Interferon action in human fibroblasts: Induction of 2′, 5′-oligoadenylate synthetase in the absence of detectable protein kinase activity

Summary In several strains of human fibroblasts, treatment with human - or -interferon (IFN) resulted in an induction of 2, 5-oligoadenylate synthetase but no protein kinase activity. The antiviral and anticellular effects of IFN are elicited in these cells in the absence of detectable IFN-induced protein kinase activity.With 2 Figures     

Three distinct loci on human chromosome 21 contribute to interferon-α/β responsiveness

The species specificity of interferons (IFNs) depends on restricted recognition of these ligands by multisubunit cell surface receptors. Expression of the human receptor subunit IFNAR in mouse cells conferred sensitivity only to one subtype of human IFN, IFN-B. Other genes on human chromosome 21 were required for responses to other subtypes of type I IFN. In contrast, IFNAR expression in hamster cells did not confer sensitivity to any human IFN tested, including IFN-B. Using human-hamster somatic cell hybrids, we mapped theIfnabr gene, encoding a ligand-binding subunit of the IFN-/ (type I) receptor, to human chromosome 21.Ifnabr colocalized withIfnar to the distal region of q22.1. The presence of a chromosomal fragment encoding IFNABR and IFNAR was also not sufficient to confer sensitivity to human IFN. In contrast, hybrids carrying in addition the region 21q22.2 showed a full response to human IFN-B, suggesting that a gene located in this region encodes a third factor required for type I IFN receptor activity.     

Transient expression of myosin heavy chain MHCIα in rabbit muscle during fast-to-slow transition

The expression of an -cardiac-like myosin heavy chain, MHCI, was investigated at both the mRNA and protein levels in rabbit tibialis anterior muscle undergoing fast-to-slow transition by continuous chronic low-frequency stimulation (CLFS). According to sequence analyses of the PCR product, the MHCI isoform was found to be identical to the -cardiac MHC expressed in rabbit atrium. In muscles at different degrees of transformation, the upregulation of MHCI mRNA preceded that of the MHCI mRNA. At more advanced stages of the transformation, MHCI mRNA decayed while MHCI mRNA persisted at high levels. The expression of MHCI, therefore, was transitory. Studies at the protein level were based on immunoblotting using a monoclonal antibody (F88 12F8,1), characterized to be specific to MHCI in rabbit muscle. These studies revealed a similar relationship between initial increase and successive decline of the MHCI protein as seen at themRNA level. Immunohistochemistry of 30-day stimulated muscle revealed that up to 65% of the fibres expressed the MHCI isoform in combination with other adult MHC isoforms. The most frequent patterns of coexistence were MHCIIa+MHCI + MHCI (28%), MHCI+MHCI (18%), and MHCIIa + MHCI (11%). According to these combinations, the upregulation of MHCI may be assigned as an intermediate step in the transformation of existing fibres during theMHCIIa MHCI transition. A small fraction of fibres contained, in addition to the MHCI + MHCI and MHCIIa + MHCI combinations, developmental myosin, suggesting that MHCI was also expressed in regenerating fibres originating from satellite cell-derived myotubes.     

Identification of alpha-cardiac myosin heavy chain mRNA and protein in extraocular muscle of the adult rabbit

Summary Extraocular muscles contain both fast-twitch and multiply-innervated, tonic-contracting fibres. In rat, these fibres collectively express numerous myosin heavy chain isoforms including fast-type embryonic and neonatal, adult slow twitch type I and fast twitch type II, and a fast isoform unique to extraocular muscle. Immunocytochemical and Western blotting results are presented which suggest that, in rabbit, an additional species, the -cardiac myosin heavy chain, is present. The immunoreactive species is found in all rabbit extraocular muscles and in the rotatory extraocular muscles is expressed in almost all fibres which do not contain a fast myosin heavy chain. Positive identification of this isoform as the -cardiac myosin heavy chain was obtained by sequencing a cloned PCR product derived from extraocular muscle mRNA unique to the 3-end of rabbit -cardiac myosin heavy chain mRNA. This is the first unequivocal demonstration of -cardiac myosin heavy chain expression in extraocular muscle.     

