation of Lin-Sca-1+ progenitors in response to granulocyte/macrophage colony-stimulating factor (GM-CSF) (threefold), whereas both IL-4 and IL-13 enhanced G-CSF-induced colony formation (threefold), and neither of the two significantly affected CSF-1 and IL-3-induced proliferation. Finally, whereas stimulation of Lin-Sca-1+ progenitors by SCF + G-CSF resulted in the formation of 90% granulocytes, the addition of IL-13 resulted in the production of macrophages exclusively. This novel effect on differentiation was directly mediated, shared with IL-4, and could not be observed on Lin-Sca-1- progenitor cells. Collectively, these findings indicate a novel role of IL-13 in early myelopoiesis, partially overlapping but also different from that of IL-4.
Animals↗
Rat platelets contain glycosylated and non-glycosylated forms of platelet factor 4. Identification and characterization by mass spectrometry.
Platelet factor 4 is a heparin-binding protein released from the alpha granules of activated platelets. This study describes the purification and identification of two forms of rat platelet factor 4, the previously characterized non-glycosylated form of 7 kDa and an additional glycosylated form of molecular mass 9 kDa. The two proteins both neutralized the antithrombin-III-dependent inhibitory activity of heparin. Although their amino acid composition was found to be the same, in the N-terminal sequence of the 9-kDa protein, the second threonine residue could not be detected and a difference of 976Da was determined by mass spectrometry. After digestion with O-glycanase and sialidase, the two proteins showed the same molecular mass. Overall consideration of these data led to identification of the higher-molecular-mass protein as a glycosylated form of rat platelet factor 4 with O-glycosylation at the second N-terminal amino acid, while the structure of the oligosaccharide core was established by mass spectrometry and sugar differentiation with lectins. The two forms of platelet factor 4 are both present in platelets and secreted after platelet activation.
Amino Acid Sequence↗
Human interleukin-13 activates the interleukin-4-dependent transcription factor NF-IL4 sharing a DNA binding motif with an interferon-gamma-induced nuclear binding factor.
The effects of interleukin-13 (IL-13) and interleukin-4 (IL-4) on cellular functions were shown to be quite similar. We provide evidence that in monocytes as well as in T lymphocytes both IL-4 and IL-13 activate the same recently identified transcription factor NF-IL4 which binds to the specific responsive element IL-4RE. In addition, we show that a nuclear factor activated by interferon-gamma also interacts with the IL-4RE. It differs from NF-IL4 in the electrophoretic mobility of the complex with DNA, in its DNA-binding specificity and in the proteins interacting with the DNA sequence. Sensitivity against various enzyme inhibitors suggests that components of the signal transduction pathway are shared by all three cytokines.
Animals↗
Interleukin-13 induces expression and release of interleukin-1 decoy receptor in human polymorphonuclear cells.
The aim of this study was to examine whether interleukin-13 (IL-13), a cytokine with anti-inflammatory activities, affected expression of interleukin-1 (IL-1) receptors (R) in human polymorphonuclear cells (PMN). Treatment with IL-13 augmented both type I and type II (decoy) R transcripts, with the latter being by far the most represented. The transcriptional inhibitor actinomycin D blocked the induction of IL-1 R mRNAs by IL-13. Nuclear run-off experiments demonstrated an augmented transcriptional rate of IL-1 decoy R in IL-13-treated B lymphoblastoid cells. The protein synthesis inhibitor cycloheximide blocked type I R expression but superinduced decoy R expression. IL-13 augmented the binding of radiolabeled IL-1 beta on the PMN surface with an increased number of IL-1 receptors and no change in Kd values. IL-13 induced the surface expression of IL-1 decoy R and the release by PMN of an IL-1-binding protein identified as a soluble version of the IL-1 decoy R. These results show that PMN is an important target for IL-13 and that induction of expression and release of the IL-1 decoy R, in concert with inhibition of cytokine synthesis, may represent an important mechanism by which IL-13 blocks IL-1, a central mediator of inflammatory reactions.
Blotting, Northern↗
Interleukin-13 stimulates interleukin-6 production by human keratinocytes. Similarity with interleukin-4.
