Development and application of pathovar-specific monoclonal antibodies that recognize the lipopolysaccharide O antigen and the type IV fimbriae of Xanthomonas hyacinthi.

The objective of this study was to develop a specific immunological diagnostic assay for yellow disease in hyacinths, using monoclonal antibodies (MAbs). Mice were immunized with a crude cell wall preparation (shear fraction) from Xanthomonas hyacinthi and with purified type IV fimbriae. Hybridomas were screened for a positive reaction with X. hyacinthi cells or fimbriae and for a negative reaction with X. translucens pv. graminis or Erwinia carotovora subsp. carotovora. Nine MAbs recognized fimbrial epitopes, as shown by immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy; however, three of these MAbs had weak cross-reactions with two X. translucens pathovars in immunoblotting experiments. Seven MAbs reacted with lipopolysaccharides and yielded a low-mobility ladder pattern on immunoblots. Subsequent analysis of MAb 2E5 showed that it specifically recognized an epitope on the O antigen, which was found to consist of rhamnose and fucose in a 2:1 molar ratio. The cross-reaction of MAb 2E5 with all X. hyacinthi strains tested showed that this O antigen is highly conserved within this species. MAb 1B10 also reacted with lipopolysaccharides. MAbs 2E5 and 1B10 were further tested in ELISA and immunoblotting experiments with cells and extracts from other pathogens. No cross-reaction was found with 27 other Xanthomonas pathovars tested or with 14 other bacterial species from other genera, such as Erwinia and Pseudomonas, indicating the high specificity of these antibodies. MAbs 2E5 and 1B10 were shown to be useful in ELISA for the detection of X. hyacinthi in infected hyacinths.

Partial characterization of fimbriae of Xanthomonas campestris pv. hyacinthi.

Xanthomonas campestris pv. hyacinthi is a plant-pathogenic bacterium that causes yellow disease in Hyacinthus. X. c. pv.hyacinthi produces monopolarly attached fimbriae with a diameter of approximately 5 nm and a length of at least 6 micron. Fimbriae were purified by acid precipitation and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No hemagglutinating activity of purified fimbriae was found when the fimbriae were tested with several types of erythrocytes. The fimbrial protein subunit had a relative molecular mass of 17 kDa; an isoelectric point was found at pH 4.1. Analysis of the N-terminal amino acid sequence of the fimbrial subunit indicated that X. c. pv. hyacinthi expresses type 4 fimbriae. A polyclonal rabbit antiserum was raised against the purified fimbriae. This antiserum recognized fimbriae of X. c. pv. hyacinthi in immunogold electron microscopy and immunoblotting experiments. Immunofluorescence studies showed that X. c. pv. hyacinthi cells as well as purified native fimbriae were attached to stomata of hyacinth leaves, suggesting a role for these surface antigens in the first stages of yellow disease.

Monoclonal antibodies against rat glutathione S-transferase isoenzymes 2-2 and 3-3.

Monoclonal antibodies were prepared against rat glutathione S-transferase isoenzymes 2-2 and 3-3. Three clones were capable of specifically differentiating their respective antigens from other rat isoenzymes as well as human isoenzymes, in ELISA and on Western blot. One clone produced antibodies specific for isoenzyme 2-2, and 2 hybridomas were specific for isoenzyme 3-3. Balb/c mice did not respond to immunization with glutathione S-transferase isoenzymes 1-1 and 4-4. However, an immune response was obtained in some other strains of mice, with differential H-2 haplotypes, notably CBA/BrARij mice and CBA/CaHRij-T6 mice for isoenzyme 1-1 and CBA/BrARij mice for isoenzyme 4-4, which offers perspectives for obtaining additional specific monoclonal antibodies against these glutathione S-transferases.

Different roles for calcium and cyclic AMP in the action of PTH: studies in bone explants and isolated bone cells.