A comparative immunohistological study of cerebellar enolases

Summary A comparative immunohistological study of the neurone-specific enolase and enolase, demonstrates the exclusive neuronal localization of enolase and its absence from glial cells. In contrast, enolase is located in astroglial cells. The validity of enolase as a neuronal marker and enolase as an astrocytic marker, is confirmed both by a double labelling technique, using antibodies to and to revealed with fluorescence or peroxidase in the same tissue sections, and by immunoelectronmicroscopy.     

In vivo neutralization of tumor necrosis factor-alpha and interferon-gamma abrogates resistance to Yersinia enterocolitica infection in mice

Cytokines are important mediators of the inflammatory host response against infectious agents. In this study, the role of tumor necrosis factor-alpha (TNF-) and interferon-gamma (IFN-) in the elimination of a primary infection with highly virulent Yersinia enterocolitica serotype 0:8 strain WA-P has been investigated in C57BL/6 mice. The injection of anti-TNF- or anti-IFN- antibodies (serotherapy) prior to the intravenous challenge of a sublethal dose of Y. enterocolitica caused an increased bacterial net-growth in the spleens, although this effect was more pronounced for anti-TNF- treatment. The later treatment with anti-TNF- or anti-IFN- antibodies on day 3 post infection likewise abrogated resistance to Y. enterocolitica and, subsequently, led to death from progressive infection. Our data demonstrate for the first time that the endogenous production of both the cytokines TNF- and IFN- is required for the restriction of a primary Y. enterocolitica infection in mice.     

Phosphorylation-regulated low-conductance Cl− channels in a human pancreatic duct cell line

A low-conductance Cl^– channel has been identified in the apical membrane of the human pancreatic duct cell Capan-1 using patch-clamp techniques. Cell-attached channels were activated by the vasoactive intestinal polypeptide (VIP, 0.1 mol/l), dibutyryl-adenosine 3,5-cyclic monophosphate (db-cAMP, 1 mmol/l), 8-bromo adenosine 3,5-cyclic monophosphate (8-BrcAMP, 1 mmol/l), 3-isobutyl-1-methyl-xanthine (IBMX, 100 mol/l) and forskolin (10 mol/l). No channel activity was observed in non-stimulated control cells. In both cell-attached and excised inside-out patches, the channel had a linear current/voltage relationship and a unitary conductance of 9 pS at 23°C and 12 pS at 37°C. Its opening probability was not voltage dependent although pronounced flickering was induced at negative potentials. Anionic substitution led to the selectivity sequence Cl^–>I^–>HCO₃ ^–>gluconate. In insideout excised patches, the channel activity declined spontaneously within a few minutes. Reactivation of silent excised channels was achieved by adding protein kinase A (PKA, in the presence of ATP, cAMP and Mg²⁺). Conversely, active channels were silenced in the presence of alkaline phosphatase. The PKA-activated Cl^– channel was 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS, 100 mol/l) and 4-acetamido-4-isothiocyanatostilbene-2, 2-disulphonic acid (SITS, 100 mol/l) insensitive, but was blocked by diphenylamine-2-carboxylic acid (DPC, 100 mol/l). These results demonstrate that the apical low-conductance Cl^– channel in Capan-1 is regulated on-cell by VIP receptors via cAMP and off-cell by PKA and phosphatases. They provide evidence that this channel is closely related to the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel.     

Genetic Analysis of Pestiviruses at the 3′ End of the Genome

Specific PCR primers were selected for each pestivirus genotype which flanked the 3-part of the NS5B gene and more than three quarters of the 3-UTR. PCR products were sequenced in both directions using an automatic sequencing device and analyzed by computer package program DNASTAR. A comparative analysis of the 3 untranslated region (3-UTR) of 82 viruses, representing the four genotypes of the Pestivirus genus, provided a similar phylogenetic grouping as other genomic regions. Intertypic recombination was not observed, but Border disease virus (BDV) and Bovine viral diarrhoea virus type 1 (BVDV I) showed great intragenotypic variability. In most pestiviruses the stop codon is TGA, but BDV isolates were found to have either a TAG or a TAA stop codon. Various deletions and insertions were observed in the 3-UTR region. Viruses of the BVDV Ib group contained a characteristic deletion of 41 nucleotides. Compared to the 5-UTR, the 3-UTR was less conserved. The first 50–60 nucleotides were particularly variable, whilst the most conserved part was found at the 3 end of the studied region. All 82 viruses contained AT-rich stretches, the positions and sizes of which differed between the genotypes.     

