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PubMed1999

Serum and urinary concentrations of heparan sulfate in patients with diabetic nephropathy.

BACKGROUND: Heparan sulfate (HS) contributes to the negative charge in the glomerular basement membrane (GBM), which may maintain the GBM charge barrier. Changes in sulfation and/or the concentration of HS may be associated with the development of diabetic nephropathy. METHODS: Using two different antibodies specific for HS chains, one that reacts with the N-sulfated sequences in HS chains (10E4) and the other that reacts with neo-epitope of HS, which occurs after heparitinase digestion of HS chains (3G10), we examined the serum and urinary concentrations of HS by enzyme-linked immunosorbent assay and performed immunohistochemical staining of glomeruli in diabetic patients with and without nephropathy. RESULTS: The level of urinary excretion of 10E4 binding HS/creatinine clearance was significantly reduced in diabetic patients when compared with that in nondiabetic subjects (P < 0.0001), and the level was more decreased in patients with overt nephropathy than in patients without overt nephropathy. No differences or only small differences were found between these groups in serum and urinary 3G10-binding HS and in serum 10E4-binding HS. Immunohistochemical staining with these antibodies was consistent with the findings in the urine. CONCLUSIONS: The results suggest that a decreased HS N-sulfation exists in the urine, which may reflect a structural change or an altered processing of HS within the GBM. Because N-sulfation plays a key role in determining the extent of sulfation within the HS chains, the decreased urinary 10E4-binding HS may have potential implications with regard to the development of diabetic nephropathy.

Adult↗
PubMed1998

Generation and application of type-specific anti-heparan sulfate antibodies using phage display technology. Further evidence for heparan sulfate heterogeneity in the kidney.

Detailed analysis of various heparan sulfate (HS) species is seriously hampered by a lack of appropriate tools, such as antibodies. We adopted phage display technology to generate anti-HS antibodies. A "single pot" semisynthetic human antibody phage display library was subjected to four rounds of selection on HS from bovine kidney using panning methodology. Three different phage clones expressing anti-HS single chain variable fragment antibodies (HS4C3, HS4D10, and HS3G8) were isolated, with an amino acid sequence of the complementarity-determining region 3 of GRRLKD (VH3 gene, DP-38), SLRMNGCGAHQ (VH3 gene, DP-42), and YYHYKVN (VH1 gene, DP-8), respectively. The antibodies react with HS and heparin, but not with DNA or other glycosaminoglycans. Kd values for HS are about 0.1 microM. The three antibodies react differently toward various HS preparations and show different staining patterns on rat kidney sections, indicating recognition of different HS molecules. This also holds for two described mouse anti-HS IgMs (JM403 and 10E4; both generated by conventional hybridoma technique) and indicates the presence of at least 5 different HS species in the kidney. O- and N-sulfation are important for binding of HS to HS4C3 and HS3G8. The three single chain antibodies, but not JM403, block a basic fibroblast growth factor binding site of HS. It is concluded that phage display technology presents a powerful technique to generate antibodies specific for HS epitopes. This is the first time this technique has been successfully applied to obtain directly antibodies to (poly)saccharides.

Amino Acid Sequence↗
PubMed2002

Heparan sulfate 3-O-sulfotransferase isoform 5 generates both an antithrombin-binding site and an entry receptor for herpes simplex virus, type 1.

