Modulation of hematopoietic colony formation of stem cells in peripheral blood by anti-TGF-β in patients with severe immunosuppression

Summary The influence of transforming growth factor- (TGF-) on hematopoiesis has been evaluated by adding blocking antibodies against TGF- to colony forming assays (CFU-c). When optimum concentrations of recombinant growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-3 (IL-3) were added to stem cells from the peripheral blood of healthy individuals and certain patients with tumors or HIV infection, the anti-TGF- capable of blocking 5 ng/ml of active TGF- had no significant influence on erythroid or myeloid colony formation. However, in certain immunosuppressed individuals, anti-TGF- resulted in a significant decrease of erythroid colony formation and slight suppression of myeloid colony formation. The significant inhibition of hematopoiesis by plasma of HIV patients could be due to the presence of active forms of TGF-. The results of the blocking experiments are consistent with the concept that TGF- in low concentrations is essential for erythropoiesis and myelopoiesis but that higher levels of TGF- primarily inhibit erythropoiesis in vitro. TGF- serves as a coordinating factor when efficient recruitment of granulocytes and monocytes is more essential than erythropoiesis and stem cell growth.Abbreviations BFU-E burst forming unit-erythroid - CFC colony forming cells - CFU-GEMM colony forming unit-granulocyte/erythroid/macrophage/megacaryocyte - CFU-GM colony forming unit-granulocyte/macrophage - EPO erythropoietin - GM-CSF granulocyte/macrophage-colony stimulating factor - HIV human immunodeficiency virus - IL-1 interleukin-1 - IL-3 interleukin-3 - IMDM Iscove's Modified Dulbecco's medium - PBS phosphate buffered saline - TGF- transforming growth factor- - TNF- tumor necrosis factor-     

Bicarbonate permeability of epithelial chloride channels

To investigate the relation of arachidonate metabolism to the induction of fever by interleukin-1, indomethacin was administered in either an intracerebro-ventricular (icv) or a subcutaneous (sc) route in conscious rabbits. Fever induced by icv administration of recombinant human interleukin-1 (rhIL-1) was depressed by either icv or sc pretreatment with indomethacin. Fever induced by intravenous (iv) administration of rhIL-1 was significantly inhibited, though initial small increase in colonic temperature still remained, and was completely depressed by combination of icv and sc pretreatment with indomethacin. Intracerebroventricularly administered recombinant rabbit IL-1 (rrIL-1) induced dose-dependent increases in colonic temperature, which was depressed by sc pretreatment with indomethacin. There is little species specificity between human and rabbit IL-1, in terms of the pyrogenic potency and the inhibitory effect of sc indomethacin on fever induced by icv IL-1. Further, fever caused by icv administration of sodium arachidonate was significantly depressed by sc pretreatment with indomethacin. These results show that the inhibitory effect of indomethacin, administered either icv or sc, on IL-1-induced fever is similar to that of IL-1-induced fever reported previously [11]. This suggests that the site of arachidonate metabolism significantly involved in the mechanism of fever induction by IL-1 is easily accessible to the brain from the blood.     

Trennung der intestinalen β-Galactosidasen beim lactose-toleranten Erwachsenen durch Ultrazentrifugation im Dichtegradienten

Zusammenfassung Die intestinalen-Galactosidasen von 4 lactose-toleranten, erwachsenen Mitteleuropäern wurden im Saugbiopsie-Gewebe nach Solubilisierung mit Triton X-100 in einem linearen Mannitol-Gradienten (5–20%) auf der Ultrazentrifuge bei 4°C und 44000 U/min getrennt. Bei 12stündiger Zentrifugation fanden sich 3 Fraktionen, von denen die beiden schneller sedimenticrenden Lactose spalten. Alle 3 Fraktionen hydrolysieren p-Nitrophenyl--Galactosid. Die 3 isolierten-Galactosidasen entsprechen wahrscheinlich der neutralen Bürstensaum-Lactase, der sauren lysosomalen Lactase und einer cytoplasmatischen Hetero--Galactosidase.     

