Antibodies to human adhesion molecules and von Willebrand factor: In vitro cross-species reactivity in the xenotransplantation setting

Abstract: Endothelial cell activation is thought to play an important role in xenograft rejection through cell retraction and expression of pro-coagulant and pro-inflammatory factors. Identification of antibodies recognizing porcine endothelial molecules would be useful to study and manipulate the inflammatory response to a xenograft. The aim of this study was to investigate the cross-reactivity of antibodies directed against human adhesion molecules and von Willebrand factor (vWF). Binding of monoclonal antibodies (mAbs) directed against human CD31, CD44, CD49, CD54, CD62E, CD102, and CD106 was evaluated on resting and activated endothelial cells from human and pig by flow cytometry. Among 30 antibodies tested, 4 were shown to react with pig cells. Two of them, directed against human CD62E (E-selectin) and rabbit CD 106 (VCAM-1) reacted strongly with activated and/or resting pig cells, whereas two others, directed to human CD31 (PECAM) and CD44 (H-CAM), bound weakly to pig cells. In addition, we analyzed the cross-reactivity of five polyclonal or monoclonal antibodies to human or pig vWF with human, baboon, rhesus, pig, and rat vWF. Binding of antibodies was tested by ELISA by using platelet lysates as source of vWF from the different species. Four anti-human or porcine vWF antibodies exhibited a broad reactivity with vWF from all species, whereas one anti-human vWF antibody was specific for primate vWF. In this study, we identified a small number of cross-reacting antibodies that may prove useful to study in vitro and in vivo xenogeneic responses. However, the weak antibody cross-reactivity observed with most porcine molecules points out the necessity of producing species-specific antibodies to study the immune response to xenografts or for use as specific immunosuppressive therapeutic reagents.     

Development and validation of a Western immunoblot assay for detection of antibodies to porcine endogenous retrovirus

BACKGROUND: Reports that pig endogenous retrovirus (PERV) infects human cells in vitro have heightened the importance of molecular and serologic monitoring of xenograft recipients for evidence of infection with PERV. We report the development and validation of a PERV-specific Western immunoblot assay for the diagnostic testing of porcine xenografts recipients. This assay is based upon the serological cross-reactivity observed between PERV variants capable of infecting human cells in vitro and other mammalian C type retroviruses. METHODS AND RESULTS: Strong reactivity between PERV expressing embryonic pig kidney PK-15 cells and antisera raised against whole virus preparations of murine leukemia virus, gibbon ape leukemia virus (GALV), and simian sarcoma-associated virus was demonstrated by an immunofluorescence assay, suggesting specific antigenic cross-reactivity between this group of viruses and PERV. Western immunoblot analysis demonstrated that anti-GALV antisera reacted with three proteins in PK-15 cells having molecular masses of 30, 55, and 66 kDa. Antisera specific for the Gag proteins of either GALV or simian sarcoma-associated virus reacted with the 30-kDa (major) and 55-kDa (minor) proteins present in PK-15 cells and in PERV-infected 293 human kidney cells, likely representing reactivity to the processed and precursor forms of the PERV Gag protein, respectively. No reactivity was seen in uninfected 293 cells. Analysis of plasma samples from 200 United States blood donors and from 58 human immunodeficiency virus-1, 18 human immunodeficiency virus-2, 13 human T-cell lymphotrophic virus-I, 21 human T-cell lymphotrophic virus-II, and 15 cytomegalovirus infected controls were negative. CONCLUSIONS: As this assay is based on PERV antigen derived from infected human cells, it clearly has the capacity to detect a serologic response towards PERV variants that have zoonotic potential and will allow for the accurate determination of PERV-specific seroreactivity in porcine xenograft recipients.     