Heparan sulfate 3-O-sulfotransferase transfers sulfate to the 3-OH position of a glucosamine residue of heparan sulfate (HS) to form 3-O-sulfated HS. The 3-O-sulfated glucosamine residue contributes to two important biological functions of HS: binding to antithrombin and thereby carrying anticoagulant activity, and binding to herpes simplex viral envelope glycoprotein D to serve as an entry receptor for herpes simplex virus 1. A total of five HS 3-O-sulfotransferase isoforms were reported previously. Here we report the isolation and characterization of a novel HS 3-O-sulfotransferase isoform, designated as HS 3-O-sulfotransferase isoform 5 (3-OST-5). 3-OST-5 cDNA was isolated from a human placenta cDNA library and expressed in COS-7 cells. The disaccharide analysis of 3-OST-5-modified HS revealed that 3-OST-5 generated at least three 3-O-sulfated disaccharides as follows: IdoUA2S-AnMan3S, GlcUA-AnMan3S6S, and IdoUA2S-AnMan3S6S. Transfection of the plasmid expressing 3-OST-5 rendered wild type Chinese hamster ovary cells susceptible to the infection by herpes simplex virus 1, suggesting that 3-OST-5-modified HS serves as an entry receptor for herpes simplex virus 1. In addition, 3-OST-5-modified HS bound to herpes simplex viral envelope protein glycoprotein D. Furthermore, we found that 3-OST-5-modified HS also bound to antithrombin, suggesting that 3-OST-5 also produces anticoagulant HS. In summary, our results indicate that a new member of 3-OST family generates both anticoagulant HS and an entry receptor for herpes simplex virus 1. These results provide a new insight regarding the mechanism for the biosynthesis of biologically active HS.

Animals↗
PubMedDate not supplied

Internal mammary artery harvesting using the harmonic scalpel.

Internal mammary artery (IMA) harvesting using the harmonic scalpel (HS) was recently introduced. We studied 541 IMAs harvested by the same surgeon through a standard median sternotomy in 472 coronary bypass patients; 252 (47%) with the HS, while 289 (53%) were with electrocautery (EC). Patient demographics included mean ages: 67 years HS vs. 65 years EC (p = NS); male:female ratio: 3:1; and insulin dependent diabetes mellitus (IDDM): 11% HS vs. 12.5% EC (NS). Mean ultrasonic IMA flow at a mean SBP of 70 mm Hg in 10 consecutive patients of each group were: preharvest, HS 11.9 +/- 2.3 ml/min vs. EC 8.5 +/- 1.6 ml/min (p = 0.256); postharvest, HS 35.7 +/- 10.7 ml/min vs. EC 22 +/- 2.9 ml/min (0.235); and postcardiopulmonary bypass (post-CPB), HS 47.8 +/- 6.2 ml/min vs. EC 41.7 +/- 2.5 ml/min (0.381). Histologic samples of 50 consecutive IMAs showed no evidence of vessel injury in either group. Clinical results revealed postoperative bleeding in 6/217 (2.7%) HS vs. 7/255 (2.7%) EC (p = 0.783), none attributed to bleeding from the IMA; phrenic paresis: 0/217 in HS but 1/255 (0.4%) in EC (p = 0.960); sternal wound infection: 5/217 (2.3%) HS vs. 6/255 (2.4%) EC (p = 0.787); postoperative IABP: 6/217 (2.7%) HS vs. 5/255 (2%) EC (p = 0.859); mortality: 2/217 (0.9%) HS vs. 2/255 (0.8%) EC (p = 0.710). Hemodynamic, histologic, and clinical results were comparable in both groups. The authors believe the HS is safe and effective for IMA harvesting.

Adult↗
PubMed2003

High-salt-induced increase in blood pressure: role of capsaicin-sensitive sensory nerves.

OBJECTIVE: To test the hypothesis that sensory afferents are significant functional components in preventing salt-induced increases in blood pressure. DESIGN AND METHODS: Neonatal Wistar rats were subcutaneously injected with 50 mg/kg capsaicin or vehicle on the first and second days of life. After weaning, male rats were divided into three groups and treated for 4 weeks with: control plus normal (0.5%, CON-NS) or high (4%, CON-HS) sodium diet, and capsaicin pretreatment plus HS diet (CAP-HS). Mean arterial pressure (MAP) and its response to bolus injection of calcitonin gene-related peptide (CGRP) and its antagonist, CGRP(8-37), were measured by carotid arterial catheterization. Radioimmunoassay was used to measure CGRP levels in plasma and dorsal root ganglia (DRG). Expression of components of the CGRP receptor, calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1), was determined by the use of Western blot analysis. RESULTS: Baseline MAP was increased in CAP-HS compared with CON-HS and CON-NS rats, and it was higher in CON-HS than in CON-NS rats. MAP response to exogenous CGRP was enhanced in CAP-HS and CON-HS than in CON-NS rats, but MAP response to CGRP(8-37) was increased only in CON-HS rats. Plasma CGRP levels were not different among three groups, but CGRP content in DRG was decreased in CAP-HS compared with CON-HS and CON-NS rats. CRLR expression in mesenteric resistance arteries was upregulated in CAP-HS and CON-HS compared with CON-NS rats, but RAMP1 content was not different among these groups. CONCLUSION: Chronic high-salt intake upregulates expression of mesenteric CGRP receptors without altering CGRP levels in plasma and DRG. Increased expression of mesenteric CGRP receptors may play a counter regulatory role in attenuating salt-induced increases in blood pressure.