Glucocorticoids modulate TGF-beta production

TGF- is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGB- isoforms affect TGF- production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFL-1) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TFG-1, -2, and -3. TGF- mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TGF-2 and -3 stimulated TGF-1 production significantly (p < 0.01 relative to control). TGF-1 stimulated TGF-2 production (p < 0.01 relative to control). TGF-3 was undetectable. Glucocorticoids significantly inhibited TGF-1 and -2 production and reduced expression of the upregulated TGF-1 and -2 mRNA induced by exogenous TGF-1, -2 or -3 (p < 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF--stimulated cells, it was reduced by glucocorticoids. Thus, TGF- isoforms may stimulate production of various TGF- isoforms in the lung. Glucocorticoids then may block TGF- production by modulating mRNA levels and c-Jun.     

Modulation of Nav1.5 by β1- and β3-subunit co-expression in mammalian cells

Cardiac sodium channels (Na_v1.5) comprise a pore-forming -subunit and auxiliary -subunits that modulate channel function. In the heart, 1 is expressed throughout the atria and ventricles, whilst 3 is present only in the ventricles and Purkinje fibers. In view of this expression pattern, we determined the effects of 3 and 1 co-expression alone, and in combination, on Na_v1.5 stably expressed in Chinese hamster ovary cells. The current/voltage relationship was shifted –5 mV with either 1 or 3 co-expression alone and –10 mV with co-expression of both 1 and 3. In addition, 3 and 1/3 co-expression accelerated macroscopic current decay. There were significant hyperpolarizing shifts in equilibrium gating relationships with co-expression of 1 and 3 alone and in combination. Co-expression of 1/3 together resulted in a greater hyperpolarizing shift in channel availability, and an increase in the slopes of equilibrium gating relationships. Co-expression of 3 and 1/3, but not 1, slowed recovery from inactivation at –90 mV. Development of inactivation at –70 and –50 mV was accelerated by -subunit co-expression alone and in combination. -Subunit co-expression also reduced the late Na current measured at 200 ms. In conclusion, -subunits modulate Na_v1.5 gating with important differences between co-expression of 1 and 3 alone and 1/3 together.     

Glucocorticoids modulate TGF-beta production by human fetal lung fibroblasts

TGF- is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGF- isoforms affect TGF- production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFLF) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TGF-1, -2, or -3. Post-culture media were collected for ELISA assays of TGF-1, -2, and -3 . TGF- mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TFG-2 and -3 stimulated TGF-1 production significantly (p < 0.01 relative to control). TGF-1 stimulated TGF-2 production (p < 0.01 relative to control). TGF-3 was undetectable. Glucocorticoids significantly inhibited TGF-1 and TGF-2 production and reduced expression of the up-regulated TGF-1 and TGF-2 mRNA induced by exogenous TGF-1, -2, or -3 (p < 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF--stimulated cells, it was reduced by glucocorticoids. Thus, TGF- isoforms may stimulate production of various TGF- isoforms in the lung. Glucocorticoids then may block TGF- production by modulating mRNA levels and c-Jun.     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.     

Bile acids and their amidates inhibit 11β-hydroxysteroid dehydrogenase obtained from rat kidney

Recently it has ben demonstrated that interaction of corticosteroids with extraadrenal target cells can effectively be modulated by metabolic transformation of the steroid hormone. As far as 11-hydroxylated glucocorticoids are concerned 11-hydroxysteroid dehydrogenase (11-HSD) is the most important enzyme charged with target cell metabolism. Inhibition of 11-HSD function either by genetically transmitted deficiency or by exogenous enzyme inhibitors causes severe pathophysiological derangements, which result in a syndrome of apparent mineralocorticoid excess. In the present paper we have tested whether or not endogenous inhibitors of this enzyme system might exist. The effects of the main naturally occurring mono-, di-, and trihydroxylated bile acids in man on 11-HSD have been studied in in vitro experiments. Using rat renal microsomes it could be demonstrated that unconjugated bile acids of all three classes as well as the corresponding glycine and taurine amidates effectively inhibit oxidative as well as reductive activity of 11-HSD, with lithocholic acid and chenodeoxycholic acid being the most potent compounds. It is concluded that bile acids are potent endogenous inhibitors of 11-HSD and, therefore, could participate in abnormalities of cortisol metabolism observed in liver cirrhosis and extrahepatic biliary obstruction and, possibly, after ingestion of bile acids.Abbreviations CA cholic acid - CDCA chenodeoxycholic acid - DCA deoxycholic acid - GCA glycocholic acid - GCDCA glycochenodeoxycholic acid - GLCA glycolithocholic acid - 11-HSD 11-hydroxysteroid dehydrogenase (EC 1.1.1.146) - IC₅₀ molar concentration of bile acid at 50% inhibition of enzyme activity - LCA lithocholic acid - TDCA taurodeoxycholic acid - TLCA taurolithocholic acid - TUDCA tauroursodeoxycholic acid - UDCA ursodeoxycholic acid Supported by the Deutsche Forschungsgemeinschaft Hi 97/16 1-4. Parts of this study have been presented at the 21st meeting of the Gesellschaft für Nephrologie [23]     