Functional recovery after simultaneous transplantation with neuro-epithelial stem cells and adjacent mesenchymal tissues into infarcted rat brain

Summary ?Background. Cerebral infarction results in impairment of motor and cognitive functions. We performed intracranial transplantation of multipotential neuro-epithelial stem cells with mesenchyme into experimentally large ischemic lesions to study their potential to relieve deficits. Methods. Wistar albino rats were subjected to transient middle cerebral artery occlusion for 60 minutes, producing an extensive ischemic lesion in the ipsilateral striatum and adjacent cerebral cortex. The rat mesencephalic neural plate at the early somite stage (embryonic day 10.5) together with adjacent ventral mesenchyme was used as donor material. We performed histological and immunohistochemical studies, with antibodies against tyrosine hydroxylase, and dopamine- and adenosine 3′: 5′-monophosphate-regulated phosphoprotein 32 (DARPP-32; a striatal marker). Micro-angiograms were made by using Microfil silicone rubber. Morris Water-maze learning and treadmill task were employed to evaluate motor and cognitive functions. Findings. A viable non-tumoral mass was recognized in the rat striatum, up to as long as 156 days after transplantation. There were many cells positive for tyrosine hydroxylase or DARPP-32 in the graft. Some of the DARPP-32 positive cells within the graft had extended their dendrites into host tissues. In the micro-angiograms, many fine vessels were observed within the graft and dilated vessels meandered around the graft. Transplanted animals recovered significantly better in motor and cognitive functions than animals injected with only culture medium. Interpretation. Neuro-epithelial stem cells may follow several lines of differentiation; along the naturally genetically programmed line of differentiation, or along other cell lines depending on different environments. Grafting of neuro-epithelial stem cells with mesenchyme may merit a further study as a treatment for cerebral infarction. Published online June 11, 2003     

Characterization and identification of a human dentin phosphophoryn

The present study further characterizes an extract from immature, human tooth apicies from which an intact dentin phosphoprotein has been identified. Third molar apicies from developing roots were decalcified in 10% EDTA until Ca²⁺ was undetectable in the decalcifying solution. The crude extract was run on 7.5% SDS-PAGE and stained with ``Stains-All.' Four distinct bands were found and the molecular weights were 140, 60, 50, and 34 k. When run on a SDS-PAGE under nonreducing conditions the 60, 50, and 34 k bands were absent. These results suggest that the lower molecular weight bands may be subunits of the larger protein. The extract was then further purified by adding CaCl₂ and MgCl₂ to precipitate the phosphoprotein. The precipitate was subjected to a DEAE-Sepharose CL6B column and eluted by 0–0.7 M NaCl gradient solution. The amino acid composition of the purified phosphoprotein was determined and the extract was found to be rich in serine and aspartic acid residues. The N-terminal peptide Asp-Asp-Pro was identified. The sequence of the three amino acids is identical to rat incisor phosphoprotein. Received: 19 October 1995 / Accepted: 23 February 1996     

Characterization of a murine monoclonal antibodv specific for swine β1 integrin

Abstract: Murine monoclonal antibodies were raised against porcine platelets in order to provide tools for investigating interactions of human blood cells and natural antibodies with porcine tissues. Hybridomas were screened by cellular ELISA on porcine platelets and endothelial cells. Positive clones were tested by flow cytometry for reactivity with isolated endothelial cells. One clone, NaM160–1A3, produced an antibody that stained porcine but not human endothelial cells and lymphocytes. The antibody bound to a 116 kDa glycoprotein on Western blot of both platelets and endothelial cells. The antigen was purified from a platelet lysate by affinity chromatography, first on a ConA column and then on a column presenting the immobilized NaM 160–1 A3 antibody. Two glycoproteins were obtained: one (116 kDa) was recognized by the antibody and one (150 kDa) was not. The 116 kDa protein had an internal decapeptide identical with human β₁ integrin, and the 150 kDa protein had an internal amino acid sequence belonging to porcine α₂ integrin. Therefore, the NaM 160–1 A3 antibody was directed against porcine β₁ integrin and allowed the purification of the complex α₂β₁ also termed Very Late Antigen 2 (VLA-2). It did not recognize human β₁ integrin.     