Interleukin-13 (IL-13) is a recently described human lymphokine which is produced by activated T-cells. Its effect on the production of IL-6 by normal keratinocytes and keratinocyte cell lines of human origin was studied and compared to that of IL-4. IL-13, similarly to IL-4, stimulated IL-6 expression by these cells in a dose- and time-dependent manner. Contamination with endotoxin was excluded by the use of polymyxin B and heat-inactivated cytokines. Further, we showed that IL-13, like IL-4, not only stimulated IL-6 production but also was able to induce overexpression of this cytokine in response to an inflammatory signal such as lipopolysaccharide (LPS). In a previous study, we demonstrated that IL-13, by inhibiting IL-6 and other cytokines produced by monocytes, exhibited an 'anti-inflammatory profile' comparable to that displayed by IL-4. In contrast, we show here that IL-13, by stimulating IL-6 production by keratinocytes, may favour the installation of an inflammatory process at a local level and, here again, it acted like IL-4. Therefore, according to the type of target cell these two 'TH2 type' cytokines induce similar opposing effects on IL-6 production and are likely to be important cytokines in the regulation of inflammation at both systemic and local levels.
Cell Line↗
Interleukin-13 induces the production of interleukin-1 receptor antagonist (IL-1ra) and the expression of the mRNA for the intracellular (keratinocyte) form of IL-1ra in human myelomonocytic cells.
The aim of this study was to examine the expression of interleukin-1 receptor antagonist (IL-1ra) in human myelomonocytic cells treated with IL-13. IL-13 induced IL-1ra transcripts in human circulating monocytes and polymorphonuclear cells (PMN). Induction by IL-13 was not blocked, but rather superinduced, in the presence of the protein synthesis inhibitor cycloheximide. Actinomycin D blocked induction, suggesting involvement of gene transcription. The half-life of IL-1ra transcripts was prolonged by IL-13 from 1.3 hours to 4.5 hours in monocytes and to 12 hours in PMN. By reverse transcriptase-polymerase chain reaction, IL-13 was found to augment the transcripts coding for the soluble form of IL-1ra, but also to induce the expression of the intracellular (keratinocyte) form of IL-1ra, the latter being extremely low or undetectable in myelomonocytic cells. IL-13 induced production of IL-1ra in myelomonocytic cells, augmenting both cell-associated and released protein. Induction of IL-1ra by IL-13 may represent a further mechanism by which this molecule can counteract the potent proinflammatory properties of IL-1.
Cells, Cultured↗
Monocyte chemotactic protein 3 is a most effective basophil- and eosinophil-activating chemokine.
CC chemokines constitute a novel class of cytokines that attract and activate monocytes and lymphocytes, as well as basophil and eosinophil leukocytes, with distinct target cell profiles, and are believed to be involved in the regulation of different types of inflammation. The action of the recently identified monocyte chemotactic protein 3 (MCP-3) on human basophil and eosinophil function was studied and compared with that of other CC chemokines. In basophils, MCP-3, MCP-1, RANTES, and macrophage inflammatory protein (MIP)-1 alpha all induced cytosolic-free calcium concentration ([Ca2+]i) changes and, with different efficacies, chemotaxis (RANTES = MCP-3 >> MCP-1 > MIP-1 alpha), histamine release (MCP-1 = MCP-3 >> RANTES > MIP-1 alpha), and leukotriene C4 formation, after IL-3 pretreatment (MCP-1 = MCP-3 >> RANTES > MIP-1 alpha). Thus, MCP-3 was as effective as MCP-1 as an inducer of mediator release, and as effective as RANTES as a stimulus of basophil migration. In contrast to MCP-1, MCP-3 was also a stimulus for eosinophils, and induced [Ca2+]i changes and chemotaxis as effectively as RANTES, which is the most potent chemotactic cytokine for these cells. Desensitization of the transient changes in [Ca2+]i was used to assess receptor usage. In basophils, stimulation with MCP-3 prevented responsiveness to MCP-1 and RANTES, but not to MIP-1 alpha. No single CC chemokine (except for MCP-3 itself) affected the response to MCP-3, however, which was prevented only when the cells were prestimulated with both MCP-1 and RANTES. In eosinophils, by contrast, cross-desensitization between RANTES and MCP-3 was obtained. RANTES and to a lesser extent MCP-3 also desensitized eosinophils toward MIP-1 alpha. The desensitization data suggest the existence of three chemokine receptors: (a) a MCP-1 receptor expressed on basophils but not eosinophils that is activated by MCP-1 and MCP-3; (b) a RANTES receptor in basophils and eosinophils that is activated by RANTES and MCP-3; and (c) a MIP-1 alpha receptor that is activated by MIP-1 alpha, RANTES and, more weakly, by MCP-3. This study shows that MCP-3 combines the properties of RANTES, a powerful chemoattractant, and MCP-1, a highly effective stimulus of mediator release, and thus has a particularly broad range of activities toward both human basophil and eosinophil leukocytes.
Basophils↗
Interleukin 13 is a B cell stimulating factor.
