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M A Bakker

Publications and source records attributed to M A Bakker.

At least 19 recordsLinked to original sources
PubMed1998

Differential expression of agrin in renal basement membranes as revealed by domain-specific antibodies.

We determined the specificity of two hamster monoclonal antibodies and a sheep polyclonal antiserum against heparan sulfate proteoglycan isolated from rat glomerular basement membrane. The antibodies were characterized by enzyme-linked immunosorbent assay on various basement membrane components and immunoprecipitation with heparan sulfate proteoglycan with or without heparitinase pre-treatment. These experiments showed that the antibodies specifically recognize approximately 150-, 105-, and 70-kDa core proteins of rat glomerular basement membrane heparan sulfate proteoglycan. Recently, we showed that agrin is a major heparan sulfate proteoglycan in the glomerular basement membrane (Groffen, A. J. A., Ruegg, M. A., Dijkman, H. B. P. M., Van der Velden, T. J., Buskens, C. A., van den Born, J., Assmann, K. J. M., Monnens, L. A. H., Veerkamp, J. H., and van den Heuvel, L. P. W. J. (1998) J. Histochem. Cytochem. 46, 19-27). Therefore, we tested whether our antibodies recognize agrin. To this end, we evaluated staining of Chinese hamster ovary cells transfected with constructs encoding full-length or the C-terminal half of rat agrin by analysis on a fluorescence-activated cell sorter. Both hamster monoclonals and the sheep antiserum clearly stained cells transfected with the construct encoding full-length agrin, whereas wild type cells and cells transfected with the construct encoding the C-terminal part of agrin were not recognized. A panel of previously characterized monoclonals, directed against C-terminal agrin, clearly stained cells transfected with either of the constructs but not wild type cells. This indicates that both hamster monoclonals and the sheep antiserum recognize epitopes on the N-terminal half of agrin. By immunohistochemistry on rat renal tissue, we compared distribution of N-terminal agrin with that of C-terminal agrin. The monoclonal antibodies against C-terminal agrin stained almost exclusively the glomerular basement membrane, whereas the anti-N-terminal agrin antibodies recognized all renal basement membranes, including tubular basement membranes. Based on these results, we hypothesize that full-length agrin is predominantly expressed in the glomerular basement membrane, whereas in most other renal basement membranes a truncated isoform of agrin is predominantly found that misses (part of) the C terminus, which might be due to alternative splicing and/or posttranslational processing. The possible significance of this finding is discussed.

Agrin↗
PubMed1997

Hydroxyl radicals depolymerize glomerular heparan sulfate in vitro and in experimental nephrotic syndrome.

Heparan sulfate, the polysaccharide side chain of heparan sulfate proteoglycan, is important for the permselective properties of the glomerular basement membrane. In this report, we show a role for hydroxyl radicals in heparan sulfate degradation and an enhanced glomerular basement membrane permeability. First, in enzyme-linked immunosorbent assay, exposure of coated heparan sulfate (proteoglycan) to reactive oxygen species resulted in a +/-50% decrease of binding of a monoclonal antibody against heparan sulfate, whereas binding of an antibody against the core protein remained unaltered. Second, on polyacrylamide gel electrophoresis, the molecular weight of heparan sulfate exposed to radicals was reduced which indicates depolymerization. Both in enzyme-linked immunosorbent assay and gel electrophoresis, hydroxyl radicals are instrumental for heparan sulfate degradation as shown by the addition of various radical scavengers. Third, in an experimental model for human nephrotic syndrome (Adriamycin nephropathy in rats), glomerular basement membrane staining of two recently described anti-heparan sulfate antibodies (JM403 and KJ865) was reduced by 24 and 43%. Treatment of Adriamycin-exposed rats with the hydroxyl radical scavenger dimethylthiourea both reduced albuminuria by 37% (p < 0.01) and partly prevented loss of heparan sulfate staining by 53% (JM403) and 39% (KJ865) (p < 0.03). In contrast to the heparan sulfate side chains, the core protein expression and the extent of glycanation did not change in Adriamycin nephropathy. We conclude that glomerular basement membrane heparan sulfate is susceptible to depolymerization by hydroxyl radicals leading to loss of glomerular basement membrane integrity and albuminuria.