A comparative study of skeletal and cardiac tropomyosins

Summary The subunit composition, the thiol group content and the biological activities of cardiac tropomyosin (TM) of various animal species were compared. Cardiac TM from small animals such as rabbit, guinea-pig, rat and dog contain 2 SH/mole and were resolved into one band on SDS and acid urea electrophoresis and into two bands on alkaline urea electrophoresis. Chicken cardiac TM likewise gave one band and it contains 4 SH/mole. In contrast pig, sheep and human cardiac TM contain respectively 2.6, 2.4, and 2.4 SH/mole and were resolved into two bands and on the different electrophoresis systems used, with a : ratio respectively of I:4.2, I:4.6, I:4.8. The -TM components from sheep skeletal and pig and sheep cardiac muscles were more positively charged than the rabbit skeletal -TM component, as shown in alkaline urea electrophoresis system. The and combinations of dimers found for skeletal muscle by other authors, were also found for cardiac pig TM.All the TM have the same effect on the Ca²⁺-stimulated ATPase activity of desensitized actomyosin (DAM) and on the Mg²⁺-stimulated ATPase activity of DAM with troponin-complex.This work suggests that the subunits of the TM from skeletal and cardiac muscles are heterogenous in their M.W. and their charges and that in the heart as well as in skeletal muscle a relationship seems to exist between the amount of the component and the speed of contraction of the muscle: a higher amount of this component was found in the bulky hearts which are also those which contract slower.     

Integrins and extracellular matrix-proteins in the different components of the Wilms' tumour

The Wilms' tumour (WT) is composed of blastema, epithelium and mesenchyme; the epithelium and possibly also the mesenchyme develop from the blastema, parallel to embryonal development. Since interactions between cell adhesion receptors and extracellular matrix (ECM) proteins play an important role in tissue maturation, we examined the expression of the integrin subunits 1–6, 1 and 4, and of the ECM proteins fibronectin, laminin and collagen I and IV, in 20 frozen WT samples and in 5 fetal and 2 adult kidneys. The integrin and ECM protein distribution in tumour epithelium and mesenchyme showed strong similarities to that in their fetal counterparts, whereas the tumour blastema differed strongly from the fetal blastema. In the WT blastema different components were recognized. Undifferentiated blastema, characterized by expression of 3 and 6 and the virtual absence of ECM proteins. Blastema with epithelial commitment, showing increased expression of 3 and 6 and the appearance of 2 and, as a very early phenomenon, production of laminin. Blastema with mesenchymal commitment, with loss of 3 and 6 and expression of 1, 4 and 5 and presence of ECM proteins. It is speculated that the inability of the (undifferentiated) blastema to produce ECM proteins is related to its relatively high metastatic potential when compared with epithelium and mesenchyme.     

Cyclische Nucleotide als intracelluläre Überträgerstoffe bei Hormonwirkungen

Zusammenfassung Hormone dienen als extracelluläre Informationsüberträger zwischen ihrem Bildungsort, einer endokrinen Drüse, und den Zellen, deren Funktion sie regulieren. Durch die Reaktion des Hormons mit den an der Zellmembran gelegenen Receptoren wird die Aktivität der mit diesen eng verknüpften Adenyl-Cyclase beeinflußt. Die meisten Hormone erhöhen in ihrem Zielorgan die Aktivität dieses Enzyms und führen hierdurch zu einem raschen Anstieg der intracellulären Konzentration von Adenosin-3:5-monophosphat (Ado-3:5-P). Dieses cyclische Nucleotid wird durch eine spezifische Phosphodiesterase zu Adenosin-5-monophosphat abgebaut. Auch die Aktivität dieses Enzyms bestimmt die intracelluläre Ado-3:5-P-Konzentration, die im Vergleich zu der anderer Nucleotide sehr gering ist.Ado-3:5-P beeinflußt als zweiter, intracellulärer Überträgerstoff die Aktivität zahlreicher Schlüsselenzyme. Die Ado-3:5-P-Konzentration bestimmt hierdurch das Gleichgewicht verschiedener Stoffwechselwege zueinander und damit die Reaktion einer Zelle auf eine hormonale Stimulierung. An einer Reihe von Enzymen wird die durch Ado-3:5-P bedingte Aktivitäts-Änderung durch einen gleichartigen Mechanismus bewirkt. Das cyclische Nucleotid stimuliert Proteinkinasen, die eine Phosphatgruppe des ATP auf verschiedene Proteine übertragen und hierdurch deren Eigenschaften verändern können. So steigt bei Phosphorylierung durch eine Ado-3:5-P-stimulierbare Proteinkinase die Aktivität der Triglyceridlipase und der Glykogen-Phosphorylase-b-kinase an, dagegen nimmt die Aktivität der Glykogen-Synthetase ab; durch Phosphorylierung von Histonen kann deren Repressorcigenschaft vermindert und die Synthese von Enzymen gesteigert werden.In manchen tierischen Geweben wurde auch eine spezifisch durch Guanosin-3:5-monophosphat (Guo-3:5-P) stimulierbare Proteinkinase nachgewiesen. Dieses cyclische Nucleotid kommt wie Ado-3:5-P in allen Säugerorganen vor. Die Bildung von Guo-3:5-P aus GTP wird durch die Guanyl-Cyclase katalysiert, ein Ferment, das im Gegensatz zur Adenyl-Cyclase zum großen Teil nicht an die Zellmembranen gebunden ist. Die Konzentration von Guo-3:5-P in verschiedenen Geweben, im Blutplasma und im Urin wird durch Hormone beeinflußt. Es ist noch nicht bekannt, welche hormonalen Regulationen durch Guo-3:5-P vermittelt werden; dagegen ist bei vielen, rasch einsetzenden Hormonwirkungen die Beteiligung von Ado-3:5-P nachgewiesen worden.