The recently cloned human interleukin 13 (IL-13) is a novel cytokine expressed in activated T cells that has been shown to inhibit inflammatory cytokine production by lipopolysaccharide-activated monocytes. The protein encoded by the IL-13 cDNA is the human homologue of a mouse Th2-product called P600. Here, we show that IL-13 acts at different stages of the B cell maturation pathway: (a) it enhances the expression of CD23/Fc epsilon RII and class II MHC antigens on resting B cells; (b) it stimulates B cell proliferation in combination with anti-Ig and anti-CD40 antibodies; and (c) it induces IgE synthesis. Thus, the spectrum of the biological activities of IL-13 on B cells largely overlaps that previously ascribed to IL-4. The present observations suggest that IL-13 may be an important factor, in addition to IL-4, in the development of allergic diseases.
B-Lymphocytes↗
Interleukin-13 alters the activation state of murine macrophages in vitro: comparison with interleukin-4 and interferon-gamma.
Interleukin (IL)-13 is a newly described cytokine expressed by activated lymphocytes. We examined the effects of the murine recombinant cytokine on the phenotype and activation status of elicited peritoneal macrophages (M phi), concentrating on activities which are known to be modulated by interferon-gamma and IL-4. IL-13 markedly suppressed nitric oxide release and to a lesser extent secretion of the pro-inflammatory cytokine tumor necrosis factor-alpha. However, antimicrobial capacity was not completely jeopardized as the respiratory burst was unaffected, and indeed the enhanced expression of M phi mannose receptor and major histocompatibility class II, and regulation of sialoadhesin, the M phi sialic acid-specific receptor involved in hemopoietic and lymphoid interactions, suggest that these cells are not simply deactivated, but primed for an active role in immune and inflammatory responses. These activities closely mimic those of IL-4, but mediation of the effects by IL-4 was discounted by the use of a neutralizing monoclonal antibody. Thus, IL-13, like IL-4, is a cytokine which has complex effects on M phi behavior, inducing activities characteristic of both activation and deactivation.
Animals↗
The two soluble forms of the lipopolysaccharide receptor, CD14: characterization and release by normal human monocytes.
CD14, a glycolipid-anchored membrane glycoprotein, acts as a high affinity lipopolysaccharide receptor on leukocytes. We previously reported that the Mono-Mac-6 cell line releases two different soluble forms of CD14 (sCD14) (Labeta et al., Eur. J. Immunol. 1993. 23: 2144). Here we show that the two sCD14, which we now refer to as sCD14 alpha (low M(r)) and sCD14 beta (high M(r)), are also synthesized and released by normal human monocytes and present in normal plasma. Their mechanism of release was examined by using the Mono-Mac-6 cell line, chinese hamster ovary cell (CHO)/CD14+ transfectants and plasma from paroxysmal nocturnal hemoglobinuria (PNH) patients. It was found that: (1) sCD14 beta is released faster than sCD14 alpha and that the release of the latter is a lengthy process. (2) Monensin blocked the biosynthesis of membrane-bound CD14 (mCD14) and sCD14, additionally, a 50-kDa CD14 polypeptide accumulated in the cell lysate, suggesting that the different forms of CD14 may have a common precursor. (3) Monensin also blocked the release of sCD14 alpha from surface-labeled cells, suggesting that conversion of mCD14 to sCD14 alpha involves a mechanism of endocytosis followed by exocytosis. Interestingly, (4) sCD14 alpha and sCD14 beta were detected in PNH plasma, indicating that sCD14 alpha may also derive from an endogenous pathway. (5) Phospholipase C-released CD14 was identical in size to mCD14, thus differed from sCD14 beta by approximately 2000, indicating that release of sCD14 beta involves further processing. (6) CHO cells transfected with a CD14 cDNA coding for an eight C-terminal amino acids shorter product released an sCD14 beta-like form; thus absence of the eight C-terminal amino acids prevented mCD14 expression but not the secretion of sCD14 beta. The characterization of sCD14 alpha and sCD14 beta reported here may be useful for better understanding of variations in sCD14 levels in pathological conditions and the contribution of each sCD14 in sepsis and other, as yet unknown functions.
Animals↗
IL-4 and IL-13 have overlapping but distinct effects on HIV production in monocytes.