In fetal mouse calvaria forskolin (0.1-100 microM), like PTH, stimulated cyclic AMP production in a dose-dependent way. The dose-response curve for forskolin-induced bone mineral release (24 hrs), however, demonstrated a biphasic character, showing stimulation at 0.1 microM and inhibition at 5 and 10 microM. In addition, forskolin-stimulated bone resorption reached a plateau after 48 hrs of incubation, a phenomenon which did not occur with PTH. Forskolin (0.1 microM) strongly stimulated PTH-induced cyclic AMP production in fetal mouse calvaria. However, PTH-stimulated bone resorption and PTH-induced increase in cytosolic free Ca2+ in bone fetal rat cells were not stimulated by forskolin (0.1 microM). 9-(Tetrahydro-2-furyl) adenine (100 microM) completely blunted PTH-stimulated cyclic AMP response in fetal mouse calvaria. PTH-stimulated bone resorption was also completely inhibited, but only after 6 hrs and not after 24 hrs of incubation. With nifedipine and varabamil PTH-stimulated bone resorption was significantly inhibited after 24 hrs of incubation and not significantly after 6 hrs of incubation. A23187 (1 microM) significantly stimulated PTH-stimulated cyclic AMP level and increased basal cytosolic free Ca2+ concentration in cultured rat bone cells. In calvaria, however, it had no effect on either basal and PTH-stimulated cyclic AMP production or on basal and PTH-stimulated bone resorption (6 and 24 hrs). From these observations it follows that in calvaria manipulation of intracellular cyclic AMP only (partially) affects bone resorption. This observation points to a role for an additional second messenger in establishing full blown bone resorption. Some of the results are published in short elsewhere.

Enhancement of the inhibitory action of APD on the transformation of osteoclast precursors into resorbing cells after dimethylation of the amino group.

The amino-bisphosphonate APD is distinct from the bisphosphonates EHDP and Cl2MDP by a greater molar potency in vivo as inhibitor of osteoclastic bone resorption and in vitro by a pronounced inhibitory effect on the accession of osteoclast precursors to mineralized matrix. Dimethylation of the aminogroup, which increases the basic properties of this residue but precludes others, like the liability to glucuronidation or acetylation, increased the in vivo potency of this amino bisphosphonate, as well as its in vitro specificity for osteoclast-precursor accession, but decreased its cellular toxicity. The in vitro actions of dimethyl-APD were reversible with administration of PTH. It is concluded that the introduction of a basic residue in bisphosphonates may increase affinity for the specific sites on the mineralized matrix that are involved in directing the accession of precursors and their transformation into actively resorbing osteoclasts.

Two modes of action of bisphosphonates on osteoclastic resorption of mineralized matrix.

Pretreatment of a long bone explant with P-C-P can prevent the osteoclastic resorption of its mineralized matrix, when it is entirely dependent upon activation and accession of extra-osseous osteoclast precursors. When treatment of the explant is postponed until after the development of mature osteoclasts, the P-C-P dose required for an inhibitory effect is increased 100-fold for the amino bisphosphonate APD, but not for EHDP and Cl2MDP. It is concluded that high doses of all P-C-Ps inhibit the resorbing osteoclast, but that low dose of the amino P-C-P can specifically inhibit the accession of osteoclast precursors to mineralized matrix. Both actions require P-C-P binding to the mineral. The relative potencies of the P-C-Ps in the precursor-dependent system correspond to their relative potencies in vivo. This suggests that inhibition of accession underlies the high potency which the aminobisphosphonate has in vivo.

Osteoblast and osteoclast precursors in primary cultures of calvarial bone cells.

