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Biomedical subjects

A Billiau

Publications and source records attributed to A Billiau.

At least 73 records · Page 4Linked to original sources
PubMed1994

Pyrogenic activity of human native and human recombinant interleukins-1 beta: stabilization with albumin enhances the pyrogenic action of recombinant IL-1 beta delivered into the rabbit brain.

The pyrogenic potential of natural and recombinant human IL-1 beta in rabbits was found to be very similar when the substances were given intravenously. Under these conditions, stabilization of rIL-1 beta with human serum albumin (HSA) failed to affect the pyrogenic activity of recombinant IL-1 beta. When the two preparations were administered directly into the PO/AH area of the brain, recombinant IL-1 beta was less pyrogenic than its natural counterpart. This lower pyrogenicity of recombinant IL-1 beta was corrected if the injected material contained HSA, which is known to stabilize in vitro the biological activities of IL-1 beta against slow degradation. The possibility is now considered that the central and peripheral systems for IL-1 inactivation are different. The existence of an intrabrain IL-1 pool is suggested and its significance for neuroimmunomodulation is stressed.

Animals↗
PubMed1994

Glycoform heterogeneity of porcine interferon-gamma expressed in Sf9 cells.

Porcine interferon-gamma (SfPoIFN-gamma) was expressed with high efficiency in Spodoptera frugiperda (Sf9) cells by means of the baculovirus expression system. Up to 10(5) U/ml of antivirally active SfPoIFN-gamma could be tracked down in the culture medium at 64 h postinfection. Three proteins (17, 19, and 21 kDa), which under nondenaturing conditions primarily exist as mutual-dimeric combinations, were purified by immunoaffinity chromatography. Carbohydrate labeling and kinetic deglycosylation studies suggested that the 19- and 21-kDa proteins are N-glycosylated variants of a single 17-kDa protein carrying no N-linked sugars, in which one respectively two N-glycosylation sequons are occupied by glycans of 2 kDa. Both the quantitative recovery of SfPoIFN-gamma from a Con A column at 0.2 M methyl-alpha-mannopyranoside and the results of lectin blots, revealing strong affinity of the 19- and 21-kDa species for Galanthus nivalis agglutinin, support the presence of N-glycosidically linked high mannose-type chains in the carbohydrate moiety of SfPoIFN-gamma. Intriguingly, both 19- and 21-kDa glycoforms, but not their sialidase-treated derivatives, showed clear reactivity with the Sambucus nigra and Maackia amurensis agglutinins. These agglutinins specifically recognize sialic acid linked alpha(2-6) and alpha(2-3), respectively, to penultimate galactose residues. Their affinity for the larger glycoforms of PoIFN-gamma suggests that the biosynthetic pathways in Sf9 cells are able to modify oligomannose structures to complex or hybrid glycans.

Animals↗
PubMed1993

Gelatinase B is present in the cerebrospinal fluid during experimental autoimmune encephalomyelitis and cleaves myelin basic protein.

Gelatinases in inflammatory demyelinating diseases of the central nervous system (CNS) were studied using actively induced experimental autoimmune encephalomyelitis (EAE) in mice as a model system. Clinical disease scores correlated in time and in intensity with pathology parameters such as cytosis in the cerebrospinal fluid (CSF), inflammatory infiltrates, and demyelination in the CNS. Zymographic analysis was employed to measure gelatinases A and B in the CSF from individual animals. According to their apparent molecular weight (MW), gelatinases A and B appeared with a MW of 65 and 95 kDa, respectively. The 65 kDa form was present in all samples, even in those derived from non-induced animals, whereas the 95 kDa form was present only in samples from animals developing EAE. The levels of 95 and 65 kDa gelatinase correlated with the CSF cytosis. In vitro digestion of myelin basic protein (MBP) with gelatinase B and analysis of the cleavage products by protein sequence analysis pinpointed two cleavage sites in conserved regions of MBP. Gelatinase production within the CNS may constitute an important pathogenic mechanism for both the disruption of the blood-brain barrier and the destruction of myelin, as observed in several neuroinflammatory disorders.

Amino Acid Sequence↗
PubMed1993

Gene sequence, cDNA construction, expression in Escherichia coli and genetically approached purification of porcine interleukin-1 beta.