In HIV-1-infected monocytes and monocytoid cell lines, viral expression can be observed as high-level production, restricted (chronic low-level) expression, and latency (no viral expression). Interleukin-13 (IL-13) and IL-4, which have remarkedly similar deactivating effects on inflammatory monocyte functions, were studied for their regulation of HIV expression in monocytes. Pretreatment of peripheral monocytes for 48-72 h with IL-13 markedly decreased acute HIV infection, whereas IL-4 increased it. Similar effects were seen when the U1 and R-THP-1 monocytoid cell lines with restricted HIV expression were treated with these cytokines. However, when these continuously producing cell lines were chronically treated with cytokines, IL-13 increased HIV production. Neither IL-4 nor IL-13 stimulated HIV expression in latently infected cells. In chronically infected cells, several cytokines reduced viral mRNA. Both IL-4 and IL-13 increased monocyte aggregate formation, but only IL-4 ultimately stimulated cytolysis of HIV-infected monocytes as well as increased apoptosis of U1. In the presence of tumor necrosis factor alpha or IL-6, which upregulate HIV expression, IL-13 could no longer suppress HIV expression. These results indicate that IL-4 and IL-13, although closely related in modulating monocyte function, can have divergent effects on HIV expression in monocytes. Collectively, these data suggest that there exists a complex cytokine tissue environment with positive regulators of HIV expression able to override negative regulators.
Cells, Cultured↗
Self-organized neural maps of human protein sequences.
We have recently described a method based on artificial neural networks to cluster protein sequences into families. The network was trained with Kohonen's unsupervised learning algorithm using, as inputs, the matrix patterns derived from the dipeptide composition of the proteins. We present here a large-scale application of that method to classify the 1,758 human protein sequences stored in the SwissProt database (release 19.0), whose lengths are greater than 50 amino acids. In the final 2-dimensional topologically ordered map of 15 x 15 neurons, proteins belonging to known families were associated with the same neuron or with neighboring ones. Also, as an attempt to reduce the time-consuming learning procedure, we compared 2 learning protocols: one of 500 epochs (100 SUN CPU-hours [CPU-h]), and another one of 30 epochs (6.7 CPU-h). A further reduction of learning-computing time, by a factor of about 3.3, with similar protein clustering results, was achieved using a matrix of 11 x 11 components to represent the sequences. Although network training is time consuming, the classification of a new protein in the final ordered map is very fast (14.6 CPU-seconds). We also show a comparison between the artificial neural network approach and conventional methods of biosequence analysis.
Algorithms↗
Chromosomal localization of mouse and human neurotensin receptor genes.
Neurotensin is a tridecapeptide that plays several neurotransmitter or neuromodulatory roles both in the central nervous system and in the periphery. These actions are mediated by a high-affinity receptor (Ntsr). Both rat and human cDNAs encoding high-affinity receptors have been recently cloned. The availability of Ntsr probes allowed us to localize the corresponding genes on the mouse and human chromosomes. The present data demonstrate that the Ntsr gene is assigned to the H region of the mouse Chromosome (Chr) 2 and to the long arm of the human Chr 20.
Animals↗
Quantitative evaluations of gap junctions in old rat brown adipose tissue after cold acclimation: a freeze-fracture and ultra-structural study.
The morphological and functional modifications of brown adipose tissue (BAT), the tissue responsible for non-shivering thermogenesis, are well established during the phases of active stimulation (i.e. neonatal period and cold acclimation) in young animals. The 'active' brown adipocytes are filled with numerous small lipid vacuoles and large mitochondria packed with cristae rich in the protonophore uncoupling protein (UCP), whereas the 'quiescent' cell shows larger, confluent vacuoles and smaller mitochondria with rarefied cristae poor of the uncoupling protein. It is well known from literature that also gap junctions (gjs), responsible for the electrical coupling among adjacent adipocytes, modify their size following the physiological stimulus in young animals. This is in agreement with the morphology of the functionally active brown adipocyte, i.e. the multilocular, UCP-positive cell. Although the presence of the BAT in old animals is well documented, less is known about its reactivity to physiological stimuli. The present work demonstrates that after cold acclimation brown adipocytes of old rats (2 years) change their ultrastructure in a similar way as in young rats. A quantitative analysis of gap junction areas on replicas obtained by the freeze fracture technique, showed that gj increase in size (mean area 53.2 vs 110.4 x 10(-3) microns2, p = 0.003). All these morphological modifications are quite similar to those observed in BAT of young and young adult rats, supporting the hypothesis of a physiological role of brown adipose tissue at every age.
Acclimatization↗
Topography of the Leydig cell mitochondrial peripheral-type benzodiazepine receptor.