Bone cells obtained by digestion of fetal mouse or chicken calvaria were tested for their ability to form or resorb bone in vitro. The isolated cells were precultured for 6 days and subsequently cocultured for 11 days with periosteum-free noninvaded fetal mouse long bone rudiments. Bone formation and resorption during coculture were evaluated by histology and 45Ca release from prelabeled bones. The calvarial origin of cells in cocultures was traced by labeling the cells with 3H-thymidine before coculture, followed by autoradiography. Many osteoblasts and osteoclasts as well as fibroblasts developed from mouse periosteal cells released late in the sequential digestion procedure and previously denoted as "osteoblastlike" (BL). No or few osteoblasts and osteoclasts but many fibroblasts developed from early released cell fractions that have previously been denoted as "osteoclastlike" (CL). Only osteoblasts and fibroblasts but not osteoclasts developed from chicken calvarial cell fractions. The osteoblasts developed primarily from cell fractions from the inner layer of the periosteum, previously denoted as "osteoblastlike" (OB). Cells obtained from the outer layer of the periosteum (PF) gave rise mainly to fibroblasts. These studies show that osteoblast and osteoclast precursor cells are maintained in monolayer cultures of periosteal cell fractions. However, sequential digestion of mouse calvaria does not lead to separation of the two types of bone cells. Rather, osteoclast and osteoblast precursors are released jointly, from the periosteal cell layers closest to the bone surface. In the chicken cell fractions osteoclast precursors are absent after preculture, resulting in a more homogeneous population of osteoblast and fibroblast but not osteoclast precursors.

[The use of monoclonal antibodies in plant production]. / Toepassing van monoclonale antilichamen in de plantaardige produktie.

Due to the high specificity of monoclonal antibodies (MCAs) the most important application of MCAs in the plant production may be diagnosis. Specific MCAs have been produced against plant pathogens including viruses, bacteria, fungi and nematodes and against plant substances. In general, however, MCAs are still less suitable than polyclonal antisera for application as diagnostic tools in plant extracts.

The effect of calcium and 3',5' cAMP on the induction of ornithine decarboxylase activity in bone and bone cells.

The interrelationship between mediators of hormone action (cAMP and calcium) and induction of ornithine decarboxylase (ODC) activity was investigated in bone and bone cells. Stimuli that enhanced the cAMP level in both osteoblastlike (BL) and osteoclastlike (CL) cells and in intact calvaria only induced ODC activity in the isolated cell populations. Moreover, addition of fresh medium induced ODC activity in BL cells and not in calvaria, without an effect on the cAMP level. The tumor-promoting agent 12-O-tetradecanoyl-phorbol-13-acetate (TPA), however, induced in both systems ODC activity, without an effect on the cAMP concentration. From these data it was concluded that induction of ODC activity may not be mediated by cAMP. The role of Ca in ODC induction was also investigated. Experiments with BL cells, incubated in media with various Ca concentrations, as well as experiments with the Ca blocker D600 showed that basal and hormone-induced ODC activity was dependent on the intracellular Ca concentration. A regulatory role of cAMP, however, in concert with Ca, cannot be excluded.

Bone formation by isolated calvarial osteoblasts in syngeneic and allogeneic transplants: light microscopic observations.

Taking advantage of recently developed methods for osteoblast isolation, we used these cells to study bone morphogenesis in syngeneic and allogeneic intramuscular transplants. Syngeneic osteoblasts from fetal rat calvaria produced small islands of bone by the third day after transplantation. These islands increased in size and began to fuse after about 14 days. At the surface of the woven bone laid down first, lamellar bone developed. The amount of this bone increased, and in 56-day-old transplants solid blocks of bone were present. Osteoclasts were scarce, and the woven bone remained unresorbed. Bone marrow was absent. The structure of bone in transplants differed from that of mature calvarial bones in which only remnants of woven bone remained and bone marrow was well developed. The scarcity of osteoclasts in transplants could be caused by their relative paucity among the injected cells, since these cells responded strongly to added parathyroid hormone by increased production of cyclic adenosine monophosphate (cAMP) but only weakly to calcitonin. Osteoblasts isolated from the surface of calvarial lamellar bone of 28-day-old rats formed woven bone similar to the bone formed by fetal cells. This suggests that the type of bone produced does not depend on the intrinsic properties of the osteoblasts. Bone formed in an allogeneic system was surrounded by infiltrations containing lymphocytes, macrophages, and osteoclastlike cells in 14-day-old transplants. Osteoblasts at the bone periphery were destroyed and bone matrix was resorbed by infiltrating cells. Numerous bone lacunae were enlarged, suggesting the occurrence of osteocytic osteolysis. Isolated osteoblasts cultured for three population doublings or longer did not form bone after transplantation, although they retained some reactivity toward parathyroid hormone.