Evidence for the presence of pro-γ-melanotropin,the NH2-terminal fragment of the corticotropin-β-lipotropin precursor,in corticotropin-producing tumours

Summary Five corticotropin-producing tumours were examined for peptides related to the corticotropin--lipotropin precursor. Two were basophil pituitary adenomas and three were bronchial carcinoids. The cells of the two pituitary adenomas stained with antisera against -endorphin and against pro--melanotropin, the NH₂-terminal fragment of the corticotropin--lipotropin precursor, but not with antisera against -melanotropin or -lipotropin. The corticotropin-storing tumor cells of the bronchial carcinoids stained with antisera against -endorphin, -lipotropin or pro--melanotropin. Only one of the three bronchial carcinoids contained cells reacting with the antiserum against -melanotropin. Although the two types of corticotropin-storing tumours (pituitary adenoma and bronchial carcinoid) differed with respect to -lipotropin content, the over-all picture indicates that the proteolytic processing of the corticotropin precursor proceeds along similar lines in tumour cells and in pituitary corticotrophs.An acetic acid extract of one of the bronchial tumours was subjected to gel chromatography and immunochemical analysis of material related to pro--melanotropin. The immunoreactive material displayed a considerable size heterogeneity, with the predominant components having a molecular weight larger than that of authentic pro--melanotropin.     

Enzymaktivitätsveränderungen der β-Hexosaminidase im Urin bei Nierenkranken

Zusammenfassung Bei Nierenkranken, nicht renal Erkrankten, essentiellen Hypertonikern und gesunden Probanden wurde die Enzymaktivität der-Hexosaminidase und zum Vergleich der-Galaktosidase im 24 h-Urin bestimmt. Gegenüber allen anderen Gruppen wiesen Nierenkranke eine signifikant höhere Enzymaktivität der-Hexosaminidase bei jedem Patienten auf, während-Galaktosidase nur im Mittelwert erhöhte Werte zeigte. Die Ursache der Enzymerhöhung wird diskutiert.     

Factors influencing serum neopterin and β2-microglobulin levels in a healthy diverse population

Sera and questionnaire data from a population-based random sample of healthy adults was used to evaluate factors influencing neopterin and 2-microglobulin (2m) values. Both neopterin and 2m levels increased with age and were higher among white than blacks (mean values for whites and blacks: neopterin, 5.06 vs 4.49 nmol/L; 2m, 1.36 vs 1.28 mg/L). Gender differences were noted for 2m but not neopterin values (2m males vs females: 1.37 vs 1.29 mg/L). Neopterin values were lower among current smokers than among nonsmokers (4.32 vs 5.16 nmol/L) and were higher among users of antihistamines (5.46 among users vs 4.65 nmol/L among nonusers). Neopterin and 2m were correlated in this healthy adult population (adjustedr=0.53,P=0.001), yet no other interrelationships with numerous biologic markers except between 2m and serum-soluble interleukin-2 receptor levels (adjustedr=.41,P=0.05) were observed. These findings provide important baseline information to consider before planning or evaluating studies utilizing neopterin or 2m levels.     