Animals↗
PubMed1997

Antiproteinuric effect of ciclosporin A in adriamycin nephropathy in rats.

Ciclosporin A (CsA) can reduce proteinuria in various forms of human and experimental glomerulopathies. This antiproteinuric effect can be the result of a decrease of immunological damage, a decrease in the glomerular filtration rate (GFR), or a change in the permselective properties of the glomerular capillary wall. In this study we investigated the effect of CsA on Adriamycin-induced nephropathy in rats. A single intravenous injection of Adriamycin (5 mg/kg body weight) induced a severe nephrotic syndrome with a massive albuminuria (+/- 400 mg/24 h from 3 weeks onwards) and a hypoalbuminemia (+/- 7 mg/ml after 5 weeks). The IgG/albumin selectivity index was 0.16 +/- 0.05, indicating a preferential loss of albumin. A 5-day treatment with CsA reduced the albumin excretion by almost 50% (from 336 +/- 91 to 178 +/- 58 mg/24 h; p = 0.002) and induced an increase in the serum albumin level (from 7.1 +/- 4.1 to 12.8 +/- 3.2 mg/ml; p = 0.002) in contrast to the vehicle olive oil (OO). CsA also decreased the GFR by 40% (from 0.74 +/- 0.11 to 0.41 +/- 0.11 mg/ml/100 g body weight; p = 0.002). Albuminuria corrected for the GFR (fractional excretion of albumin, FE(alb)) was still significantly lower in CsA-treated than in OO-treated animals (FE(alb) CsA: 1.35 +/- 0.88, FE(alb) OO: 3.17 +/- 2.29%; p = 0.0005). This suggests that other factors are also involved in the reduction of albuminuria. To exclude that CsA has an effect on the tubular reabsorption of albumin, we evaluated the blockade of the tubular reabsorption by lysine and found no difference in albuminuria between the CsA- and OO-treated groups. These experiments suggest that the antiproteinuric effect of CsA is not (only) due to a decrease in the GFR, but also to a decrease of the enhanced permeability of the glomerular capillary wall for albumin.

Albuminuria↗
PubMed1997

Vessel wall heparan sulfate and transcapillary passage of albumin in experimental diabetes in the rat.

BACKGROUND: To investigate whether albuminuria in streptozotocin-induced diabetes mellitus in the rat is a symptom of a more generalized vessel wall permeability, and whether changes in vessel wall heparan sulfate (HS) are associated with alterations in vessel wall permeability. METHODS: The transcapillary escape rate of albumin (TERalb) was calculated from the disappearance rate from the circulation of i.v. injected radiolabeled albumin, together with the regional clearance of albumin (RCalb) in several tissues using a double isotope technique. These measurements were performed in seven rats one year after diabetes induction and in seven sex- and age-matched control rats. To evaluate the association between vessel wall HS and the transcapillary passage of albumin, we determined the content of basement membrane HS in tissue homogenates of heart, liver, kidneys, and lungs with a sensitive inhibition-ELISA using a monoclonal antibody (JM-403), which specifically recognizes basement membrane HS. RESULTS: Diabetic rats developed albuminuria (31.7 +/- 10.8 mg/24 h) in contrast to control animals (2.2 +/- 1.5 mg/24 h; P = 0.0006). TERalb was increased from 13.3 +/- 1.7 in control rats to 15.6 +/- 2.6%/h in diabetic rats, P = 0.02. RCalb was significantly increased in heart, liver, skeletal muscle and aorta, unchanged in kidneys and skin, and significantly decreased in lung tissue. We found a decrease in HS content in heart tissue of diabetic rats, and a correlation between HS content and RCalb (r = -0.72, P = 0.004), in contrast with an increase in lung HS content that correlated with a decrease in RCalb (r = -0.64, P = 0.014). No changes in HS content were found in kidney and liver tissue. CONCLUSIONS: These data indicate that in one-year diabetic rats albuminuria coincides with an increased TERalb and RCalb in most, but not all tissues, and that alterations in basement membrane HS content correlate with changes in the RCalb, which suggests a functional relationship.

Animals↗
PubMed1996

Elastase, but not proteinase 3 (PR3), induces proteinuria associated with loss of glomerular basement membrane heparan sulphate after in vivo renal perfusion in rats.