---

Abkürzungen Ado-3:5-P Adenosin-3:5-monophosphat - dAdo-3:5-P Desoxy-adenosin-3:5-monophosphat - Guo-3:5-P Guanosin-3 : 5-monophosphate - Nuc-3:5-P Nucleosid-3:5-monophosphat - NTP Nucleosidtriphosphat - NMP Nuclcosid-5-monophosphat - dATP Desoxyadenosintriphosphat - P_i anorganisches Phosphat - PP_i anorganisches Pyrophosphat - DNS Desoxyribonucleinsäure - RNS Ribonucleinsäure - r-RNS ribosomale RNS - m-RNS Boten-RNS - Glykogen-Synthetase UDP-Glucose--1,4-glucan--4-glucosyltransferase - ICSH interstitial-cell-stimulating hormone     

Immunoreactivity of the JK-132 monoclonal antibody directed against basement membrane collagen in normal and diabetic glomeruli

The possible involvement of basement membrane-associated collagen (recognized by the monoclonal antibody JK-132) in the evolution of diabetic nephropathy was studied in kidney specimens from seven patients with noninsulin-dependent diabetes mellitus, and its distribution was compared with those of antibodies against 1 to 4 chains of type IV collagen. JK-132, a monoclonal antibody against basement membrane-associated collagen, reacted immunohistochemically exclusively with the mesangial matrix of the glomerular capillary. In contrast, antibodies to the 1 and 2 chains (IV) reacted strongly with mesangial matrix, and less strongly with the glomerular basement membrane (GBM). Antibodies to the 3 and 4 chains (IV) reacted mainly with GBM. In diabetes, JK-132 reacted most extensively with the expanded mesangial matrix, its staining intensity increasing with progression of the diabetic glomerulosclerosis. Antibodies to the 1 and 2 chains (IV) reacted prominently with the expanded mesangial matrix but less strongly with the GBM. Antibodies to the 3 and 4 chains reacted intensely with the thickened GBM. These results suggest that basement membrane-associated collagen differs from 1 to 4 chains of type IV collagen and that basement membrane-associated collagen is a good marker of mesangial expansion in diabetic nephropathy.     

Transforming growth factor β upregulates the integrin-mediated adhesion of human prostatic carcinoma cells to type I collagen

