Monoclonal antibodies reveal a weak interaction between the F18 fimbrial adhesin FedF and the major subunit FedA.

F18+ Escherichia coli can cause post-weaning diarrhoea and oedema disease in pigs. These diseases are responsible for substantial economic losses, but a vaccine is not available. A good knowledge of the characteristic of the fimbriae is useful for the development of a vaccine composed of the fimbrial virulence factor. F18 fimbriae are composed of the major subunit FedA and the minor subunits FedE and the adhesin FedF. In the present study monoclonal antibodies (mAbs) against FedA and FedF were produced. In addition to their diagnostic value, these mAbs revealed a weaker interaction between FedA and FedF compared to the subunit-subunit interactions in other fimbriae, like type 1 and P pili. Further experiments are needed to investigate if this weak interaction could be one of the reasons for the slow colonisation of the small intestinal mucosa by F18+ E. coli.

Elucidation of the molecular mechanisms of two nanobodies that inhibit thrombin-activatable fibrinolysis inhibitor activation and activated thrombin-activatable fibrinolysis inhibitor activity.

UNLABELLED: Essentials Thrombin-activatable fibrinolysis inhibitor (TAFI) is a risk factor for cardiovascular disorders. TAFI inhibitory nanobodies represent a promising step in developing profibrinolytic therapeutics. We have solved three crystal structures of TAFI in complex with inhibitory nanobodies. Nanobodies inhibit TAFI through distinct mechanisms and represent novel profibrinolytic leads. SUMMARY: Background Thrombin-activatable fibrinolysis inhibitor (TAFI) is converted to activated TAFI (TAFIa) by thrombin, plasmin, or the thrombin-thrombomodulin complex (T/TM). TAFIa is antifibrinolytic, and high levels of TAFIa are associated with an increased risk for cardiovascular disorders. TAFI-inhibitory nanobodies represent a promising approach for developing profibrinolytic therapeutics. Objective To elucidate the molecular mechanisms of inhibition of TAFI activation and TAFIa activity by nanobodies with the use of X-ray crystallography and biochemical characterization. Methods and results We selected two nanobodies for cocrystallization with TAFI. VHH-a204 interferes with all TAFI activation modes, whereas VHH-i83 interferes with T/TM-mediated activation and also inhibits TAFIa activity. The 3.05-Å-resolution crystal structure of TAFI-VHH-a204 reveals that the VHH-a204 epitope is localized to the catalytic moiety (CM) in close proximity to the TAFI activation site at Arg92, indicating that VHH-a204 inhibits TAFI activation by steric hindrance. The 2.85-Å-resolution crystal structure of TAFI-VHH-i83 reveals that the VHH-i83 epitope is located close to the presumptive thrombomodulin-binding site in the activation peptide (AP). The structure and supporting biochemical assays suggest that VHH-i83 inhibits TAFIa by bridging the AP to the CM following TAFI activation. In addition, the 3.00-Å-resolution crystal structure of the triple TAFI-VHH-a204-VHH-i83 complex demonstrates that the two nanobodies can simultaneously bind to TAFI. Conclusions This study provides detailed insights into the molecular mechanisms of TAFI inhibition, and reveals a novel mode of TAFIa inhibition. VHH-a204 and VHH-i83 merit further evaluation as potential profibrinolytic therapeutics.

Inhibition of and sensitization to the lethal effects of tumor necrosis factor.

The extension of the positive results obtained with tumor necrosis factor (TNF) in the locoregional treatment of cancer to systemic treatments requires the selective inhibition of its shock-inducing properties. In this paper, recent data regarding the mechanisms by which infections and tumors render mice extremely sensitive to the lethal effects of TNF as well as regarding the inhibition of the dose-limiting toxicities, hypotension and hepatotoxicity, are summarized. An interleukin-12 (IL-12) driven induction of interferon-gamma (IFN-gamma), probably in synergism with endogenous TNF, was found to mediate infection-induced sensitization. The sensitization induced by tumors develops independent of the IL-12/IFN-gamma axis but ultimately leads to a common step, which can be inhibited by alpha-CD11a and is specific for sensitization. Hypotension can be inhibited by methylene blue (MB), an inhibitor of the nitric oxide (NO)-induced activation of the cytosolic guanylate cyclase, without the indispensable protective properties of NO being affected. Finally, two acute phase proteins, alpha 1-acid-glycoprotein (AGP) and alpha 1-antitrypsin (AT), were able to protect against the TNF-induced liver failure. None of these three inhibitors seems to affect the antitumor effects of TNF.