Elastase, but not PR3, induces proteinuria associated with loss of glomerular basement membrane (GBM) heparan sulphate after in vivo renal perfusion in rats. PR3 and elastase are cationic neutral serine proteinases present in the azurophilic granules of polymorphonuclear leucocytes. Release of these proteolytic enzymes along the glomerular capillary wall may induce glomerular injury. Here, we investigated the effects of PR3 and elastase on the induction of proteinuria and glomerular injury after renal perfusion of these enzymes in Brown-Norway rats. Perfusion of active elastase induced a dose-dependent proteinuria 24h after perfusion, while inactivated elastase did not. Perfusion of comparable amounts of active PR3 did not induce proteinuria. Light and electron microscopy showed no morphological abnormalities in any experimental group. However, immunohistology revealed that proteinuria occurring after perfusion of active elastase was associated with a strong reduction in intraglomerular expression of the heparan sulphate side chain and, to a lesser extent, of the protein core of heparan sulphate proteoglycans (HSPG). In vitro, both elastase and PR3 digested HSPG. However, PR3 bound to a lesser extent to HSPG than elastase. We conclude that elastase, but not PR3, induces proteinuria after in vivo renal perfusion. This differential effect probably relates to different binding to the GBM of those enzymes due to differences in their isoelectric points. Degradation of heparan sulfate proteoglycans, leading to the disappearance of their side chains that contribute to the polyanionic structure of the GBM, appears to be involved in the induction of proteinuria after perfusion of elastase.

Animals↗
PubMed1995

Presence of N-unsubstituted glucosamine units in native heparan sulfate revealed by a monoclonal antibody.

Immunohistochemical application of antibodies against heparan sulfate proteoglycan core protein and heparitinase-digested heparan sulfate stubs showed the presence of heparan sulfate proteoglycan in all basement membranes of the rat kidney. However, a monoclonal antibody (JM-403) against native heparan sulfate (van den Born, J., van den Heuvel, L. P. W. J., Bakker, M. A. H., Veerkamp, J. H., Assmann, K. J. M., and Berden, J. H. M. (1992) Kidney Int. 41, 115-123) largely failed to stain tubular basement membranes, suggesting the presence of heparan sulfate chains lacking the specific JM-403 epitope. Heparan sulfate preparations from various sources differed markedly with regard to JM-403 binding, as demonstrated by liquid phase inhibition in enzyme-linked immunosorbent assay, the interaction decreasing with increasing sulfate contents of the polysaccharide. Mapping of the JM-403 epitope indicated that it was dominated by one or more N-unsubstituted glucosamine unit(s), since treatments that destroyed or altered the structure of such units in heparan sulfate preparations (cleavage at N-unsubstituted glucosamine units with HNO2 at pH 3.9 and N-acetylation with acetic anhydride, respectively), abolished antibody binding. Conversely, immunoreactivity could be induced in a (D-glucuronyl-1,4-N-acetyl-D-glucosaminyl-1,4) polysaccharide by the generation of N-unsubstituted glucosamine N-unsubstituted glucosamine in a JM-403-binding heparan sulfate (preparation HS-II from human aorta) was demonstrated by an approximately 3-fold reduction in molecular size following HNO2 (pH 3.9) treatment. Further characterization of the epitope recognized by JM-403, based on enzyme-linked immunosorbent assay inhibition tests with chemically/enzymatically modified polysaccharides, indicated that one or more N-sulfated glucosamine units are invariable present, whereas L-iduronic acid and O-sulfate residues appear to inhibit JM-403 reactivity. It is concluded that the epitope contains one or more N-unsubstituted glucosamine and D-glucuronic acid units and is located in a region of the heparan sulfate chain composed of mixed N-sulfated and N-acetylated disaccharide units.

Animals↗
PubMed1995

Selective proteinuria in diabetic nephropathy in the rat is associated with a relative decrease in glomerular basement membrane heparan sulphate.