Animals↗
PubMed2006

Characteristics and long-term outcome of 15 episodes of systemic lupus erythematosus-associated hemophagocytic syndrome.

Reactive hemophagocytic syndrome (HS) occurs mainly in the setting of serious infections and lymphomas. HS can occur in the course of 2 active systemic diseases, without simultaneous infection: adult Still disease and systemic lupus erythematosus (SLE). Observations of specific lupus-associated HS are rare, and the long-term outcome of these patients with active SLE is unknown. We retrospectively studied 15 episodes of SLE-associated HS in 12 patients (10 women, 2 men) and noted the long-term outcome. HS occurred at a mean age of 25 years. All patients were febrile with >or=2 cytopenias, and bone marrow aspiration indicated hemophagocytosis. HS revealed SLE in 9 patients and recurred in 3. The main features of SLE-associated HS were a low frequency of hepatosplenomegaly, a high frequency of heart involvement (5 pericarditis, 4 myocarditis requiring transfer to intensive care unit), and a low C-reactive protein level (mean, 15 mg/L). Cutaneous-mucous symptoms of SLE, arthritis, and nephritis were present respectively in 8 (53%), 6 (40%), and 4 (27%) episodes, but symptoms of SLE were absent in 4 episodes at admission. All patients had anti-nuclear antibodies when the HS occurred. Anti-double-stranded DNA antibodies were present in 12 episodes. Treatment was steroids in 14 cases but cyclophosphamide was the only treatment able to control HS in 2 cases. All the cases of SLE-associated HS were controlled by the immunosuppressive regimen. Intravenous immunoglobulins seemed poorly effective. No infectious agent was found. Clinical presentations of the 23 patients with SLE-associated HS described in the literature were reviewed and were similar to those of the current series. The mean follow-up was 88 months (range, 7-240 mo). One patient died at 15 months (sepsis). Among the 5 patients with a follow-up >8 years, 4 always had active disease. During the follow-up of SLE, immunosuppressive drugs were added in 8 patients (cyclophosphamide in 7, azathioprine in 3, mycophenolate mofetil in 2) with significant adverse drug reactions. In the long-term, SLE-associated HS seems to define a severe SLE form with frequent flares, possible HS recurrences, and the need for prolonged immunosuppression.

Adolescent↗
PubMed2004

Enteral administration of high-fat nutrition before and directly after hemorrhagic shock reduces endotoxemia and bacterial translocation.

OBJECTIVE: To determine whether potential enhancement of endotoxin neutralization via high-fat enteral nutrition affects endotoxemia and bacterial translocation after hemorrhage. SUMMARY BACKGROUND DATA: Endotoxin and bacterial translocation due to gut barrier failure are important initiating events in the pathogenesis of sepsis after hemorrhage. Systemic inhibition of endotoxin activity attenuates bacterial translocation and distant organ damage. Triacylglycerol-rich lipoproteins constitute a physiological means of binding and neutralizing endotoxin effectively. We hypothesized that enhancement of triacylglycerol-rich lipoproteins via high-fat enteral nutrition would reduce endotoxemia and prevent bacterial translocation. METHODS: A rat model of nonlethal hemorrhagic shock was used. Hemorrhagic shock (HS) rats were divided into 3 groups: rats starved overnight (HS-S); rats fed with a low-fat enteral diet (HS-LF), and rats receiving a high-fat enteral diet (HS-HF). RESULTS: Circulating triacylglycerol and apolipoprotein B, reflecting the amount of triacylglycerol-rich lipoproteins, were elevated in HS-HF rats compared with both HS-S rats (P <or= 0.005 and P <or= 0.05, respectively) and HS-LF rats (P <or= 0.005 and P <or= 0.05). Circulating endotoxin was lower in HS-HF rats (7.2 +/- 10.2 pg/ml) compared with both HS-S rats (29.1 +/- 13.4 pg/ml, P <or= 0.005) and HS-LF rats (29.9 +/- 5.2 pg/ml, P <or= 0.005). In line, bacterial translocation was lower in HS-HF rats (incidence 4/8 rats; median 3 [range 0-144] cfu/g) compared with both HS-S rats (8/8; 212 [60-483] cfu/g; P = 0.006), and HS-LF rats (8/8; 86 [30-209] cfu/g; P = 0.002). CONCLUSION: This study is the first to show that high-fat enteral nutrition, leading to increased plasma triacylglycerol and apolipoprotein B levels, significantly decreases endotoxemia and bacterial translocation after hemorrhage.