Differential response of a(2)-macroglobulin-deficient mice in models of lethal TNF-induced inflammation.

Tumor necrosis factor (TNF) is an essential mediator in the pathogenesis of Gram-negative septic shock. Injection of TNF into normal mice leads to systemic, lethal inflammation, which is indistinguishable from lipopolysaccharide (LPS)-induced lethal inflammation. alpha(2)-macroglobulin (A2M) is a major positive acute phase protein with broad-spectrum protease-inhibitory activity. Mouse A2M-deficient (MAM-/-) mice were significantly protected against lethal systemic inflammation induced by TNF. The protection is not due to faster clearance of the injected TNF. The induction of tolerance to TNF-induced lethality by repetitive administration of small doses of human TNF for five consecutive days was equally efficient in both mutant mice compared to wild-type mice. In D-(+)-galactosamine (GalN)-sensitized mice, TNF induces lethal inflammatory hepatitis. MAM(-/-) mice are equally sensitive to the lethal combination of TNF/GalN. Furthermore, interleukin-1-induced desensitization to TNF/GalN was not impaired in MAM(-/-) mice. We conclude that MAM plays a mediating role in TNF-induced lethal shock and that MAM deficiency does not reduce changes in efficiency of tolerance and desensitization to TNF and TNF/GalN-induced lethality, respectively.

Tumor necrosis factor-induced cytotoxicity is not related to rates of mitochondrial morphological abnormalities or autophagy-changes that can be mediated by TNFR-I or TNFR-II.

Tumor necrosis factor (TNF) may cause apoptosis or necrosis and induces mitochondrial changes that have been proposed to be central to cytotoxicity. We report similar patterns of TNF-induced mitochondrial morphological alterations and autophagy in cell types with differing sensitivity to TNF-induced cytotoxicity. Specific ligation of TNFR-I or TNFR-II induces different rates of apoptosis and mitochondrial morphological change, but similar rates of autophagy. These changes do not invariably lead to cell death, and survival or progression to apoptosis or necrosis following TNF exposure may depend in part on the extent of mitochondrial damage and/or the autophagic capacity of the cell.

Scanning electron microscopy of the extracellular matrices of rat molar tooth germs in organ culture in vitro.

Second upper molars from 3-day postnatal rats were cultured for 2 weeks and compared with in-vivo specimens from 7-day postnatal rats. Several preparatory techniques were applied to expose the extracellular matrices, the three-dimensional structure of which were examined by SEM. The combination of phosphate-buffered saline and ultrasonics as preparation for the observation of the enamel, hypochlorite treatment to study the predentine, the freeze-fracture technique for the dentinal tubules and oxygen-plasma-ashing for the mineralization front of dentine gave best results. Enamel formed in vitro was prismatic similar to in vivo. The fissures were devoid of enamel and the enamel-free areas at the cusp tips were larger than in vivo. In the cervical area in vitro, the enamel stopped abruptly instead of gradually decreasing. The predentine and the dentine were normal in structure.

Light and transmission electron microscopy of the effects of calcium, magnesium and phosphate on dentine and enamel formed by rat molars in vitro.

The effect of addition of eight different combinations of Ca, Mg and P supplements (control, Ca, Mg, P, CaMg, CaP, PMg and CaMgP) on three-day-old rat maxillary second molars, explanted at the premineralizing stage and cultured for two weeks, was studied. Light-microscopy sections, cut parallel to the occlusal plane, were divided into four sectors and given a score according to an ordinal scale for dentine and enamel depending on the regularity of these matrices. An analysis of variance on these scores revealed a significant favourable effect of Mg, CaMg and CaMgP and an adverse effect of Ca on enamel. A favourable effect on dentine regularity was obtained after addition of Ca or Mg. Ultrastructurally, enamel changes such as amorphous enamel matrix, voids and disturbance in rod-interrod pattern were seen after addition of Ca, P, CaP. Thin enamel with less tight packing of crystals was observed after CaMg addition. A thick layer of enamel with highly-organized rod-interrod pattern was seen with Mg, PMg and CaMgP addition. It is suggested that Mg plays an important role in the interaction with Ca and P for the harmonious development of enamel and dentine in vitro.