In the present study we investigated whether glomerular hyperfiltration and albuminuria in streptozotocin-induced diabetic nephropathy in male Wistar-Münich rats are associated with changes in the heparan sulphate content of the glomerular basement membrane. Rats with a diabetes mellitus duration of 8 months, treated with low doses of insulin, showed a significant increase in glomerular filtration rate (p < 0.01) and effective renal plasma flow (p < 0.05), without alterations in filtration fraction or mean arterial blood pressure. Diabetic rats developed progressive albuminuria (at 7 months, diabetic rats (D): 42 +/- 13 vs control rats (C): 0.5 +/- 0.2 mg/24 h, p < 0.002) and a decrease of the selectivity index (clearance IgG/clearance albumin) of the proteinuria (at 7 months, D: 0.20 +/- 0.04 vs C: 0.39 +/- 0.17, p < 0.05), suggesting loss of glomerular basement membrane charge. Light- and electron microscopy demonstrated a moderate increase of mesangial matrix and thickening of the glomerular basement membrane in the diabetic rats. Immunohistochemically an increase of laminin, collagen III and IV staining was observed in the mesangium and in the glomerular basement membrane, without alterations in glomerular basement membrane staining of heparan sulphate proteoglycan core protein or heparan sulphate. Glomerular basement membrane heparan sulphate content, quantitated in individual glomerular extracts by a new inhibition ELISA using a specific anti-glomerular basement membrane heparan sulphate monoclonal antibody (JM403), was not altered (median (range) D: 314 (152-941) vs C: 262 (244-467) ng heparan sulphate/mg glomerulus). However, the amount of glomerular 4-hydroxyproline, as a measure for collagen content, was significantly increased (D: 1665 (712-2014) vs C: 672 (515-1208) ng/mg glomerulus, p < 0.01). Consequently, a significant decrease of the heparan sulphate/4-hydroxyproline ratio (D: 0.21 (0.14-1.16) vs C: 0.39 (0.30-0.47), p < 0.05) was found. In summary, we demonstrate that in streptozotocin-diabetic rats glomerular hyperfiltration and a progressive, selective proteinuria are associated with a relative decrease of glomerular basement membrane heparan sulphate. Functionally, a diminished heparan sulphate-associated charge density within the glomerular basement membrane might explain the selective proteinuria in the diabetic rats.

Albumins↗
PubMed1995

Reduction of heparan sulphate-associated anionic sites in the glomerular basement membrane of rats with streptozotocin-induced diabetic nephropathy.

Heparan sulphate-associated anionic sites in the glomerular basement membrane were studied in rats 8 months after induction of diabetes by streptozotocin and in age- adn sex-matched control rats, employing the cationic dye cuprolinic blue. Morphometric analysis at the ultrastructural level was performed using a computerized image processor. The heparan sulphate specificity of the cuprolinic blue staining was demonstrated by glycosaminoglycan-degrading enzymes, showing that pretreatment of the sections with heparitinase abolished all staining, whereas chondroitinase ABC had no effect. The majority of anionic sites (74% in diabetic and 81% in control rats) were found within the lamina rara externa of the glomerular basement membrane. A minority of anionic sites were scattered throughout the lamina densa and lamina rara interna, and were significantly smaller than those in the lamina rara externa of the glomerular basement membrane (p<0.001 and p<0.01 for diabetic and control rats, respectively). Diabetic rats progressively developed albuminuria reaching 40.3 (32.2-62.0) mg/24 h after 8 months in contrast to the control animals (0.8 (0.2-0.9) mg/24 h, p<0.002). At the same time, the number of heparan sulphate anionic sites and the total anionic site surface (number of anionic sites x mean anionic site surface) in the lamina rara externa of the glomerular basement membrane was reduced by 19% (p<0.021) and by 26% (p<0.02), respectively. Number and total anionic site surface in the remaining part of the glomerular basement membrane (lamina densa and lamina rara interna) were not significantly changed. We conclude that in streptozotocin-diabetic rats with an increased urinary albumin excretion, a reduced heparan sulphate charge barrier/density is found at the lamina rara externa of the glomerular basement membrane.

Albuminuria↗
PubMed1995

Decrease of heparan sulfate staining in the glomerular basement membrane in murine lupus nephritis.