The role ofγδ T cells in the normal and disordered immune system

Summary A small population of T cells does not express the conventional T cell receptor characterized by the and polypeptide chains (TCR) but instead, two polypeptides termed and (TCR). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR remains unclear. In selected sites such as skin or gut TCR predominates in mice which might suggest a role of T cells in the first line of defense against infection, T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for T cells since MHC-nonrestricted and also CD4 and CD8 negative T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the T cells. In various diseases slightly increased numbers of T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of T cells.List of abbreviations C constant region (immunoglobulin or TCR gene segment) - CD4 cluster of differentiation 4 (mainly on helper cells) - CD8 cluster of differentiation 8 (mainly on cytotoxic cells) - D diversity region (immunoglobulin or TCR gene segment) - DNA desoxyribonucleic acid - IL2 interleukin 2 - J joining region (immunoglobulin or TCR gene segment) - kD kiloDalton - MHC major histocompatibility complex - NK natural killer (cells) - RA rheumatoid arthritis - TCR T cell receptor - V variable region (immunoglobulin or TCR gene segment)     

Oral Administration of Gammalinolenic Acid, an Unsaturated Fatty Acid with Anti-inflammatory Properties, Modulates Interleukin-1β Production by Human Monocytes

Administration of gammalinolenic acid (GLA), an unsaturated fatty acid, reduces joint inflammation in patients with rheumatoid arthritis. Addition of GLA in vitro suppresses release of interleukin-1 (IL-1) from human monocytes stimulated with lipopolysaccharide (LPS). LPS-induced IL-1 release is followed by IL-1-induced IL-1 release, an amplification process termed autoinduction. We show here, using IL-1 stimulation to simulate autoinduction, that administration of GLA to healthy volunteers and to patients with inflammatory arthritis reduces LPS-induced IL-1 secretion mainly by reducing autoinduction of IL-1. GLA reduces LPS-induced pro-IL-1 mRNA modestly and IL-1-induced pro-IL-1 gene expression markedly. In addition to reducing amplification of IL-1, GLA increases the amount of IL-1 receptor antagonist (IL-1Ra) secreted from stimulated cells, thereby facilitating an increase in the secreted IL-1Ra/IL-1 ratio. IL-1 is important to host defense, but the amplification mechanism may be excessive in genetically predisposed individuals. Thus, reduction of IL-1 autoinduction may be protective in some patients with endotoxic shock and with diseases characterized by chronic inflammation.     

Molecular size-dependent leakage of intracellular molecules from frog skeletal muscle fibers permeabilized with β-escin

The permeability of -escin-treated cell membrane was characterized in terms of the permeant molecular size, by monitoring the leak of cytoplasmic molecules in frog skeletal muscle fibers. With a low concentration of -escin (5 M), most of the cellular ATP was lost within 30–40 min (as revealed by rigor force generation), whereas a fluorescence-labeled dextran injected into the cytoplasm (10 kDa) and cytoplasmic proteins (14–80 kDa) slowly leaked out of the cell. A high concentration of -escin (50–100 M) accelerated the leak of large molecules. Therefore, low concentrations of -escin may be used as a means of permeabilizing the cell membrane to relatively small molecules, while retaining a major fraction of the cellular macromolecules.Abbreviations MOPS 3-[N-morpholino]propanesulfonic acid - KMS potassium methanesulphonate - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - EGTA ethylene glycol-bis( -amino-ethyl ether)N,N,N,N-tetraacetic acid - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Theβ−tubulin genes ofDrosophila auraria are arranged in a cluster

When the 1-, 2- and 3-tubulin-specific DNAs fromDrosophila melanogaster were used as probes to recognize tubulin-specific sequences in the chromosomes ofDrosophila auraria, they were found to hybridize to the same polytene band in region 32C of the 2L polytene chromosome. Three overlapping clones were isolated from a EMBL 3 genomic library ofD. auraria, and they all contain -tubulin-specific sequences based on hybridization and partial-sequencing experiments of subcloned fragments. These clones hybridize in situ to the same polytene chromosome band in region 32C and they represent an approximately 35-kb fragment of genomic DNA.     