Animals↗
PubMed1996

Transcription of the mutL repair, miaA tRNA modification, hfq pleiotropic regulator, and hflA region protease genes of Escherichia coli K-12 from clustered Esigma32-specific promoters during heat shock.

The amiB-mutL-miaA-hfq-hflX-hflK-hflC superoperon of Escherichia coli contains genes that are important for diverse cellular functions, including DNA mismatch repair (mutL), tRNA modification (miaA), pleiotropic regulation (hfq), and proteolysis (hflX-hflK-hflC). We show that this superoperon contains three E simga(32)-dependent heat shock promoters, P(mutL)HS,P(miaA)HS, and P1(hfq)HS, in addition to four E sigma(70)-dependent promoters, P(mutL), P(miaA), P2(hfq), and P3(hfq). Transcripts from P(mutL)HS and P(miaA)HS were most prominent in vivo during extreme heat shock (50 degrees C), whereas P1(hfq)HS transcripts were detectable under nonshock conditions and increased significantly after heat shock at 50 degrees C. The P(mutL)HS, P(miaA)HS, and P1(hfq)HS transcripts were not detected in an rpoH null mutant. All three promoters were transcribed by E sigma (32) in vitro at 37 degrees C and contain -35 and -10 regions that resemble the E sigma(32) consensus. In experiments to assess the possible physiological relevance of the P(mutL)HS and P(miaA)HS promoters, we found that E. coli prototrophic strain MG 1655 increased in cell mass and remained nearly 100% viable for several hours at 50 degrees C in enriched media. In these cells, a significant fraction of mutL and hfq-hflA region transcripts were from P(mutL)HS and P1(hfq)HS, respectively, and the amounts of the miaA, hfq, hflX, hflK, and hflC transcripts increased in comparison with those in nonstressed cells. The cellular amounts of MutL and the hfq gene product (HF-I protein) were maintained during heat shock at 44 or 50 degrees C. Consistent with their expression patterns, miaA and hfq were essential for growth and viability, respectively, at temperatures of 45 degrees C and above. Together, these results suggest that there is a class of E sigma(32) promoters that functions mainly at high temperatures to ensure E. coli function and survival.

Adenosine Triphosphatases↗
PubMed2005

Hypertonic saline enhances neutrophil elastase release through activation of P2 and A3 receptors.

Hypertonic saline (HS) holds promise as a novel resuscitation fluid for the treatment of trauma patients because HS inhibits polymorphonuclear neutrophil (PMN) activation and thereby prevents host tissue damage and associated posttraumatic complications. However, depending on conditions of cell activation, HS can increase PMN degranulation, which could exacerbate tissue damage in trauma victims. The cellular mechanism by which HS increases degranulation is unknown. In the present study, we tested whether HS-induced ATP release from PMN and feedback via P1 and/or P2 receptors may be involved in the enhancement of degranulation by HS. We found that HS enhances elastase release and ERK and p38 MAPK activation when HS is added after activation of PMN with formyl peptide (fMLP) or phorbol ester (PMA). Agonists of P2 nucleotide and A3 adenosine receptors mimicked these enhancing effects of HS, whereas antagonists of A3 receptors or removal of extracellular ATP with apyrase diminished the response to HS. A1 adenosine receptor antagonists increased the enhancing effect of HS, whereas A1 receptor agonists inhibited elastase release. These data suggest that HS upregulates degranulation via ATP release and positive feedback through P2 and A3 receptors. We propose that these feedback mechanisms can serve as potential pharmacological targets to fine-tune the clinical effectiveness of HS resuscitation.