Immune response against BCG 65 kDa protein antigen in bladder cancer with BCG instillation therapy. Analyses carried out with the gene of this protein to develop a monoclonal antibody]

The BCG 65 kDa protein is part of the 60 kDa heat-shock protein (hsp) family. It is clear that hsp has a huge homology among mammalian and bacterial cells. In our study, we have observed the relevance of this protein in the anti-tumor response. Antibody production against the BCG 65 kDa protein was determined by solid phase ELISA using the sera of 51 bladder cancer patients who have undergone BCG instillation therapy. The BCG 65 kDa protein was made by E. coli transfected with pTB12 (plasmid DNA) which encodes this protein. It was clearly observed that the level of serum antibody titer to this protein was raised by the above instillation therapy. In order to investigate the role of this protein in anti-tumor response, we developed monoclonal antibodies against BCG 65 kDa protein. Four monoclonal antibodies were developed (B-20, B-97, B-108, B-167). We also estimated the epitope defined by each monoclonal antibody by using the truncated protein which was produced by E. coli transfected with the deletion mutants of pTB12. At this stage, we proceeded with observation of the cross epitope between mammalian cancer cells and BCG after using these monoclonal antibodies. There is no cross epitope defined by B-20, B-97 and B-167 in mammalian cells. However, the epitope defined by B-108 exists in normal tissue as well as in bladder cancer cells.     

Kinetics and character of xenoantibody formation in diabetic patients transplanted with fetal porcine islet cell clusters

Abstract: Porcine fetal islet-like cell clusters (ICC) were transplanted to 10 renal transplant patients suffering from long-standing type I diabetes. Since they had renal grafts, they were given immunosuppression with cyclosporine, prednisolone and azathioprine. Eight patients had the ICC injected intraportally and two had the ICC placed under the renal graft capsule. At the time of the xenotransplantation, ATG or 15-deoxyspergualin was given as an adjunct. Evidence of engraftment, as reflected by excretion of small amounts of porcine C-peptide into the urine, was observed in four of the patients. In all patients, irrespective of the type of immunosuppression given and whether graft function was established or not, specific xenoantibodies were formed. Titer increases in lymphocytotoxic antibodies occurred after 10 to 14 days with peak reactivity after 30 to 50 days. High titers of ADCC activity measured against porcine lymphoblasts were found in all patients. Titers were maintained at a high level for at least 100 days. Antibody titers were monitored against pig lymphocytes, erythrocytes and against pig membrane antigens solubilized from peripheral blood mononuclear cells, platelets and erythrocytes. Both IgM and IgG antibodies were formed with identical kinetics. There was no increase in the titers of alloantibodies, as evidenced by panel-reactive lymphocytotoxic antibodies. In all patients an increase in titers of isohemagglutinins was recorded, especially against blood group B antigens. Absorption studies showed that xenoreactivity present in healthy individuals could not be blocked by absorption with human RBC. However, in all transplanted patients, xenoreactivities against pig antigens were inhibited by absorption with human RBC, in particular with B-type RBC. These data show that the increase in isohemagglutinins was probably due to cross-reactivity with xenogeneic antigens. Using an ELISA assay, increased antibody titers were also recorded against purified pig MHC class I antigens. However, as is shown in the accompanying paper (next issue), these antibodies were probably not directed against protein determinants on the MHC molecules but rather against glycosylated side chains of these molecules.     