Effect of different combinations of calcium, magnesium and phosphate on the inorganic composition of rat molars in vitro.

Second upper molars from 3-day-old rats were cultured by the Trowell method for 14 days. One of each pair of molars was kept as an uncultured control; the other was cultured. Explants were exposed to eight different combinations of Ca, Mg and P additions to BGJb medium. This resulted in eight groups of explants (control, Ca, Mg, P, CaMg, CaP, PMg and CaMgP) and their eight uncultured contralateral groups. The additions were calculated to double the original measured media concentration. Cultured and uncultured germs were analysed for dry weight (D), ash weight (A), Ca, Mg and P content. The organic fraction (D-A) was calculated. The analysis of covariance by means of multiple regression revealed that Ca-addition to the culture medium stimulated D, A, Ca and P in the explants; P-addition was stimulatory for D, A, D-A and P whereas Mg addition was inhibitory for A, D-A and Ca. A positive interaction for all the tooth-germ variables was demonstrated after CaMg addition; an antagonistic effect was found for the tooth-germ variables D, A, Ca and P after CaP addition. The value of the tooth-germ variables at the time of explantation (covariate) had no significant effect on the value for the variables of the explants (except on their P content). The highest absolute values for all the variables were obtained after CaMg and CaMgP additions. Furthermore, taking into consideration morphological results, the addition of CaMgP can be recommended as medium supplement in the organ culture of rat tooth germs.

Localization of lead and fluoride in cultured tooth germs by laser microprobe mass analysis.

Trace elements can influence dental health, possibly by altering tooth resistance during preeruptive development. Therefore, it was investigated whether lead and fluoride would be incorporated into the calcifying matrices or the cellular parts of tooth germs in vitro. Using laser microprobe mass analysis, the localization of lead and fluoride was studied in the different layers or tooth germs that had been cultured in a medium to which PbCl2 of NaF had been added in different concentrations. Both elements could only be detected in the dentine layer. Hence, the enamel organ in the secretory stage of tooth development excludes lead and fluoride from the enamel, even when enamel formation by the ameloblasts is visibly disturbed. Furthermore, there seemed to be a process of saturation in the accumulation of lead and fluoride in the dentine.

Characterization of sialyltransferase mutants using surface plasmon resonance.

Sialyltransferases are enzymes responsible for the important sialylation of glycoconjugates. Since crystal structures are not available, other tools are needed to study enzymatic mechanisms. As a model, we used human alpha2,6-sialyltransferase. A putative acceptor-binding domain containing the small and the very small sialyl motifs was randomly mutated. This resulted in enzymes with altered enzymatic activity. Affinity chromatography demonstrated that their binding to donor substrate was maintained. To illustrate the role of the mutated domain in acceptor binding, a method based on surface plasmon resonance was set up. Only at low salt and high acceptor concentration was association of wild-type ST6GalI with asialofetuin demonstrated. As expected, this interaction was affected by cytidine 5'-monophospho-N-acetylneuraminic acid, the donor substrate, which proves the specificity of the interaction. Different types of mutants were found. For some, the drop in activity could be explained by loss in affinity for the acceptor. For others, the catalytic center, but not the acceptor-binding site, was affected. Neither acceptor binding nor catalytic activity were limited to the sialyl motifs. To our knowledge, this is the first example in which surface plasmon resonance is successfully used to demonstrate the binding of a glycosyltransferase to its natural acceptor.

Effects of pulp removal and organ culture on adult rat incisor odontoblasts.