Recently we found in biopsies of human lupus nephritis a nearly complete loss of heparan sulfate (HS) staining in the glomerular basement membrane (GMB). To clarify the relationship between HS staining and albuminuria in lupus nephritis, we studied MRL/lpr mice with short (< 7 days) or prolonged duration of albuminuria (14-21 days) and compared these with mice of different ages without albuminuria. Kidney sections were stained for mouse immunoglobulin (Ig), HS, heparan sulfate proteoglycan (HSPG)-core protein and laminin in immunofluorescence. In mice with prolonged albuminuria HS staining in the glomerular capillary loops had almost completely disappeared, whereas staining was unaltered in non-albuminuric mice (P = 0.001). In mice with short duration of albuminuria, there was a tendency toward a decrease of HS staining (P = 0.06). The expression of HSPG-core protein and other extra cellular matrix (ECM) components was unaltered in all groups. HS staining correlated inversely with albuminuria (rs = -0.55; P < 0.001) and with staining of Ig deposits in the capillary loops (rs = -0.74; P < 0.001). Despite the nearly complete loss of HS staining in the GBM in mice with prolonged albuminuria, there was no change in glomerular HS content as assessed by agarose electrophoresis and HS inhibition ELISA. We conclude that the development of albuminuria in MRL/lpr mice is accompanied by a loss of HS staining in the GBM, probably due to the masking of HS by deposits of Ig. In vitro studies revealed that autoantibodies complexed to nucleosomal antigens can inhibit the binding of the anti-HS monoclonal antibody to HS. Whether this also occurs in vivo remains to be determined.

Albuminuria↗
PubMed1994

Monoclonal antibodies against the protein core and glycosaminoglycan side chain of glomerular basement membrane heparan sulfate proteoglycan: characterization and immunohistological application in human tissues.

We raised monoclonal antibodies (MAb) against the core protein and the heparan sulfate (HS) side chain of heparan sulfate proteoglycan (HSPG) from glomerular basement membranes (GBM). Anti-HSPG-core MAb were obtained after immunization of mice with HSPG purified from human GBM and the anti-HS MAb after immunization of mice with HSPG from rat glomeruli, which crossreacted with human HS and GBM HSPG. The specificity of the MAb was demonstrated by ELISA studies, Western blotting, inhibition experiments, and indirect immunofluorescence (IF) on kidney cryostat sections pre-treated with glycosaminoglycan (GAG)-degrading enzymes. Indirect IF on normal human kidney tissue showed prominent GBM staining for both MAb, with variable staining of the other renal basement membranes (BMs). By indirect immunoelectron microscopy (IEM), most intense staining was observed at the endothelial side of the GBM for both MAb, although the staining patterns were not identical. Both MAb were used to localize HSPG in human tissues by indirect IF. They bound to antigens present in the BMs of most tissues examined, including those of epithelia and endothelia. Differences between both MAb were observed for BMs of muscle cells, since the anti-HSPG core protein MAb (JM-72) staining was negative, whereas the anti-HS MAb (JM-403) clearly stained these structures. Comparison of our staining patterns in human tissues with the distribution of other anti-BM HSPG antibodies suggests that there are at least two types of BM HSPG, which have common epitopes on the HS side chains recognized by JM-403.

Animals↗
PubMed1993

6-Mercaptopurine: cytotoxicity and biochemical pharmacology in human malignant T-lymphoblasts.