A genomic clone (PIL3) covering the 8.8-kb prointerleukin-1 beta ('catabolin') gene of the domesticated swine (Sus scrofa domestica) was isolated from a genomic library and characterized by nucleotide sequencing. Typical features of the gene include a seven-exon structure, with the highest degree of nucleotide and amino acid conservation among human and porcine genes being found in the receptor-binding portion encoded by exons six and seven. Three 250-bp repetitive elements with a > 75% similarity to the pig repetitive element-1 family sequence are located in untranslated gene segments. Southern-hybridization experiments disclosed extensive genomic heterogeneity of the porcine interleukin-1 beta (IL-1 beta) gene region, suggesting a duplication of at least the 3' half of the gene in the porcine genome. Since similar hybridization patterns were observed for wild boar (Sus scrofa) genomic DNA, it was concluded that this gene rearrangement had preceded domestication of the wild swine. In addition, the cDNA for processed porcine IL-1 beta was constructed through polymerase-chain-reaction-mediated exon fusion by overlap extension starting from the genomic template. Recombinant IL-1 beta was expressed in Escherichia coli as a fusion protein containing an N-terminal hexahistidine tag followed by a factor-Xa-cleavage site. The protein was efficiently purified through adoption of a scheme that consisted of four alternating cycles of immobilized metal-ion-affinity chromatography and size-exclusion chromatography. 13.8 mg highly purified recombinant porcine IL-1 beta was obtained starting from a 900-ml thermo-induced E. coli culture (final endotoxin concentration < 0.22 ng/ml). The protein behaved homogeneously as a monomeric species, which was reactive in Western-blot experiments with an anti-(human-IL-1 beta) serum and which appeared to induce gelatinase B in MDBK cells in a dose-dependent fashion.

Amino Acid Sequence↗
PubMed1993

Human and bovine granulocyte chemotactic protein-2: complete amino acid sequence and functional characterization as chemokines.

Tumor cells are capable of simultaneously producing a number of related inflammatory peptides, now classified as chemokines. We have isolated a new human granulocyte chemotactic protein (GCP-2), coproduced with interleukin-8 (GCP-1/IL-8) by osteosarcoma cells. Furthermore, the bovine homologue of human GCP-2 was purified from kidney tumor cells using the same isolation procedure. Both chemokines occur in at least four NH2-terminally truncated forms. These 5-6 kDa proteins do not differ in potency and efficacy as granulocyte chemotactic factors using a standard in vitro migration assay. The complete primary structures of human and bovine GCP-2 were disclosed by sequencing peptide fragments derived from the natural proteins. On the basis of the conservation of four cysteine residues, the two molecules are to be classified within the C-X-C chemokine family, including IL-8. Human and bovine GCP-2 are 67% similar at the amino acid level. Their sequences show only weak similarity with that of IL-8, and human GCP-2 does not cross-react in a radioimmunoassay for IL-8. Human and bovine GCP-2 are specific granulocyte chemotactic factors in that they do not attract human monocytes. Bovine GCP-2 is not species specific since it is at least as active as human GCP-2 on human granulocytes. Both chemokines can also activate postreceptor mechanisms leading to release of gelatinase B by granulocytes. This is indicative for a possible role in inflammation and tumor cell invasion.

Amino Acid Sequence↗
PubMed1993

Refolding and single-step purification of porcine interferon-gamma from Escherichia coli inclusion bodies. Conditions for reconstitution of dimeric IFN-gamma.