Adenosine Triphosphate↗
PubMed2005

Conception rates in clomiphene citrate cycles with and without hormone supplementation: a pilot study.

OBJECTIVE: A trial was conducted to examine the effects of a timed sequence of hormone supplementation (HS) with oral estradiol (E(2)) and vaginal progesterone (P) following clomiphene citrate (CC) therapy to determine if this regimen can increase pregnancy rates in CC cycles. METHODS: This study was a randomized, open-label study. Seventy-one oligo-ovulatory women were randomized into one of two groups; those who received CC plus HS (n = 34) and those who received no HS (n = 37). All subjects received 100 mg CC orally from cycle days 3 to 7. Subjects randomized to HS started oral E(2) at a dose of 1.5 mg BID on cycle day 8. All subjects monitored urine luteinizing hormone (LH) levels starting on cycle day 10; additionally, intercourse was encouraged starting on cycle day 10. Subjects receiving HS discontinued E(2) with LH surge and started using vaginal progesterone gel (Prochieve 8%) daily for 2 weeks starting 3 days after LH surge. All subjects had a pregnancy test (Assure) 2 weeks after LH surge. If pregnancy occurred, the subject continued using vaginal progesterone gel daily for an additional 10 weeks. RESULTS: Sixty-five (65) subjects (31 CC plus HS and 34 no HS) completed the study. Fifty of the 65 subjects (77%) (23 [74%] CC plus HS, 27 [79%] no HS) who completed the study ovulated. The mean (range) progesterone (P) concentration for these 50 subjects was 1662.6 (340 to 5690) ng/dL, and mean and median P levels were slightly higher, but not statistically significant, in the HS group compared with the no HS group. Pregnancy rates were clinically, but not statistically, different between the treatment groups. Of the 50 responders, 12% became pregnant (17% CC plus HS, 7% no HS). CONCLUSION: These findings show a potential supportive effect on pregnancy rates in the CC plus oral estradiol and vaginal progesterone gel group compared to CC alone that needs to be confirmed in a larger study.

Adult↗
PubMed1989

3,5,3'-Triiodothyronine determines the viability of GC cells after heat shock.

The heat shock (HS) response is a characteristic disruption of protein synthesis which occurs in cells exposed to a variety of noxious stimuli. The effects of HS on thyroid hormone-responsive GC cells were studied in an attempt to devise an in vitro model for the adaptive changes in thyroid hormone action caused by nonthyroidal disease. HS enhanced GC cell synthesis of 70 K and 90 K proteins in a manner previously described as characteristic of the HS response in many tissues. A step-wise decrease in GC cell viability occurred when cells were exposed to 45 C for 10 to 35 min. HS (45 C, 20 min) resulted in a rapid decrease in binding of T3 to nuclear receptors. Two hours after HS, analysis of T3 binding to isolated nuclei showed a 50% fall in binding capacity (240 fmol/100 micrograms DNA) compared to non-HS control cells (540 fmol/100 micrograms DNA); no difference in dissociation constant (Kd) was observed. The effect of thyroid hormone on cell viability after HS was then determined. Thyroid hormone depletion (less than or equal to 0.02 nM T3) resulted in significantly (P less than 0.05) enhanced cell viability compared to cells cultured with physiological T3 (0.2 nM) after incubation at 45 C for intervals of 10-35 min. This inverse relationship between medium T3 content and cell tolerance of HS occurred over a wide range of T3 concentrations. Mean cell viability after exposure to 45 C for 20 min was 44 +/- 3% in T3-depleted cultures (less than or equal to 0.02 nM), 27 +/- 1% to 32 +/- 5% in cultures containing 0.07-0.5 nM T3, and 13 +/- 3% in cultures containing 5 nM T3. Our results thus characterize the response to HS in GC cells and the relationship of this response to medium T3. Similar to the effect of various nonthyroidal diseases on rat hepatocytes in vivo, HS resulted in a decrease in T3 nuclear receptors. Similar to the adverse effect of thyroid hormone on morbidity in animals with experimental diseases or injury, GC cell viability after HS was inversely related to medium T3 content. Thus the HS response in GC cells may be a valuable in vitro model relevant to the effect on thyroid hormone action caused by nonthyroidal disease.