The effects of prolonged exposure to 2 and 10 microM 6-mercaptopurine (6MP) in the human lymphoblastic T-cell line MOLT-4 were studied with respect to cell-kinetic parameters, phosphoribosyl pyrophosphate (PRPP) and purine ribonucleotide levels, formation of 6MP-nucleotides, especially methyl-thio-IMP (Me-tIMP), DNA and RNA synthesis ([32P] incorporation), and [8-14C]6MP incorporation into newly synthesized DNA and RNA. The results provided new insights into the complex mechanism of action of 6MP in human malignant lymphoblasts. Exposure to 2 microM 6MP resulted in a rapid inhibition of purine de novo synthesis (PDNS) by increased levels of Me-tIMP, resulting in increased PRPP levels and decreased purine ribonucleotides, affecting cell growth and clonal growth, and less cell death. DNA synthesis decreased, associated with an increasing delay of cells in S phase. Incorporation of thioguanine nucleotides into newly synthesized DNA resulted in an increasing arrest of cells in G2 + M phase. RNA synthesis, initially decreased, recovered partially, associated with a recovery of purine ribonucleotides. New formation of 6MP-nucleotides (tIMP) was only detected within the first 24 hr, and 6MP levels in the culture medium were already undetectable after 6 hr of exposure to 2 microM, indicating a high rate of incorporation and complete conversion of 6MP within this period. Incorporation of 6MP-nucleotides into DNA was 5 times as high as incorporation into RNA. Exposure to 10 microM 6MP resulted in early cytotoxicity at 24 hr, associated with a complete inhibition of PDNS by a large pool of Me-tIMP and lower levels of purine ribonucleotides as compared to 2 microM 6MP. A more severe delay of cells in S phase was associated with an inhibition of DNA synthesis to 14% of control within the first 24 hr, and an arrest in G2 + M phase. Further increasing levels of Me-tIMP caused an arrest of cells and late cytotoxicity in S phase at 48 hr, preventing further progression into G2 + M phase. Our data suggest that inhibition of PDNS due to Me-tIMP is a crucial event in the mechanism of 6MP cytotoxicity. It is responsible for decreased RNA synthesis and decreased availability of natural deoxyribonucleotides, causing a delay of DNA synthesis in S phase. This enhances incorporation of 6MP as thioguanine nucleotides into DNA in the S phase and subsequent late cytotoxicity in the G2 phase. However, with high concentrations of 6MP, the large pool of Me-tIMP causes severe reduction of natural deoxyribonucleotides in lymphoblasts with an active PDNS.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbon Radioisotopes↗
PubMed1993

Distribution of GBM heparan sulfate proteoglycan core protein and side chains in human glomerular diseases.

Using monoclonal antibodies (mAbs) recognizing either the core protein or the heparan sulfate (HS) side chain of human GBM heparan sulfate proteoglycan (HSPG), we investigated their glomerular distribution on cryostat sections of human kidney tissues. The study involved 95 biopsies comprising twelve different glomerulopathies. Four normal kidney specimens served as controls. A homogenous to linear staining of the GBM was observed in the normal kidney with anti-HSPG-core mAb (JM-72) and anti-HS mAb (JM-403). In human glomerulopathies the major alteration was a segmental or total absence of GBM staining with anti-HS mAb JM-403, which is most pronounced in lupus nephritis, membranous glomerulonephritis (GN), minimal change disease and diabetic nephropathy, whereas the HSPG-core staining by mAb JM-72 was unaltered. In addition we found HSPG-core protein in the mesangial matrix when this was increased in membranoproliferative GN Type I, Schönlein-Henoch GN, IgA nephropathy, lupus nephritis, diabetic nephropathy and in focal glomerulosclerosis. Also staining with the anti-HS mAb JM-403 became positive within the mesangium, although to a lesser extent. Furthermore, amyloid deposits in AL and AA amyloidosis clearly stained with anti-HSPG-core mAb JM-72, and to a lesser degree with anti-HS mAb JM-403. Finally, in membranous GN (stage II and III), the GBM staining with anti-HSPG-core mAb JM-72 became irregular or granular, probably related to the formation of spikes. In conclusion, major alterations were observed in the glomerular distribution of HS and HSPG-core in various human glomerulopathies. The mAbs can be useful to further delineate the significance of HSPG and HS for glomerular diseases.

Antibodies, Monoclonal↗
PubMed1992

A monoclonal antibody against GBM heparan sulfate induces an acute selective proteinuria in rats.

After immunization of mice with partially-purified heparan sulfate proteoglycan (HSPG) isolated from rat glomeruli, a monoclonal antibody (mAb JM-403) was obtained, which was directed against heparan sulfate (HS), the glycosaminoglycan side chain of HSPG. In ELISA it reacted with isolated human glomerular basement membrane (GBM) HSPG, HS and hyaluronic acid, but not with the core protein of human GBM HSPG, and not with chondroitin sulfate A and C, dermatan sulfate, keratan sulfate and heparin. Furthermore, it did not bind to laminin, collagen type IV or fibronectin. Specificity of JM-403 for HS was also suggested by results of inhibition studies, which found that intact HSPG and HS, but not the core protein, inhibited the binding of JM-403 to HS. In indirect immunofluorescence on cryostat sections of rat kidney, a fine granular to linear staining of the GBM was observed, along with a variable staining of the other renal basement membranes. Pretreatment of the sections with heparitinase completely prevented the binding of mAb JM-403, whereas pretreatment with chondroitinase ABC or hyaluronidase had no effect. The precise binding site of mAb JM-403 was investigated by indirect immunoelectron microscopy. It revealed a diffuse staining of the whole width of the GBM. One hour after intravenous injection of JM-403 into rats, the mAb was detected along the glomerular capillary wall in a fine granular pattern, which shifted towards a more mesangial localization after 24 hours. No binding was observed anymore by day 15. Intravenous injection induced a dose-dependent, transient and selective proteinuria that was maximal immediately after the injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Production and characterization of a monoclonal antibody against human glomerular heparan sulfate.