Prostate cancer frequently metastasizes to bone, and we propose that this process may be facilitated by the adhesion of metastatic cells to bone-derived type I collagen. We examined collagen receptor function and regulation in osteotropic PC-3 human prostatic carcinoma cells. PC-3 cell adhesion to immobilized human type I collagen was promoted by Mn and Mg ions and was RGD-independent. Antibodies directed against 1 or 2 integrin subunits inhibited adhesion to collagen by 90% and 53%, respectively, suggesting involvement of the 21 receptor. Anti-1 or anti-3 antibodies had no effect on adhesion. Flow cytometry and immunoprecipitation of [S]methionine-labeled cells demonstrated that 21 was the major collagen receptor expressed by PC-3 cells. The pretreatment of PC-3 cells with transforming growth factor-1 (TGF-1), a major bone-derived growth factor, caused a rapid (2 h) 2-fold increase in the de novo synthesis of 2 and 1 integrin subunits, and also increased by 2- to 3-fold the adhesion and spreading of PC-3 cells on collagen. We conclude that 21 is the major collagen receptor employed by PC-3 cells, and that 21 upregulation by TGF- is associated with an increased adhesion and spreading on collagen. The data suggest that exposure of metastatic PC-3 cells to the high levels of TGF- in bone may promote their ability to adhere to bone-derived collagen, which may thereby facilitate the localization of metastatic cells in the skeleton.     

Interferon-α activates cytotoxic function but inhibits interleukin-2-mediated proliferation and tumor necrosis factor-α secretion by immature human natural killer cells

Natural killer (NK) cells play an important role in host defense mechanisms against infection and neoplasia. Interferon- (IFN-) has been shown to activate NK cells and to augment their cytotoxic activity, albeit its role in the maturation pathway of NK cells has not been elucidated. The present study examined whether IFN- activates the immature NK subset (Free cells) to become cytotoxic and also ascertained whether IFN- uses the same pathway of activation as that mediated by interleukin-2 (IL-2). Incubation of sorted Free cells overnight with IFN- resulted in augmentation of their cytotoxic function against NK sensitive target cells. The enhanced cytotoxic activity was not accompanied by a new recruitment of NK-target binder cells but by an increase in the frequency of killer cells in the conjugate fraction. Activation of the Free subset by IFN- resulted in upregulation of CD69, CD11b, and CD2 surface expression and stimulated secretion of IFN-. Unlike IL-2, IFN- did not stimulate the Free cells to proliferate or secrete TNF- and activation of cytotoxicity and modulation of surface antigens by IFN- were independent of TNF-. The failure of IFN- to stimulate secretion and proliferation by Free cells appeared to be mediated by negative signals. This was corroborated in experiments demonstrating that when Free cells were cultured with both IFN- and IL-2, a significant inhibition was observed for both the IL-2 dependent secretion of TNF- and proliferation. These results demonstrate that IFN- serves as both an activator and a regulator of NK function. Further, activation of the immature Free NK cells by IL-2 and IFN- proceeds by TNF--dependent and independent pathways, respectively. The findings also support our contention that the mechanism of activation of the cytotoxic machinery of NK cells is not linked to the mechanism of activation of cytokine secretion and/or proliferation.Abbreviations used IFN interferon - IL interleukin - PBL peripheral blood leukocytes - PE phycoerythrin - PE-GAM PE-conjugated Fab2 goat anti-mouse IgG - NK natural killer - NRS normal rabbit serum - TNF tumor necrosis factor - FCS fetal calf serum - FITC fluorescein isothiocyanate - PBS phosphate-buffered saline - MACS magnetic cell sorting - ELISA enzyme-linked immunosorbent assay - BSA bovine serum albumin - PKC protein kinase C - mAb monoclonal antibody - PBMC peripheral blood mononuclear cells - BCLL B-chronic lymphocytic leukemia - E effector - T target     

Die Nebennierenrindenfunktion unter Applikation von 9α-Fluorcortisol

Zusammenfassung Untersuchungen über den Einfluß von 9-Fluorcortisol auf die Nebennierenrindenfunktion ergaben — in Verbindung mit in der Literatur mitgeteilten Werten — eine dosisabhängige Einschränkung der Ausscheidung von Nebennierenrindenhormonen. Die Ansprechbarkeit der Nebennierenrinde auf exogenes ACTH bleibt erhalten. Es ist daher eine Hemmung der hypophysären ACTH-Sekretion anzunehmen, die durch die Struktur des synthetischen Steroids erklärbar ist. — In geringer Dosierung, wie sie als Erhaltungsdosis bei Langzeittherapie verabfolgt wird, verursacht 9-Fluorcortisol keine wesentliche Einschränkung der Hormonausscheidung.

---

Effect of 9-fluorocortisol on adrenocortical function

Summary Investigation of adrenal cortical function during administration of 9-fluorcortisol revealed—in connection with results obtained from the literature—a dose-related inhibition of the secretion of adrenocortical hormones. Adrenal cortical response to exogenous ACTH remains unaffected. An inhibition of hypophyseal ACTH-secretion is therefore assumed, caused by the structure of the synthetic steroid. At low dosage, as applied in long-term treatment, no significant alteration of steroid excretion patterns was observed.