Animals↗
PubMed1988

An evaluation of cimetidine and ranitidine in the pain relief and acute healing of duodenal ulcer disease.

In a multicenter, single-blind, randomized trial, the effectiveness in pain relief and healing of three regimens of H2-receptor antagonists was compared in 338 patients with endoscopically confirmed active duodenal ulcers, 112 receiving cimetidine 300 mg four times daily (QID), 110 receiving cimetidine 800 mg at bedtime (HS), and 116 receiving ranitidine 300 mg HS. Evaluation of the immediate symptomatic response during the first 24 hours of therapy showed that a greater proportion of patients with nighttime pain were improved on HS regimen and a greater portion of patients with daytime pain were improved on QID regimen. With continued treatment the differences between a once-daily and a four-times-daily regimen disappeared and the HS regimen provided as much daytime pain relief as the QID regimen. For the two once-daily regimens where ranitidine and cimetidine were directly compared, a greater proportion of patients on cimetidine 800 mg HS had an immediate symptomatic improvement than on ranitidine 300 mg HS. When those who smoked were evaluated separately this difference was also evident. During the first 24 hours, a total of 68% of the smokers in the cimetidine HS treatment groups had improvement in daytime pain versus 44% of patients in the ranitidine group at day one. Nighttime pain relief in smokers on day one was apparent in 78% of the patients receiving the cimetidine HS regimen, in contrast to 67% of ranitidine-HS-treated patients. At the completion of a four-week course of treatment, both physicians' and patients' global assessments of the reduction in the severity and frequency of ulcer-related symptoms significantly favored cimetidine HS over ranitidine HS. These results after four weeks of treatment were corroborated by the endoscopy data, which showed 71% of patients in the cimetidine HS group were healed, compared with 63% in the ranitidine HS group. The 300 mg QID cimetidine regimen produced a 69% healing rate. For those patients whose ulcers were unhealed by the end of four weeks of treatment, a further course of treatment produced almost 9C% healing in all treatment groups and symptomatic relief in virtually all patients. None of the regimens was associated with unexpected adverse effects and all patients tolerated the treatments well. The results of this study confirm the efficacy and safety of single bedtime doses of cimetidine or ranitidine, as well as the four-times-daily cimetidine regimen, in the acute treatment of duodenal ulcers.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMed1985

Close and focal contact adhesions of fibroblasts to a fibronectin-containing matrix.

The fibroblast cell line Balb/c 3T3 makes both close and tight-focal adhesive contacts with the plasma fibronectin (pFn)-coated tissue culture substratum. Detachment of these cells mediated by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid leaves tight-focal contacts and a subset of close contacts as substratum-attached material (SAM). The enrichment of heparan sulfate (HS) proteoglycan (HS-PG) in SAM under specific attachment conditions, as well as the recent demonstration of HS binding to pFn or cellular Fn, has evolved a series of experiments with the selective HS-binding protein platelet factor-4 to analyze the requirement of the HS-binding activity of pFn in the formation of these two types of adhesive contacts. In addition, the cell-binding domain (CBD) of pFn, which recognizes an unidentified cell surface receptor, has been isolated free of HS-binding domains after proteolytic cleavage of pFn. These functional studies indicate that the binding of pFn on the substratum to cell surface HS-PG is necessary and sufficient to generate close contacts with transmembrane signaling of this proteoglycan to reorganize lengthy microfilament bundles in the cytoplasm. Cells spread incompletely on CBD alone, form only close contacts, and reorganize highly concentrated actin filaments only in their spiky projections. Furthermore, the formation of tight-focal contacts with associated stress fibers appears to require both reactions of pFn, binding to cell surface HS-PG and to its unidentified receptor. The importance of HS-PG in these functional studies has led to its better biochemical characterization in SAM. Initially formed close contacts of these cells contain principally a large HS-PG that aggregates into high-molecular-weight complexes by some unknown mechanism. With time, there are two different mechanisms of catabolism of HS-PG, one of which includes the liberation of single-chain HS. The importance of these changes in the HS-PG in SAM are now being analyzed with regard to the formation and disappearance of close and tight-focal adhesions.