After immunization with heparan sulfate proteoglycan (HSPG) isolated from human glomeruli, two mouse monoclonal antibodies (mAbs) against heparan sulfate (HS) were obtained. Both mAbs were of the IgM isotype and showed identical specificity. One of these, mAb JM-13 is described in detail. In enzyme-linked immunosorbent assay and Western blotting, reactivity was found with human glomerular basement membrane HSPG and HS. No binding occurred to the core protein of HSPG obtained after removal of HS with trifluoromethanesulfonic acid. In enzyme-linked immunosorbent assay, mAb JM-13 did neither bind to other proteoglycans, nor to other basement membrane components like collagen type IV, laminin, or fibronectin. In indirect immunofluorescence on cryostat sections of human kidneys, a restricted staining of tubular basement membranes was observed along with staining of the vascular basement membranes. In the glomerulus, a weak, fine granular staining was seen along the capillary wall and in the mesangium. MAb JM-13 bound also to the basolateral cell membranes of proximal tubular cells, to the cell membranes of cultured human and rat glomerular visceral epithelial cells, rat mesangial cells, human hepatocytes in culture, and in liver cryostat sections, indicating also a recognition of cell surface-associated HS. Pretreatment of the sections with heparitinase abolished binding of JM-13, whereas treatment with chondroitinase ABC had no effect. Inhibition studies in enzyme-linked immunosorbent assay as well as in indirect immunofluorescence corroborated the HS specificity of mAb JM-13. In conclusion, mAb JM-13 binds to an epitope on the HS chains of glomerular, tubular, and cell surface-associated HSPG.

Animals↗
PubMed1988

Purine de novo synthesis as the basis of synergism of methotrexate and 6-mercaptopurine in human malignant lymphoblasts of different lineages.

Methotrexate (MTX) causes an inhibition of purine de novo synthesis (PDNS), resulting in increased intracellular availability of 5-phosphoribosyl-1-pyrophosphate (PRPP) in human malignant lymphoblasts with an active PDNS. Normal bone marrow cells and peripheral blood lymphocytes lack this capacity. The increased levels of PRPP can be used for enhanced incorporation of 6-mercaptopurine (6MP), indicating a potential time-, sequence- and dose-dependent synergism of both drugs. The effects of 0.02 microM and 0.2 microM MTX on the PDNS of MOLT-4 (T-), RAJI (B-) and KM-3 (non-B-non-T-) human malignant lymphoblasts were studied with respect to PRPP levels, aminoimidazolecarboxamide ribonucleosidemonophosphate (AICAR) levels and the incorporation of labeled glycine into purine metabolites. These results were correlated with the activity of the PDNS (labeled glycine incorporation) and the purine salvage pathway (labeled hypoxanthine incorporation) in untreated cells. Inhibition of PDNS by 0.02 microM MTX was complete in KM-3 cells with a moderately active PDNS and salvage pathway. RAJI cells, with a relatively low PDNS and high salvage pathway, demonstrated an incomplete, but increasing inhibition of PDNS, whereas inhibition of PDNS in MOLT-4 cells with both pathways active was minimal and recovered in time. Treatment with 0.2 microM MTX resulted in a complete inhibition of PDNS in all cell lines. After treatment with MTX an enhanced incorporation of labeled hypoxanthine and 6MP was noticed, confirming the potential rescue from MTX cytotoxicity by hypoxanthine and a potential synergism of MTX and 6MP on cytotoxicity. The enhanced incorporation of 6MP was more obvious in RAJI and KM-3 cells in comparison with MOLT-4 cells. These data demonstrate the important role of both the activities of the PDNS and the purine salvage pathway in malignant lymphoblasts of different subclasses with respect to the synergism of MTX and 6MP.