---

---

Astonin-H, Hersteller: Fa. E. Merck A.G., Darmstadt.

---

In der Arbeit werden folgende Abkürzungen verwendet: 17-KS=17-Ketosteroide; 17-OH-CS=17-Hydroxycorticosteroide; F=Cortisol=Pregn-4-en-11,17,21-triol-3,20-dion; E=Cortison=Pregn-4-en-17,21-diol-3,11,20-trion; THF=Tetrahydrocortisol=5-Pregnan-3,11,17,21-tetrol-20-on; allo-THF=allo-Tetrahydrocortisol=5-Pregnan-3,11,17,21-tetrol-20-on; THE=Tetrahydrocortison=5-Pregnan-3,17,21-triol-11,20-dion; Andro=Androsteron=5-Androstan-3-ol-17-on; Ätio=Ätiocholanolon=5-Androstan-3-ol-17-on; DHA=Dehydroepiandrosteron=Androst-5-en-3-ol-17-on.

---

Herrn Prof. Dr. med. H. Franke zum 60. Geburtstag gewidmet.     

Specific alterations in the expression of α3β1 and α6β4 integrins in highly invasive and metastatic variants of human prostate carcinoma cells selected by in vitro invasion through reconstituted basement membrane

Highly invasive cell subpopulations from a human prostate carcinoma cell line, PC-3, were selected for by allowing the parental PC-3 cells to invade through reconstituted basement membrane, Matrigel. These cells were collected, cultured and then selected further by repeated invasion through the in vitro invasion chamber. The invasive subpopulations (I-PC3 (2) and (3)) were found to be approximately 15-fold more invasive in vitro than the parental cells, had a distinct rounded morphology in culture, and proliferated more rapidly than the parental cells. When injected either subcutaneously or intraperitoneally into immunocompromised SCID mice, the I-PC3 cells were found to form tumors at the primary sites and to be highly invasive and metastatic. In contrast, the parental PC-3 cells formed tumors at the site of inoculation in these mice but failed to invade or metastasize. The I-PC3 cells attached equally as well as PC-3 cells to fibronectin, laminin, collagen type IV and vitronectin, but unlike the parental PC-3 cells these invasive variants failed to spread on any of these substrates. On Matrigel, the PC-3 cells became highly organized, whereas the I-PC3 cells remained rounded, clumped together and penetrated into the Matrigel. Biochemical analysis of the expression of adhesion proteins and integrins demonstrated that whereas the parental cells synthesized and secreted substantial amounts of fibronectin, the I-PC3 cell variants did not secrete any fibronectin. Although both PC-3 and I-PC3 cells expressed equivalent levels of cell surface v 3, 21 and 51 integrins, the expression of the 31 integrin, which is expressed at very high levels on the parental PC-3 cells, was drastically reduced on the invasive I-PC3 cells. This decrease in expression of 3 occurred also at the level of mRNA expression. Finally, whereas the PC-3 cells express 61, in the invasive I-PC3 cells the a6 subunit was associated mostly with the 4 subunit. Since the 64 integrin is analogous to the A9 tumor antigen which is associated with aggressive human squamous cell carcinomas, the apparent overexpression of 64 may also participate in the aggressive behavior of these variant prostate carcinoma cells. Alterations in the expression of the 31 and 64 integrins may thus allow these cells to become more invasive, and lead to an increased propensity for metastasis.     

Molecular localization of human IgG anti-F(ab′)2 reactivity with variable- and constant-region λ light-chain epitopes