抗输卵管抗体鉴定及种属交叉反应性研究

本文报告了抗人和不同种属哺乳动物(小鼠、牛、猪)输卵管多克隆抗体的初步鉴定及种属交叉反应性的研究结果。结果表明:(1)不同种属动物输卵管中存在着较多的共同抗原或共同抗原决定簇;(2)抗猪和牛输卵管抗体与人输卵管抗原有明显的交叉反应性。提示人和猪以及人和牛输卵管抗原中存在较多类似的抗原决定簇。鉴于透明带抗原有卵巢源和输卵管源,因此本研究结果为人和猪透明带抗原中存在着明显交叉性提拱了部分依据。     

Interleukin 1 and the glomerular mesangium. III. IL-1-dependent stimulation of mesangial cell protein kinase activity

Interleukin 1 (IL-1) exerts a number of biologic actions upon cultured glomerular mesangial cells (MC). These include stimulation of cellular proliferation and induction of prostaglandin and type IV collagenase secretion. It was determined that this activity, as with other polypeptide growth factors, was associated with the activation of specific MC plasma membrane protein kinases. Plasma membranes from cycling MC were incubated with purified IL-1 and (32P) ATP in the absence of calcium and cyclic nucleotides. Macrophage IL-1 stimulated the rapid phosphorylation of several plasma membrane proteins, the most significant of which were 52-55 kd, 46 kd, and 20 kd in size. Macrophage IL-1 induced specific membrane phosphorylation in concentrations as low as 1.5 x 10(-12) M, an effect obtained with equivalent concentrations of purified MC IL-1. The 46 kd phosphoprotein, which was the most prominent, was alkali-resistant and contained phosphotyrosine when examined by phosphoamino acid analysis. The 52-55 kd and 20 kd phosphoproteins were alkali-labile and contained phosphoserine. The 46 kd phosphoprotein was the major phosphoprotein recovered from Con A-Sepharose and IL-1 affinity columns. Induction of plasma membrane-associated protein kinase activity may represent one mechanism whereby IL-1 initiates mesangial cellular activation.     

Role of endothelial cells in IgA nephropathy

Summary: Various polyspecific autoantibodies have been described in the sera of patients with IgA nephropathy, including anti-IgG, anti-IgA, antigalactosyl, antifibronectin and antimesangial antibodies. We and other authors have demonstrated the presence of antivascular endothelial cell (VEC) antibodies, in up to 32% of patients with IgA nephropathy. These antibodies were directed against molecules on the endothelial cell surface. Some of these antibodies were directed against HLA class I antigens; the majority, however, were reactive against antigens present on unstimulated endothelial cells, as prior stimulation of the cells with interferon-γ or interleukin 1 did not result in an increase in anti-VEC activity of the sera. The clinical significance of these anti-VEC antibodies in IgA nephropathy is unknown. A significant correlation was demonstrated between these antibodies and the presence of heavy proteinuria. A significant association was also shown between the presence of these antibodies and the histopathologic markers of activity such as crescents and focal and segmental necrotizing lesions, as well as immunoglobulin and C3 deposition in the vessel walls.     

Biological and immunological studies on human tumor-associated cellular proteins detected by two-dimensional gel electrophoresis

Two tumor-associated cellular proteins, 82k/6.3 (MW/pI) and 61k/7.5, which were detected by two-dimensional gel electrophoresis, were studied by biochemical and immunological methods. In two-dimensional gel electrophoresis, 82k/6.3 and 61k/7.5 were rich in colon cancer tissue compared with normal colon mucosa, and they were also detected in fetal intestines. This shows that both proteins might be involved in category of oncofetal proteins. The localization of 82k/6.3 and 61k/7.5 was investigated by subcellular fractionation. They were rich in microsomal fraction, but not found in both nuclear and mitochondrial fractions. In binding reaction with seven kinds of lectins, 82k/6.3 reacted with RCAI, DBA and WGA, where 61k/7.5 reacted with RCAI, DBA, WGA, UEAI and SBA. Transferrin reacted with only RCAI. Each hybrid producing monoclonal antibody against 82k/6.3 or 61k/7.5 was generated by fusing spleen cells of BALB/c mice immunized by the two proteins and mouse myeloma cells. Each monoclonal antibody was specified in enzyme-linked immunoassay. In indirect immuno-fluorescent studies, monoclonal antibodies against 82k/6.3 and 61k/7.5 reacted with cytoplasma and membrane of human cancer cells. This result strongly suggests the localization of the two proteins demonstrated by subcellular fractionation.     