Aminoimidazole Carboxamide↗
PubMed1988

Effects of methotrexate on purine and pyrimidine metabolism and cell-kinetic parameters in human malignant lymphoblasts of different lineages.

MOLT-4 (T-), RAJI (B-), and KM-3 (non-B-non-T-, common ALL) malignant lymphoblasts demonstrated significant differences in their activities of purine de novo synthesis (PDNS) and purine salvage pathway and in their cell-kinetic parameters. Incubations with concentrations of methotrexate (0.02 and 0.2 microM), which can be maintained during many hours in the oral maintenance therapy of acute lymphoblastic leukemia, indicated large differences between the three cell lines with respect to the inhibition of PDNS, depending on the concentration of methotrexate (MTX) and on the activities of the two pathways. These dose- and cell line-dependent differences corresponded to the perturbations of cell-kinetics and purine and pyrimidine (deoxy)ribonucleotide pools in the three cell lines. Exposure of MOLT-4 cells to 0.02 microM MTX resulted in an incomplete inhibition of DNA synthesis in early S phase, as shown by DNA-flow cytometry and increase of dCTP levels, which recovered spontaneously after 48 hr. Almost no impairment of RNA synthesis occurred (unbalanced growth). In RAJI cells, exposed to 0.02 microM MTX, DNA synthesis was delayed in the S phase, not arrested, and RNA synthesis was not impaired, also indicating an unbalanced growth pattern, which, however, did not recover in time. KM-3 cells were arrested in G1 phase and subsequently in early S phase after incubation with 0.02 microM MTX, and perturbations of ribonucleotides indicated a complete inhibition of RNA synthesis, resulting in a balanced growth pattern. Cytotoxicity was more pronounced in KM-3 cells. The reliability of the soft agar colony forming assay after low dose MTX treatment is discussed. Exposure of MOLT-4 and KM-3 cells to 0.2 microM MTX resulted in a complete inhibition of DNA synthesis, with cessation of cell progression through all parts of the cell cycle and arrest in G1 phase. RAJI cells showed an increasing accumulation of cells in G1 phase without complete cessation of cell cycle progression. Perturbations of ribonucleotide pools suggested an inhibition of RNA synthesis in all cell lines, indicating a balanced growth pattern in KM-3 cells and MOLT-4 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Cycle↗
PubMed1986

Sequence-, time- and dose-dependent synergism of methotrexate and 6-mercaptopurine in malignant human T-lymphoblasts.

Methotrexate (MTX) and 6-mercaptopurine (6MP) are common drugs in the oral maintenance therapy of acute lymphoblastic leukemia (ALL). On the basis of their biochemical effects on cell metabolism, a sequence-dependent synergism might be anticipated. In order to investigate this hypothesis, MOLT-4 human malignant T-lymphoblasts were incubated with various concentrations of MTX. The time at which maximal increase of intracellular 5-phosphoribosyl-1-pyrophosphate (PRPP) levels was found correlated with the concentrations of MTX used. Determination of aminoimidazolecarboxamide ribonucleoside monophosphate (AICAR) levels and labeled glycine incorporation into purine metabolites revealed an incomplete inhibition of purine de novo synthesis after incubation with 0.02 microM MTX, and a complete inhibition with 0.2 microM MTX. After prolonged periods of incubation, glutamine exhaustion of the medium caused inhibition of purine de novo synthesis in MTX-untreated cells, with a concomitant increase of PRPP levels. Addition of glutamine to the medium prevented this phenomenon. The increased availability of PRPP after pretreatment with MTX can be used for enhanced intracellular incorporation of hypoxanthine and 6MP in their respective nucleotides. The time- and dose-dependent effects of MTX on PRPP levels correlated with the enhanced incorporation of hypoxanthine and 6MP. The data presented in this study demonstrate that a synergistic action of the combination of MTX and 6MP can be anticipated in malignant lymphoblasts with an active purine de novo synthesis depending on the concentration of MTX and on the time and sequence of administration of both drugs.

Aminoimidazole Carboxamide↗