Animals↗
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↗
PubMed2001

Heparan sulfate proteoglycan expression in the optic chiasm of mouse embryos.

Previous studies have demonstrated that heparan sulfate (HS) proteoglycans (PGs) regulate neurite outgrowth through binding to a variety of cell surface molecules, extracellular matrix proteins, and growth factors. The present study investigated the possible involvement of HS-PGs in retinal axon growth by examining its expression in the retinofugal pathway of mouse embryos by using a monoclonal antibody against the HS epitope. Immunoreactive HS was first detected in all regions of the retina at embryonic day (E) 11. The staining was gradually lost in the central regions and restricted to the retinal periphery at later developmental stages (E12--E16). Prominent staining for HS was consistently found in the retinal fiber layer and at the optic disk, indicating a possible supportive role of HS-PGs in axon growth in the retina. At the ventral diencephalon, immunostaining for HS was first detected at E12, before arrival of any retinal axons. The staining matched closely the neurons that are immunopositive for the stage-specific embryonic antigen 1 (SSEA-1). At E13 to E16, when axons are actively exploring their paths across the chiasm, immunoreactivity for HS was particularly intense at the midline. This characteristic expression pattern suggests a role for HS-PGs in defining the path of early axons in the chiasm and in regulating development of axon divergence at the midline. Furthermore, HS immunoreactivity is substantially reduced at regions flanking both sides of the midline, which coincides spatially to the position of actin-rich growth cones from subpial surface to the deep regions of the optic axon layer at the chiasm. Moreover, at the threshold of the optic tract, immunoreactive HS was localized to deep parts of the fiber layer. These findings indicate that changes in age-related fiber order in the optic chiasm and optic tract of mouse embryos are possibly regulated by a spatially restricted expression of HS-PGs.

Animals↗
PubMed1993

Monoclonal antibodies to heparan sulfate inhibit the formation of thrombin-antithrombin III complexes.

As components of the cell surface and extracellular matrix, heparan sulfate proteoglycans play an important role in the function of most organs, including the vasculature. Autoimmunity to heparan sulfate (HS) glycosaminoglycan epitopes may play a role in tissue injury. Tight skin (TSK) mice suffer a disease resembling scleroderma, an autoimmune disease associated with alterations in the extracellular matrix of the skin and other organs, as well as vascular injury. Studies of autoimmunity to HS were performed in TSK mice employing a solid-phase radioimmunoassay for the detection of anti-HS antibody. An increase in the clonal frequency of hybridomas secreting anti-HS antibody was noted in TSK mice compared to that in control mice. Liquid-phase competitive inhibition specificity studies of IgM and IgG anti-HS monoclonal antibodies demonstrated little cross-reactivity with other glycosaminoglycans. Some cross-reactivity, albeit at lower affinity, with phosphorylated antigens including DNA, cardiolipin, and RNA polymerase were noted. Negatively charged sulfate groups and carboxyl groups played a role in the immunodominant site. Monoclonal antibodies to HS inhibited the binding of HS to antithrombin III and the formation of thrombin-antithrombin III complexes. These results demonstrate autoimmunity to HS in TSK mice. The results also indicate that HS is a high-affinity antigen of pathological significance for anti-DNA and anti-phospholipid antibodies. Thrombosis induced by polyclonal anti-phospholipid antibodies may be mediated by a subset of these antibodies with high affinity for HS. Anti-HS antibodies may promote a procoagulant state by the blockade of HS binding to antithrombin III, inhibiting the accelerated formation of thrombin-antithrombin III complexes.

Animals↗
PubMed1999

Transient exposure to tumor necrosis factor-alpha inhibits collagen accumulation by cultured hypertrophic scar fibroblasts.