Native MA-10 mouse Leydig tumor cell mitochondrial preparations were examined by transmission electron (TEM) and atomic force (AFM) microscopic procedures in order to investigate the topography and organization of the peripheral-type benzodiazepine receptor (PBR). Mitochondria were immunolabeled with an anti-PBR antiserum coupled to gold-labeled secondary antibodies. Results obtained indicate that the 18,000 MW PBR protein is organized in clusters of 4-6 molecules. Moreover, on many occasions, the interrelationship among the PBR molecules was found to favor the formation of a single pore. Taking into account recent observations that the 18,000 MW PBR protein is functionally associated with the pore forming 34,000 MW voltage-dependent anion channel (VDAC) these results suggest that (i) the mitochondrial PBR complex could function as a pore, thus allowing the translocation of cholesterol and other molecules to the inner mitochondrial membrane, and (ii) the native receptor is a multimeric complex of an approximate 140,000 MW composed on an average of five 18,000 PBR subunits, one 34,000 VDAC subunit, and associated lipids.
Animals↗
Role of aging on electrical, mechanical, and coronary modification induced by ouabain and epinine in isolated rat heart.
OBJECTIVE: The contractile response to digitalis and beta adrenoceptor agonists is lower in the senescent than in the adult myocardium, while the development of ventricular arrhythmias is increased. The aim of this study was to examine the effects of aging on cardiac response to digitalis and an adrenergic agonist used clinically. METHODS: The electrical and mechanical responses were tested in isolated and perfused hearts from 3-24 month old rats receiving 15 min infusion of digitalis drug (ouabain, 6 x 10(-5) M) alone, and after 5 min of beta adrenoceptor agonist drug (epinine, 1.5 x 10(-7) M). RESULTS: Ouabain action was associated with a rise in left ventricular end diastolic pressure (p < 0.01) which increased progressively with aging, and with an elevation of left ventricular developed pressure (p < 0.01) which decreased progressively with aging. Epinine induced a reduction of left ventricular end diastolic pressure (p < 0.01) and a rise in left ventricular developed pressure (p < 0.01) but both effects decreased progressively with aging. Ouabain reduced coronary flow and this decrease was more pronounced with aging (p < 0.01), while epinine caused an increase (p < 0.01) that diminished in older hearts. Ouabain given after epinine resulted in a greater increase in left ventricular end diastolic pressure than epinine (p < 0.01) but lower than that caused by ouabain alone (p < 0.01), a greater increase in left ventricular developed pressure than epinine and ouabain (p < 0.01), and a smaller reduction of coronary flow rate than ouabain alone (p < 0.01). All these effects, however, diminished progressively with aging. Arrhythmia scores were higher during ouabain than in control (p < 0.01) and in epinine treated hearts (p < 0.01); pretreatment with epinine did not modify arrhythmia score during ouabain administration. The number and severity of arrhythmias, however, increased with aging in all groups. CONCLUSIONS: Aging has a negative effect on both the positive inotropic and the arrhythmogenic effects of ouabain and epinine, although these phenomena are more pronounced during ouabain administration. However, when the two drugs are given simultaneously, epinine does not modify the arrhythmogenic effect of ouabain but reduces some of its deleterious haemodynamic effects.
Aging↗
Site-directed mutagenesis of the peripheral benzodiazepine receptor: identification of amino acids implicated in the binding site of Ro5-4864.
The peripheral benzodiazepine receptor (PBR) is an 18-kDa protein present in the outer mitochondrial membrane. The human PBR can be labeled with the benzodiazepine Ro5-4864 and with the isoquinoline carboxamide PK11195. The two ligands compete with each other in binding experiments, with previous results suggesting overlapping but not identical binding sites. To define the regions of the receptor interacting with PK11195 and Ro5-4864 and to address the question of the topology of the molecule in the membrane, we generated mutant human PBRs with amino- and carboxyl-terminal deletions and with point mutations in potentially accessible cytoplasmic regions. The mutant genes were expressed in yeast and analyzed in binding experiments using radiolabeled PK11195 and Ro5-4864. The results showed that, whereas deletions in the amino-terminal sequence had marked consequences for the binding affinity of both ligands, the final 13 amino acids at the carboxyl terminus could be deleted with no effect on the binding of either Ro5-4864 or PK11195. The site-directed mutagenesis experiments pinpointed four amino acids as participating in the binding site of Ro5-4864. Three of these, Glu-29, Arg-32, and Lys-39, which are located in the first putative cytoplasmic loop, are conserved in human, bovine, rat, and mouse PBRs. The remaining residue, Val-154, which is found at the interface between the putative fifth transmembrane region and the cytoplasm, is present in the human, rat, and mouse sequences but is replaced by methionine in the bovine sequence. The exchange of Met-154 for valine in the bovine PBR introduced a binding site for Ro5-4864, which is absent in the native PBR. These four amino acids played a minor role, if any, in the binding site of PK11195. We also showed that the histidines previously suggested to be part of the binding site of PK11195 are not directly involved in the interaction of the human receptor with either PK11195 or Ro5-4864.
Amino Acid Sequence↗