Recombinant porcine interferon-gamma, overexpressed in Escherichia coli, was found to accumulate in cytoplasmic inclusion bodies. The influence of various physicochemical parameters on refolding was investigated using 6 M guanidine/HCl-solubilised inclusion bodies which had been purified by ultracentrifugation on a sucrose step gradient. It appeared that the yield of reconstitution of denatured protein reached 60-70% under optimum conditions, i.e. at an intermediary guanidine/HCl concentration of 0.5 M and at a protein concentration of 10-20 microM (0 degrees C). Since intermediary guanidine/HCl concentrations at 0.5-1.65 M increasingly promoted off-pathway formation of soluble aggregates and at 0.5-0.2 M progressively promoted precipitation, maximal recovery of biologically active protein required a twofold transition in the surrounding guanidine/HCl concentration (6 M-->0.5 M-->0 M). A single additional size-exclusion chromatographic step yielded a final product that was > 99.5% pure, had specific antiviral activity > 10(7) U/mg protein and contained < or = 25 pg/ml endotoxin. Cross-linking by means of disulfosuccinimidyl tartarate revealed that the refolded protein possessed a dimeric structure. Furthermore, we have characterized three different molecular species of recombinant porcine interferon-gamma that are formed under non-optimal refolding conditions (1 M guanidine/HCl) and that differ from each other in specific activity, size and stability. One of these converts irreversibly into dimeric interferon-gamma in a temperature-dependent manner and is therefore considered as a productive folding intermediate.

Animals↗
PubMed1993

Identification of a novel granulocyte chemotactic protein (GCP-2) from human tumor cells. In vitro and in vivo comparison with natural forms of GRO, IP-10, and IL-8.

Stimulated human osteosarcoma cells (MG-63) were used as a source of granulocyte chemotactic protein (GCP). In addition to the previously isolated GCP-1/IL-8, natural forms of GRO alpha, GRO gamma, and IP-10 were purified and identified by amino acid sequence analysis. Further, a novel GCP, GCP-2, was isolated in its natural form (6 kDa) and was found to be structurally related to the other members of the IL-8 family. GRO alpha, IP-10, and GCP-2 showed heterogeneity, in that several forms of each protein were recovered. These differed in truncation at the amino terminus. Reverse phase HPLC allowed us to separate four such different forms of GCP-2. These tumor-derived factors were compared in granulocyte activation and chemotaxis assays. IL-8 induced neutrophil gelatinase B release at 2 nM, but GRO alpha and GCP-2 showed a 5- to 10-fold lower specific activity. When the migration of granulocytes through polycarbonate micropore membranes was measured, GCP-2 and GRO alpha had a maximal chemotactic index comparable to that of IL-8. The minimal effective dose for GCP-2 and GRO alpha was 3 to 10 nM, whereas the specific activity of IL-8 was at least 10-fold higher. IP-10 was not active in this assay at doses up to 100 nM. Finally, in vivo chemotaxis was measured by using granulocyte recruitment in the rabbit skin model. After intradermal injection of 200 ng/site, GCP-2 provoked a significant granulocyte infiltration, albeit to a lesser extent than did IL-8 and GRO alpha. GCP-2 did not attract monocytes in vivo nor did it induce the cells in vitro to migrate or to produce enzyme. In conclusion, this study reveals a new member of the IL-8 family and shows that these related inflammatory mediators possess different potencies and efficacies towards granulocytes.

Amino Acid Sequence↗
PubMed1993

Increased circulating interleukin-6 (IL-6) activity in endotoxin-challenged mice pretreated with anti-IL-6 antibody is due to IL-6 accumulated in antigen-antibody complexes.

Mice pretreated with monoclonal anti-interleukin-6 (IL-6) antibody and then challenged with lipopolysaccharide (LPS), paradoxically develop higher levels of circulating biological IL-6 activity, as measured by the hybridoma growth promotion assay, than mice similarly challenged but not pretreated with antibody. Here we provide evidence that this increased biological activity was entirely accounted for by the presence of increased amounts of IL-6 protein, which could be isolated by immunoaffinity chromatography and subsequently visualized after gel electrophoresis. Chromatography on a protein G matrix and a sandwich ELISA allowed to demonstrate that all IL-6 present in the serum was in the form of antigen-antibody complexes. Serum samples of antibody-treated animals which contained the highest biological activity typically contained near equimolar concentrations of IL-6 and antibody. In vitro neutralization tests with pure antibody and IL-6 demonstrated that, with both antibodies tested, more than 1000-fold molar excess of antibody is needed for neutralization in the hybridoma growth assay. It is concluded that increased biological activity in serum of the anti-IL-6 antibody-treated mice is due to sequestration of the endogenous IL-6 in the form of antigen-antibody complexes which, due to the lack of sufficient antibody excess, produce nearly full activity in the hybridoma growth assay.