Relative purity of thrombin-based hemostatic agents used in surgery

BACKGROUND: Hemostatic agents used in surgery contain thrombin isolated from either a bovine or human source. The use of thrombin derived from a bovine source has been associated with the development of an abnormal immune response, but a study of the immunoreactivity of the various commercially available thrombin preparations has not been conducted. This study determined the relative purity of commercially available thrombin preparations, if humans have natural antibodies that recognize these preparations, and if elicited antibodies against bovine thrombin cross-react with other bovine or human hemostatic agents. STUDY DESIGN: The purity of hemostatic agents was determined by protein and substrate assays, electrophoresis, and immunoblotting. The natural antigenicity and cross-reactivity of elicited antibodies were measured by ELISA using serum samples from 82 donors from the Red Cross and serum collected from patients exposed to bovine thrombin, respectively. RESULTS: All of the bovine thrombin preparations were found to contain the xenogeneic carbohydrate galactosealpha1-3galactose. The natural antigenicity of the bovine thrombin preparations was greater than that of a human thrombin preparation and similar to that of porcine aortic endothelial cells. Antibodies elicited against bovine thrombin were found to cross-react with other bovine preparations and other xenoantigens but not with human hemostatic preparations. CONCLUSIONS: All patients have antibovine thrombin antibodies, even before exposure to bovine thrombin-containing hemostatic agents. The cross-reactivity of elicited antibovine thrombin antibodies indicates that if a patient has been sensitized to a bovine product, it is likely safer to use a human-derived product in lieu of a bovine product.     

The calcium oxalate crystal growth inhibitor protein produced by mouse kidney cortical cells in culture is osteopontin.

Urine contains proteins that inhibit the growth of calcium oxalate (CaOx) crystals and may prevent the formation of kidney stones. We have identified a potent crystal growth inhibitor in the conditioned media from primary cultures of mouse kidney cortical cells. Conditioned media, incubated with the kidney cells for 6-72 h, was assayed for crystal growth inhibition; inhibitory activity increased 15-fold by 24 h. Inhibitory activity was purified from serum-free media containing proteinase inhibitors using anion-exchange and gel-filtration chromatography. A single band of molecular weight 80,000 daltons was seen after SDS-polyacrylamide gel electrophoresis. The sequence of the N-terminal 21 amino acids of this protein matched that of osteopontin (OP), a phosphoprotein initially isolated from bone matrix. Antisera raised to fusion proteins produced by plasmids containing the N-terminal or C-terminal portions of OP cDNA also cross-reacted with the protein purified from cell culture media on western blots. The effect of the purified protein on the growth of CaOx crystals was measured using a constant composition assay. A 50% inhibition of growth occurred at a protein concentration of 0.85 micrograms/ml, and the dissociation constant of the protein with respect to CaOx crystal was 3.7 x 10(-8) M. The concentration of OP in mouse urine, measured using antibodies raised to the purified protein, was approximately 8 micrograms/ml. We conclude that OP is synthesized by kidney cortical tubule cells and functions as a crystal growth inhibitory protein in urine.     

Measurement of Intact Rat Osteocalcin in Osteoblast (ROS17/2.8) Cells and in Ovariectomized Rats with a Sandwich Enzyme Immunoassay