The morphological integrity of adult rat incisor odontoblasts was studied to determine the effects of pulp removal and subsequent incubation. From each pair of maxillary incisors one was left intact while the pulp from the other one was removed. Both series were incubated in BGJb medium according to the Trowell method. Morphometrical measurements of the thickness of the odontoblast layer and the size and shape of the odontoblast nuclei were performed on photomicrographs of transverse sections of the incisors. After pulp removal the odontoblasts seemed disorganized and their nuclei were darker and more rounded. During incubation the odontoblasts did not recover from the effect of this pulp removal and degenerative changes were frequently seen. In the culture of the complete tooth organ the morphological integrity of the odontoblasts was much better preserved. Therefore the organ culture of intact teeth seems more favourable.

Expression in plants of the cloned satellite tobacco necrosis virus genome and of derived insertion mutants.

Mechanical inoculation of cowpea leaves with cloned full-size copies of satellite tobacco necrosis virus in the presence of tobacco necrosis (helper) virus resulted in the appearance of infectious virus particles. This could be clearly demonstrated by the detection of structural changes in the progeny viral RNA after inoculation with helper virus and hybrid plasmids containing an STNV-DNA copy with small (14-mer) oligodeoxynucleotides inserted at different sites in either the coding or the non-coding region. Furthermore, although orientation and position of the viral cDNA in the different plasmids did not seem to be of major importance for the ensuing infection process, the presence of adjacent complementary homopolymeric regions (dG/dC) at both ends of the STNV nucleotide sequence was necessary. Progeny STNV was obtained which carried oligonucleotide insertions at various positions of the genome.

Controlled synthesis of the coat protein of satellite tobacco necrosis virus in Escherichia coli.

Chimeric plasmids were constructed such that the cloned complete satellite tobacco necrosis virus (STNV) RNA information came under transcriptional control of the leftward promoter (PL) of bacteriophage lambda. The promoter is fully repressed at low temperatures (28 degrees) by the thermolabile repressor product of the lambdacI857 gene, present in the bacterium on a deficient prophage or as part of another plasmid. Synthesis of the STNV coat protein in Escherichia coli could be initiated by heat induction (42 degrees). These in vivo results confirm that the 5'-untranslated region of STNV RNA, preceding the initiating AUG, provides adequate genetic information for efficient translation in the bacterial system. However, fast degradation of the bacterially synthesized STNV coat protein was observed. The use of a protease-deficient strain reduced this breakdown considerably.

Characteristics of mineralization of rat molar tooth germs in organ culture.

The behaviour of first (M1) and second (M2) upper molars of 3-day-old rats was compared histologically and by measuring several variables (dry and ash weight, Ca, P and Mg content) on cultured teeth and contralateral control teeth (not cultured). New information was gained by additional computations and calculating correlation coefficients between the variables of the control and cultured molars separately and combined. The M2 seems to perform better in culture than the M1. The M2 model revealed possibilities for further standardization.

An ultrastructural study of dentinogenesis and amelogenesis in rat molar tooth germs cultured in vitro.

Molar tooth germs from three-day-old rats were cultured successfully for fourteen days, permitting the study of the development in vitro of both extracellular matrix and cellular elements such as odontoblasts and ameloblasts. The ultrastructure of the cultured tooth germs was compared with the ultrastructure of tooth germs in vivo at a comparable developmental stage. Progenitor cells of odontoblasts and ameloblasts were found to differentiate in vitro. Odontoblasts seemed to contain more lysosome-like bodies and fewer secretory granules than in vivo. They formed normally mineralizing dentine or a thick layer of dense, unmineralized predentine with incidentally some amorphous, extracellular material. Enamel was exclusively present opposite well developed dentine. It was often hyper- or hypomineralized and enamel rods were not as regularly shaped as in vivo. In places where no enamel formation had taken place, large amounts of amorphous extracellular material were sometimes seen. From these observations it can be concluded that cellular development in cultured tooth germs appeared more or less normal, but extracellular matrix formation and mineralization were sometimes disturbed.

Identification of tumor necrosis factor (TNF) amino acids crucial for binding to the murine p75 TNF receptor and construction of receptor-selective mutants.