Animals↗
PubMed1993

Modification of the anti-CD3-induced cytokine release syndrome by anti-interferon-gamma or anti-interleukin-6 antibody treatment: protective effects and biphasic changes in blood cytokine levels.

Anti-interferon-gamma (IFN-gamma) antibodies were found to protect mice against pathological changes induced by injection of anti-CD3 antibody: incidence of diarrhea, severity of hypothermia and mortality rates were dramatically reduced. In anti-IFN-gamma antibody-treated mice, IFN-gamma blood levels were significantly reduced at 1.5 h post anti-CD3 challenge, but more elevated levels were found from 4 to 24 h. This rebound-like IFN-gamma response coincided with more profound hypoglycemia. Tumor necrosis factor and interleukin (IL)-6 levels were not affected by anti-IFN-gamma treatment. Exogenous IFN-gamma, administered within 3 h (but not later) of the anti-CD3 challenge made the syndrome worse. Furthermore, inter-mouse strain differences in sensitivity to the anti-CD3 syndrome correlated with the ability of the strain to produce IFN-gamma. Anti-IL-6 antibodies provided only marginal protection against hypothermia and mortality, but did markedly reduce hypoglycemia. Levels of biologically active IL-6 in serum were not influenced by anti-IL-6 antibody treatment during the first few hours after anti-CD3 challenge, but were significantly increased at later times. The data provide evidence that endogenous IFN-gamma is a critical element in the early phase of the anti-CD3 syndrome; endogenous IL-6, while possibly being involved in hypoglycemia, seems of lesser importance for the outcome of the syndrome.

Animals↗
PubMed1993

Plasmodium berghei: recombinant interferon-gamma and the development of parasitemia and cerebral lesions in malaria-infected mice.

Mice infected with Plasmodium berghei K173-parasitized erythrocytes develop severe hypothermia followed by death as a consequence of murine cerebral malaria early in the second week after infection. A single intraperitoneal injection of 10(5) Units of IFN-gamma given between Day 4 and Day 6 postinfection results in a transient decrease of body temperature. No effect on parasitemia and cerebral malaria is obtained by this treatment. Daily injections of relatively low doses of IFN-gamma delays the patency of the infection for 2 days. Furthermore the proliferation rate of the parasites is reduced and the development of cerebral malaria is also delayed for 2 days. The reduction of body temperature, as found in untreated infected mice, is absent. Administration of IFN-gamma by means of a continuous delivery from intraperitoneally inserted osmotic pumps (1.2 x 10(4) Units of IFN-gamma/24 hr) also delays patency and inhibits parasitemia. Body temperature decreases during infection but mice are protected against the development of cerebral malaria. In nude mice, this treatment inhibits parasitemia to the same extent. However, reduction of body temperature was also prevented. High doses of IFN-gamma delivered by osmotic pumps (2.5 x 10(4) or 10(5) Units of IFN-gamma/24 hr) appear to be lethally toxic in conventional as well as in nude mice, independently of infection. Cerebral malaria-like symptoms are found in these mice. Treatment of infected C57BL/6J mice with antibody to IFN-gamma 4 days before and after infection as well as on the day of infection enhances parasitemia but does not affect the development of murine cerebral malaria. Single injections of anti-IFN-gamma-antibody 6 hr prior to infection or 7 days after infection have no effect. In CBA/Ca mice, treatment with anti-IFN-gamma-antibody enhances parasitemia; furthermore protection against cerebral malaria was obtained in part of the mice.

Animals↗
PubMed1993

Interleukin-8 activates microtubule-associated protein 2 kinase (ERK1) in human neutrophils.