We developed a sandwich enzyme immunoassay system for intact rat osteocalcin to improve the region specificity for the detection of this molecule. We synthesized two peptides of N-terminal 20 residues and C-terminal 10 residues of rat osteocalcin. After conjugating these peptides with carrier protein, we obtained anti-N- and anti-C-terminal rat osteocalcin antibodies in rabbits raised against these two peptides, respectively. By using these antibodies, we measured intact rat osteocalcin levels in a two-site immunoassay manner. These antibodies did not show the cross-reactivity to human osteocalcin. The immunoreactive peak corresponding to the intact molecules was detected by our intact osteocalcin method after high-performance liquid chromatographic fractionation of osteocalcin fragments in plasma from uremic rats. Furthermore, the intact rat osteocalcin was stable over 8 hours at 25°C. Intact rat osteocalcin levels extracellularly secreted from ROS 17/2.8 cells were measured by this method, showing time- and dose-dependent significant increases when administered 1,25(OH)₂D₃. The inhibition for the secretion of intact osteocalcin by actinomycin D was also detected quantitatively with this method. In ovariectomized rats, intact osteocalcin levels in plasma were acutely elevated after ovariectomy, and its elevation was significantly depressed by 17β-estradiol administration. These data suggest that this sandwich method is able to measure the intact form of osteocalcin secreted by osteoblasts. As the antibodies identify the specific regions of osteocalcin molecule, this method would be useful for sensitive estimation of bone turnover for various experimental conditions in rats. Received: 12 August 1995 / Accepted: 28 November 1995     

Immunohistochemical studies with a monoclonal antibody on the distribution of phosphophoryn in predentin and dentin

Summary A monoclonal antibody was raised against phosphoryn, a unique noncollagenous phosphoprotein in dentin. Mouse myeloma NS-I cells were fused with spleen cells obtained from BALB/c mice immunized with phosphophoryn from fetal calf tooth germs. Mice inoculated with the hybridoma produced ascites fluid containing the antibody and this reacted only with a band of phosphophoryn transblotted from polyacrylamide gel. Immunohistochemical studies with the antibody showed that phosphophoryn was present in odontoblasts, odontoblastic processes and dentin, but not in the matrix of predentin, and that the phosphophoryn content of the dentin layer was high at and around the predentin-dentin junction and gradually decreased toward the enamel layer. The area corresponding to mantle dentin was not stained with the antibody.     

The Effects of Various Extracellular Matrices on Renal Cell Attachment to Polymer Surfaces During the Development of Bioartificial Renal Tubules

Extracellular matrices (ECM) are utilized for obtaining better cell attachment to polymer surfaces in cell cultures. To establish beneficial bioartificial renal tubules, tubular epithelial cells and ECM were investigated in this study. MDCK cells and KU-2 cells were seeded onto 96 well plates which had been precoated with collagen types I and IV, laminin, and fibronectin. The culture media were removed and replaced with new ones at 15, 30, 60, 90, 120, and 150 min and 24 h after start time to evaluate the incubation time effects. The degrees of cell attachment onto ECM were measured by MTT assay. In the MDCK cell culture, better cell attachment was observed between 60 min and 24 h after incubation start time with the use of laminin at a concentration of 5 μg/ml, 60 min and more after incubation start time with the use of fibronectin at the concentrations of 1 and 4 μg/ml, or 30 min and more after incubation start time with the use of fibronectin at the concentrations of 16 and 32 μg/ml. On the other hand, in the KU-2 cell culture, better cell attachment was observed between 15 and 60 min after the incubation start time or 24 h after the incubation start time with the use of laminin at a concentration of 40 μg/ml. These data suggest that various cells possibly each have a most suitable ECM kind, best concentration, and best incubation time.     

Allosensitization does not increase the risk of xenoreactivity to alpha1,3-galactosyltransferase gene-knockout miniature swine in patients on transplantation waiting lists