Human IgG antibodies reacting with antigenic determinants on F(ab)₂ fragments represent generic antiidiotypic antibodies present in the serum of normal individuals. Additionally, the titers of these antibodies in the sera of patients with systemic lupus erythematosus (SLE) are inversely related to disease activity. Because these autoantibodies recognize predominantly light chain-related epitopes, especially type, we synthesized constant (C)- and variable (V)-related overlapping 7-mer peptides on polypropylene pins to determine anti-F(ab)₂-reactive epitopes on human light chains. ELISA reactivity of affinity-purified anti-F(ab)₂ antibodies obtained from normal individuals and from patients with SLE, as well as murine anti-human light-chain monoclonal antibodies specific for C and V subgroup-related determinants, was tested using the overlapping 7-mers of human light-chain sequence. The patterns of reactivity against C-related peptides were similar in both normal and SLE-derived anti-F(ab)₂ antibodies. However, reactivity profiles against V-related peptides were distinctively different between the normal and the SLE-associated anti-F(ab)₂ autoantibodies. A decrease in reactivity among the SLE IgG anti-F(ab)₂ antibodies was noted for particular amino acid V complementarity-determining region (CDR) residues, including glycine at positions 27 and 54, alanine at 16 and 37, and tyrosine at 28 and 91. This different pattern of reactivity from normal may indicate that in SLE there is a failure of antiidiotypic control mechanisms, as reflected by a defect in production of antibody to immunodominant V CDR residues.     

Comparative evaluation of integrin α- and β-chain expression in colorectal carcinoma cell lines and in their tumours of origin

The integrin family consists of broadly expressed cell surface adhesion receptors, each member of which is composed of a non-covalently linked / heterodimer. Integrin receptors are involved in the interaction with matrix proteins and may contribute to invasion and metastasis of carcinomas. To examine the biological role integrins play in colorectal carcinoma we compared the expression of integrin - and -subunits in situ and in vitro. Eight newly established cell lines derived from immunohistochemically characterized colorectal carcinomas together with two sublines obtained after nude mouse passage and the commonly used colon carcinoma lines HT-29, SW480, SW620, and COLO 205 were investigated by immunocytochemistry and flow cytometry. The carcinomas in situ expressed 1-, 2-, 3-, 6-, v-and 1-subunits in variable amounts while being devoid of 4, 5 and 3. The individual integrin profile of the tumour in tissue was essentially maintained in vitro. However, a neo-expression of the 5 chain was found, together with an induction or increase in 1, 2, 3, v and 1 levels. No decrease in integrin subunit expression was observed. Standard-serum and serum-free medium revealed no striking differences in - and -chain expression in the cell lines HT-29 and COLO 205. In serum-free medium, SW480 showed a slight increase of 1 and 5 and a decrease of 3 and v while SW620 expressed more 1. We conclude that the great variability of adhesion receptor expression of the integrin family in colorectal carcinomas in situ is essentially maintained in vitro, although culture conditions which are only marginally influenced by serum factors unpredictably lead to some increase in expression or even induction of several integrin subunits.This work is dedicated to Prof. Wilhelm Doerr on the occasion of this 80th birthday     

Interferon (type-α and γ) production by fresh and cryopreserved human mononuclear cells

Summary A comparative study of interferon (IFN) production (type- and ) was carried out using Ficoll-hypaque purified fresh and cryopreserved mononuclear cells from eight normal healthy individuals. Newcastle disease virus-NDV (R₂B strain) was used as an inducer for type- and Staphylococcal enterotoxin-A-(SEA) for type-IFN production. There was no significant difference between the titres of type- and -IFN and lymphocyte subpopulations of fresh and cryopreserved mononuclear cells studied under identical conditions.     

NF-κB translocation and endothelial cell activation is potentiated by macrophage-released signals co-secreted with TNF-α and IL-1β

Objective and Methods: Over-expression of the immune response can lead to pathological conditions such as septic shock or chronic inflammation. Endothelial cell activation by pro-inflammatory products of activated macrophages plays a key role in these conditions. Here we examine the response of primary human endothelial cells (HUVEC) to conditioned media (CM) obtained from LPS-activated macrophages. We further characterized the translocation of NF-B in the presence of CM by studying the degradation rate of individual IB isoforms.Results: We show that, as expected, CM induced NF-B translocation, as well as adhesion capacity in HUVEC. We further show that this response is critically dependent on TNF- and IL1 naturally present in the CM. However, both the amplitude of NF-B translocation and adhesiveness observed with CM were well beyond the saturation levels attained after the sole stimulation with recombinant TNF- and IL-1, either separately or together. Our results show that CM induced a faster degradation of the IB- and IB- isoforms than the recombinant cytokines, leading to an enhanced recruitment of NF-B activity.Conclusions: The above results suggest that the physiological context of factors co-secreted by LPS-activated macrophages enhances TNF- mediated endothelial activation.Received 5 October 2003; returned for revision 2 December 2003; accepted by A. Falus 19 May 2004