The signal transduction initiated by the human cytokine interleukin-8 (IL-8), the main chemotactic cytokine for neutrophils, was investigated and found to encompass the stimulation of protein kinases. More specifically, IL-8 caused a transient, dose and time dependent activation of a Ser/Thr kinase activity towards myelin basic protein (MBP) and the MBP-derived peptide APRTPGGRR patterned after the specific concensus sequence in MBP for ERK enzymes. The activated MBP kinase was furthermore identified as an extracellular signal regulated kinase (ERK1) based on several criteria such as substrate specificity, molecular weight, activation-dependent mobility shift, and recognition by anti-ERK antibodies. For comparison, the chemotactic response of neutrophils to a stimulus of bacterial origin (fMet-Leu-Phe or fMLP) was also examined and found to involve the activation of a similar ERK enzyme. The present data clearly indicate that in terminally differentiated, non-proliferating human cells, the MBP kinase/ERK activity can serve other purposes than mitogenic signaling, and that processes such as chemotaxis, induced by bacterial peptides as well as by human cytokines like IL-8, involve the regulation of ERK enzymes.

Amino Acid Sequence↗
PubMed1993

Bacterial expression of a single-chain antibody fragment (SCFV) that neutralizes the biological activity of human interferon-gamma.

Studies with animal models have indicated that neutralizing antibodies against human interferon-gamma (HuIFN-gamma) may be used to treat a number of diseases in man. A major handicap for the implementation of this form of therapy is the immunogenicity of antibodies of non-human origin. Antibody fragments that do not contain parts of the most immunogenic regions may help to circumvent this problem. Therefore, we have constructed several antibody fragments [VH (variable fragment of the heavy chain), Fv (variable fragment) and scFv (single-chain Fv)] derived from a murine hybridoma (D9D10) which produces a neutralizing antibody against HuIFN-gamma. cDNAs encoding the variable domains of the L and H chains of D9D10 were cloned by PCR-based techniques in a suitable E. coli expression system. Bacterial clones are described that produce either VH, Fv or scFv. The Ig fragments were secreted into the periplasm and leaked into the culture supernatant. By SDS-PAGE and immunoblot analysis, the fragments were shown to be of the expected size (15, 14 and 30 kDa for VH, Fv and scFv, respectively). Functionality of the recombinant Ig fragments was tested by an ELISA for HuIFN-gamma binding and by a neutralization assay for the antiviral activity of HuIFN-gamma. Fv as well as scFv, but not VH, were found to bind to HuIFN-gamma and to neutralize its antiviral activity. Since it is found that scFv proteins are more stable at physiological temperatures than the Fv, it may have potential usefulness for the treatment of diseases in which overproduction of IFN-gamma plays a crucial role.

Amino Acid Sequence↗
PubMed1993

Immunocytochemical evidence that S-100-positive cells of the mouse anterior pituitary contain interleukin-6 immunoreactivity.

We have previously shown that bioactive interleukin-6 (IL-6) is produced by rat and mouse (anterior) pituitary cells in vitro. Since the amount produced correlated with the presence of S-100-containing folliculostellate (FS) cells, these cells were suggested to be a source of IL-6 in the anterior pituitary (AP) lobe. In the present study we used immunocytochemical techniques to confirm this presumption. Freshly isolated mouse pituitary cells were subjected to immunocytochemical procedures whereby two different (neutralizing) monoclonal antibodies (MAb) against mouse IL-6 (6B4 and 20F3) and a polyclonal antiserum raised against bovine S-100 were used as primary antibodies. Single immunostaining revealed a small portion of mouse pituitary cells (about 6.5%) to be positive for IL-6 immunoreactivity with both antibodies. Importantly, the same proportion of cells was found to be IL-6 positive if only the AP was used as the cell source. About 7.5% of the pituitary cells stained for the presence of S-100 immunoreactivity. Positive staining for IL-6 was also found in pituitary cell samples from 2-day-old monolayer cultures and from redispersed 9-day-old histotypic aggregates, which both secreted bioassayable IL-6. In contrast, no IL-6 staining was found in AtT-20 cells, an established ACTH-secreting tumor cell line of the mouse pituitary which did not secrete bioactive IL-6. The specificity of the IL-6 immunostaining was demonstrated by a total loss of staining when MAb 6B4 was omitted or replaced by irrelevant rat IgG. Conclusively, pre-adsorption of the anti-IL-6 MAb (6B4) with recombinant mouse IL-6 totally abolished staining of pituitary cells. Double immunostaining for IL-6 and S-100 revealed that most if not all of the IL-6-containing pituitary cells were positive for S-100. Few of the S-100-containing cells did not stain for IL-6. These results confirm our previous hypothesis that FS cells, characterized by immunostaining of S-100 protein, contain bioactive and immunoreactive IL-6 and therefore are very likely producers of IL-6 in the AP. Furthermore, our results suggest that IL-6 is implicated in the local regulatory role ascribed to FS cells in the pituitary gland.