BamH1 polymorphism in the Chinese,Malays, and Indians in Singapore and its application in the prenatal diagnosis of β-thalassemia

Summary The distribution of restriction fragment length polymorphism (RFLP) at theBamH1 site of the -globin gene was investigated in the Chinese, Indian, and Malay race in Singapore. The sample comprised of 183 normal individuals and 35 -thalassemia carriers in which 13 were couples with at least one -major child. The results from this study indicate thatBamH1 polymorphism will be informative in 22% of pregnancies at risk for -thalassemia major in Chinese, 19% in Malays and 7% in Indians. In prenatal diagnosis usingBamH1 polymorphism for one -major affected family, the fetus was diagnosed to be normal or -carrier. The validity ofBamH1 polymorphism in the exclusion of -thalassemia major was subsequently confirmed at birth by globin chain biosynthesis.     

Decreased serum levels of TGF-β in patients with acutePlasmodium falciparum malaria

Apart from cellular immunity and immunopathology, various cytokines have been implicated in malaria-associated immunosuppression. In this study, serum levels of transforming growth factor- (TGF-) were determined with an enzyme-linked immunosorbent assay in 37 patients with acutePlasmodium falciparum malaria prior to, during, and after therapy and in 17 healthy controls in Bangkok, Thailand. Patients were treated with artesunate and mefloquine. TGF- serum levels were found decreased prior to treatment (14±11 pg/ml versus 63±15 pg/ml in healthy controls;P<0.05). The serum concentrations of TGF- increased after initiation of treatment and were within normal range on day 21. Serum levels of both tumor necrosis factor-ga (TNF-) and soluble TNF-receptor 55 kDa were inversely correlated to serum levels of TGF- (r= –0.667 andr=}-0.592, n=37; respectively,P < 0.05 for both). No correlation between parasitemia and serum levels of TGF- could be found. The results are compatible with a decreased production and release, an enhanced clearance or utilization, or tissue accumulation of TGF- in acuteP. falciparum malaria.     

Antiviral action of interferon-β on Newcastle disease virus: selectivity to the hemagglutinin-neuraminidase gene expression

Interferon- (IFN-) strongly inhibited the expression of the hemagglutinin-neuraminidase (HN) gene of Newcastle disease virus (NDV), a paramyxovirus, in HeLa cells under the conditions where it did not affect the expression of the four upstream genes encoding the nucleocapsid protein, phosphoprotein, membrane protein and fusion protein. Even the downstream gene, encoding the large protein as well as the genome replication, appeared to be less susceptible to IFN- than the HN gene. This selective action of IFN- did not appear to be attributable to its well characterized antiviral mechanisms such as acceleration of RNA decay and translation inhibition. No similar down-regulation of a particular gene expression was found with another paramyxovirus, Sendai virus, or with a rhabdovirus, vesicular stomatitis virus, or seems to have been reported previously with any negative-strand RNA viruses. This new effect of IFN- thus suggests gene expression mechanism unique to NDV and may further lead to the discovery of a novel biochemical effect of IFN-.     

Localisation of CEA, β-HCG,SP1, and keratin in the tissue of lung carcinomas

Summary One hundred and twenty seven cases of lung tumors were studied by the immunoperoxidase technique for the presence of CEA and-HCG. Twenty-nine of these tumors were additionally stained for keratin and SP1. CEA and SP1 could be demonstrated in 80% of the studied cases, while-HCG was found in only 9%. SP1 revealed an almost identical staining pattern to CEA and keratin was found only in squamous cell carcinomas. The tissue positivity of none of these three markers correlated with tumor size, lymphnodal involvement or histological type.This study was supported by Deutsche Stiftung für Krebsforschung - Dr. Mildred Scheel-Stiftung     

Metoprolol in portal hypertension

Summary In a controlled trial, the effect of the ₁-selective blocking agent metoprolol on cirrhotic portal hypertension was investigated. A sustained reduction of portal pressure was observed in 60% of the treated patients after 1 and 2 months. No correlation between changes of portal pressure and cardiac output was established. This may indicate a direct action of-blocking substances on the splanchnic vascular system. The results suggest that treatment with metoprolol may be of value in patients with portal hypertension secondary to cirrhosis of the liver. However, to eliminate nonresponders the pressure has to be measured repeatedly.