The bioactivity of tumor necrosis factor (TNF) is mediated by two TNF receptors (TNF-Rs), more particularly TNF-RI and TNF-RII. Although human TNF (hTNF) and murine TNF (mTNF) are very homologous, hTNF binds only to mTNF-RI. By measuring the binding of a panel of mTNF/hTNF chimeras to both mTNF-R, we pinpointed the TNF region that mediates the interaction with mTNF-RII. Using site-specific mutagenesis, we identified amino acids 71-73 and 89 as the main interacting residues. Mutein hTNF-S71D/T72Y/H73 Delta/T89E interacts with both types of mTNF-R and is active in CT6 cell proliferation assays mediated by mTNF-RII. Mutein mTNF-D71S/Y72T/Delta 73H/E89T binds to mTNF-RI only and is no longer active on CT6 cells. However, the L929s cytotoxicity of this mutein (an effect mediated by mTNF-RI triggering) was also 100-fold lower than that of wild-type mTNF due to enhanced dissociation during incubation at subnanomolar concentrations. The additional mutation of amino acid 102, resulting in the mutein mTNF-D71S/Y72T/Delta 73H/E89T/P102Q, restored the trimer stability, which led to an enhanced specific activity on L929s cells. Hence the specific activity of a TNF species is governed not only by its receptor binding characteristics but also by its trimer stability after incubation at subnanomolar concentrations. In conclusion, the mutation of TNF amino acids 71-73, 89, and 102 is sufficient to obtain a mTNF mutein selective for mTNF-RI and a hTNF mutein that, unlike wild-type hTNF, also acts on mTNF-RII.

Heterotrimers formed by tumor necrosis factors of different species or muteins.

Incubation of murine tumor necrosis factor (mTNF) at subnanomolar concentrations results in partial dissociation of the trimers, coinciding with a decrease in bioactivity. Using size-exclusion chromatography, we observed that the conversion of labeled mTNF to monomers is not only prevented by coincubation with an excess of unlabeled mTNF but also with unlabeled human TNF (hTNF). Moreover, after coincubation of mTNF and hTNF four different TNF complexes were revealed by native polyacrylamide gel electrophoresis, viz. homotrimeric mTNF and hTNF, as well as two complexes with an intermediate migration pattern. Analytical gel filtration in combination with native polyacrylamide gel electrophoresis and Western blot immunodetection indicated that these new complexes consisted of heterotrimeric TNF molecules. We conclude that an exchange of monomers takes place during coincubation of two different species of TNF, which results in homotrimeric and heterotrimeric TNF. To assess receptor interaction in vitro, TNF heterotrimeric molecules were used as obtained after incubation of mTNF with labeled hTNF (which only binds to mTNF receptor I) or with labeled mutein mTNF75 (specific for mTNF receptor II). These heterotrimers were retained by both mTNF receptors, which means that the mTNF subunits incorporated in heterotrimeric complexes still can bind to both types of TNF receptor. In addition, the gradual decrease in mTNF bioactivity during preincubation at subnanomolar concentrations was prevented by the presence of mutein mTNF75, which is inactive in an L929 cytotoxicity assay, indicating that heterotrimerization can influence the overall bioactivity.

Receptor-selective mutants of tumour necrosis factor in the therapy of cancer: preclinical studies.

The use of TNF-mutants that are selective agonists of the TNF-R55 is one strategy that is being explored to broaden the therapeutic margin of TNF. Several problems still have to be overcome before they can be used in clinical trials. Regarding the sensitizing effect of some infections and some tumours, we identified IFN-gamma as a mediator in BCG- but not in tumour-induced sensitization. In both models, the vessel wall is most probably the key tissue as alpha-LFA-1 antibodies could protect against lethality. Studies in primates showed that an unexpected feature, namely, the longer half-life of such mutants, might interfere with this strategy. Recent observations also indicate that the mechanism of tolerance-induction, another way to separate antitumour and toxic effects of TNF, might reside in the functional ablation of the TNF-R75. Using IL-60/0 knockout mice, we could not find any causal role for IL-6 in TNF-mediated lethality, this in contrast to results obtained previously with neutralizing antibodies. Finally, we identified the acute phase protein alpha 1-acid glycoprotein as a protein with protective properties towards TNF-induced lethality and liver damage.