Animals↗
PubMed1993

[The pyrogenic activity of native and recombinant human interleukin-1 beta].

Comparative doses (100-180 ng/kg) of highly purified human native interleukin-1 beta (nIL-1 beta) and human recombinant IL-1 beta (rIL-1 beta) intravenously injected were found to cause similar changes in body temperature in rabbits. Under these conditions, stabilization of rIL-1 beta by human serum albumin (HSA) fails to affect rIL-1 beta pyrogenic activity. nIL-1 beta, 0.05-2.0 ng, injected into the PO/AH region of the brain causes dose-dependent fever in the animals. With intrahypothalamic nIL-1 beta (versus i.v. injection), the pyrogenic activity of rIl-1 beta is much lower than that of nIL-1 beta. Moreover, pyrogenicity appears to be dependent on the type of rIL-1 beta, namely on free or stabilized by HSA. The former has about 100-fold and the latter a 25-fold lower activity than the native cytokine (in terms of a dose-pyrogenic effect relationship). The findings are discussed in the light of the existence of various interleukin IL-1 beta pools.

Animals↗
PubMed1993

Synergistic toxicity of IFN-gamma-producing Escherichia coli K12 cells.

Genetically modified microorganisms (GMMs) are frequently used as producers of mammalian immunomodulatory proteins, e.g. interferons and interleukins. Here we have examined the question of whether such GMMs interact in a way different from that of their non-modified parent micro-organisms with mammalian antimicrobial defence systems. As a typical GMM host micro-organism we used Escherichia coli K12, and as a typical immunomodulatory protein produced by a GMM we used mouse interferon-gamma (MuIFN-gamma). Two experimental systems are described in which synergistic "toxic" biological effects are induced by a combined treatment with E. coli and MuIFN-gamma but not, or less so, by the parental strain and the recombinant protein separately. First, it is shown that the IFN-gamma-producing GMM, or mixtures of E. coli cells and IFN-gamma, are cytolytic for mouse embryo fibroblastoid cells (MEF), whereas no cell killing occurs in MEF cultures treated with control E. coli cells or in those treated with bacteria-free recombinant IFN-gamma. Second, it is demonstrated that intraperitoneal injection in mice of high but not low numbers of control E. coli K12 cells induces a shock-like mortality, whereas co-injection with IFN-gamma induces killing at low numbers. IFN-gamma-producing E. coli cells cause a mortality rate that does not differ from that of control E. coli cells, probably because in these experimental conditions the level of recombinant MuIFN-gamma per cell is insufficiently high. Taken together, these data indicate that synergistic toxic effects induced by bacteria and their recombinant products can occur and may in certain situations enhance the intrinsic toxic capacity of the GMM. Synergistic toxic effects may thus be of relevance for identifying the safety level that should be employed when working with GMMs.

Animals↗
PubMed1992

Protective effect of anti-interleukin (IL)-6 antibody against endotoxin, associated with paradoxically increased IL-6 levels.

Two rat monoclonal antibodies (6B4 and 20F3) against mouse interleukin (IL)-6 were studied for their effects on the generalized Shwartzman reaction and on cytokine production elicited by endotoxin injections. Both antibodies were found to protect mice against the generalized Shwartzman reaction. Production of interferon and tumor necrosis factor in these animals, as assessed from serum levels, were not consistently affected by the antibody treatment, although rather increased levels were occasionally noted. Paradoxically, however, endotoxin-induced serum levels of IL-6 in anti-IL-6-treated mice were consistently found to be markedly increased and also to persist for longer time periods. The more vigorous and persistent response may have been due to slower elimination, increased synthesis, or a combination of both. Endogenous production of IL-6 in mice may be sufficiently large to supersede the neutralizing potential of an excess of antibody, as was evident from the fact that ascites fluid of the anti-IL-6-producing 6B4 hybridoma was biologically active in the IL-6 assay.

Animals↗