BACKGROUND: The recent availability of alpha1,3-galactosyltransferase knockout (GalT-KO) miniature swine has eliminated anti-Gal antibodies as the major barrier to xenotransplantation, potentially bringing this modality closer to clinical application. Highly-allosensitized patients, who have poor prospects of receiving a suitable cross-match negative human organ, might be the first patients to benefit from xenotransplantation of porcine organs. However, concerns exist regarding cross-reactivity of alloreactive anti-human leukocyte antigen (HLA) antibodies against xenogeneic swine leukocyte antigen (SLA) antigens. We have investigated this question using sera from such patients on GalT-KO target cells. METHODS: Using flow cytometry and complement-dependent cytotoxicity (CDC) assays, we have tested a panel of 88 human serum samples from patients awaiting cadaveric renal allotransplantation for reactivity against: 1) human; 2) standard miniature swine; and 3) GalT-KO peripheral blood lymphocytes (PBL) and cultured endothelial cells. RESULTS: Anti-swine IgM and IgG antibody binding, as well as CDC, were significantly attenuated on GalT-KO versus standard swine. No correlation was found between the degree of anti-human panel reactive antibodies (PRA) and xenoreactivity against either standard or GalT-KO miniature swine. Treatment of sera with dithiothreitol (DTT) showed that the majority of remaining lymphocytotoxicity against GalT-KO swine was mediated by preformed IgM antibodies. Patients with high alloreactivity but low anti-GalT-KO xenoreactivity were readily identified. CONCLUSIONS: Highly allosensitized patients awaiting renal transplants appear to be at no increased risk of xenosensitization over their non-sensitized cohorts, and could therefore be candidates for xenotransplantation using GalT-KO swine donors.     

Two classes of dentin phosphophoryns,from a wide range of species,contain immunologically cross-reactive epitope regions

Summary An immunological species comparison, using a monospecific rabbit polyclonal antibody directed against rat incisor α-phosphophoryn, has been undertaken to assess the similarity in epitope regions among various dentin phosphophoryns (PP) that were prepared from human, monkey, bovine, ovine, and echinoderm teeth. Dentin extracellular matrix proteins were extracted with a standard method using 0.5 M EDTA in the presence of enzyme inhibitors. Final phosphophoryn purification was performed on DEAE ion exchange HPLC. Cross-reactivity of the polyclonal antibody was examined by enzyme-linked immunosorbant assay (ELISA) and dot-blot. The results of this investigation demonstrate a cross-reactivity of the rat-α-phosphophoryn antibody (anti-RIPP) with at least one phosphophoryn component in each dentin studied, indicating the existence of similar antigenic determinants among these proteins. It would seem that these epitope regions have been strongly conserved since the epitope region is also present in the phosphoprotein of echinoderm teeth. No crossreactivity was found with phosvitin (a phosphoserine-rich phosphoprotein), rat serum albumin, bovine serum albumin, or collagen type IV. However, a new and distinct second cross-reactive phosphophoryn, not calcium ion-precipitable, was found in the EDTA insoluble fraction from the teeth. These results indicate that dentin phosphophoryns are specific phenotypic markers for odontoblast expression. Because of the species cross-reactivity, the polyclonal anti-RIPP antibody may be a useful probe in studying the distribution of phosphophoryns in other species, such as human teeth.     

A comparative immunocytochemical study on the subcellular distributions of 44 kDa bone phosphoprotein and bone gamma-carboxyglutamic acid (Gla)-containing protein in osteoblasts

Bone gamma-carboxyglutamic acid (Gla)-containing protein (BGP or osteocalcin) and 44 kDa bone phosphoprotein (44K BPP, also called Sialoprotein I or osteopontin) have been localized at the ultrastructural level in osteoblasts from woven bones of newborn rats. Frozen, undecalcified sections of periodate-lysine-paraformaldehyde fixed specimens were incubated with affinity purified, monospecific antibodies against BGP or 44K BPP. The sites of the antigen-antibody reaction were demonstrated by the avidin-biotin-peroxidase complex method using the Hanker-Yates reagent as a peroxidase substrate. In some cases immunostaining could only be achieved after detergent treatment. The immunostained sections were then flat-embedded in Epon 812 and processed for electron microscopy. Strong specific intracellular labeling was obtained with both antibodies, but the patterns of staining differed significantly: BGP antigenicity was mainly located in the endoplasmic reticulum (ER), whereas 44K BPP behaved as a Golgi-specific antigen. In both cases, however, we found no evidence for immunostained secretory vesicles. There was no correlation between the expression of BGP by osteoblasts and the morphological aspect of these cells, their apparent degree of polarization with respect to the bone matrix, or their relation with the mineralized phase.