BACKGROUND: Hypertrophic scars (HS) are frequent consequences of deep dermal injury, such as deep partial-thickness burns and abrasions, and are characterized by overproduction of collagen. In vitro studies have shown that cultured HS fibroblasts produce elevated levels of collagen and insulin-like growth factor-binding protein 3 (IGFBP-3). Additionally, histological studies have indicated HS contain fewer tumor necrosis factor alpha (TNF-alpha)-positive infiltrating cells and express lower levels of TNF-alpha mRNA, suggesting TNF-alpha, which can inhibit collagen expression in some systems, may function to deactivate the wound healing process in scars. HS also exhibit increased levels of transforming growth factor beta (TGF-beta), a factor that stimulates collagen and extracellular matrix deposition by fibroblasts and also stimulates IGFBP-3 expression. In some systems, IGFBP-3 mediates the effects of TGF-beta. The present study sought to determine the effects of continuous and transient TNF-alpha exposure on collagen and IGFBP-3 expression by cultured HS fibroblasts and to investigate the role of IGFBP-3 in collagen accretion by HS fibroblasts. MATERIALS AND METHODS: Superficial and deep dermal HS fibroblasts from four patients were cultured. Fibroblasts were cultured in serum-free medium and exposed to 0-2 ng/ml TNF-alpha for 0, 1, 4, or 72 h. After 72 h of culture, medium samples were processed for Western blot analysis of type I collagen accumulation or for ligand blot analysis of IGFBP-3 accumulation. The effects of an anti-IGFBP-3 neutralizing antibody on collagen accumulation were also assessed. RESULTS: Treatment of superficial and deep HS fibroblasts with TNF-alpha resulted in dose-dependent decreases in accumulation of both type I collagen and IGFBP-3 in the culture medium (P < 0.01). However, using the anti-IGFBP-3 neutralizing antibody, a causal relationship between decreased IGFBP-3 and decreased collagen accumulation could not be demonstrated. Transient exposure of cultured HS fibroblasts to TNF-alpha for as little as 1 h was as effective as continuous exposure to TNF-alpha for 72 h in inhibiting collagen accumulation. CONCLUSIONS: These results support the hypothesis that TNF-alpha functions as a wound healing deactivation signal that is deficient in HS. Although TNF-alpha inhibited accretion of both collagen and IGFBP-3, the role of IGFBP-3 in HS remains unresolved. This study suggests that transient TNF-alpha exposure may be used to inhibit collagen overaccumulation in HS and that the timing of TNF-alpha exposure following dermal injury may not be critical for this inhibition.

Adult↗
PubMed1995

Basic fibroblast growth factor-heparan sulphate complex in the human dialysis-related amyloidosis.

A major constituent of the amyloid fibrils in dialysis-related amyloidosis is beta 2-microglobulin (beta 2-MG). Heparan sulphates (HS) co-localize with the amyloid fibrils and monocytes/macrophages are commonly found around amyloid deposits, but the role of HS in amyloidogenesis is not yet defined. HS have variable saccharide sequences and can interact specifically with basic fibroblast growth factor (bFGF), a potent chemotactic factor for the monocyte/macrophage. The present investigation was undertaken to look for a functional link between co-localized HS and the pathogenesis of dialysis-related amyloidosis. Using amyloid-enriched ligament, immunohistochemical localization was tested for beta 2-MG, endogenous bFGF, and bFGF-binding portions of HS. For the detection of bFGF-binding portions of HS, the ligament sections were incubated with exogenous bFGF and then with anti-bFGF antibody. The specificity of the interaction between bFGF and HS was established by confirming a concomitant loss of immunoreactivity during selective removal of HS with heparitinase. beta 2-MG, endogenous bFGF, and bFGF-binding portions of HS were detected between bundles of collagen. Endogenous bFGF and bFGF-binding portions of HS were not detected in more advanced amyloid lesions, whereas beta 2-MG and other portions of HS were detected. We propose that beta 2-MG, endogenous bFGF, and bFGF-binding portions of HS form a complex and localize in the early amyloid lesions of dialysis-related amyloidosis.

Amyloidosis↗