Synovial fluid chondroitin and keratan sulphate epitopes, glycosaminoglycans, and hyaluronan in arthritic and normal knees

OBJECTIVES—To determine concentrations of chondroitin sulphate (CS) and keratan sulphate (KS) epitopes, glycosaminoglycans (GAGs) and hyaluronan (HA) in knee synovial fluid (SF) from normal subjects and patients with osteoarthritis (OA) or rheumatoid arthritis (RA), to test whether these variables may be used as markers of the OA process.
METHODS—OA was subdivided into large joint OA (LJOA), nodal generalised OA (NGOA), and OA with calcium pyrophosphate crystal deposition (CPA). Clinical assessment of inflammation (0-6) was undertaken on OA and RA knees. Knee SF was examined by enzyme linked immunosorbent assay for: CS epitopes, using monoclonal antibodies 3-B-3 and 7-D-4; KS epitope using monoclonal antibody 5-D-4; and HA, using biotinylated HA binding region of cartilage proteoglycan. Total sulphated GAGs were measured by dye binding with 1:9 dimethylmethylene blue.
RESULTS—Increased SF 3-B-3 concentrations and 3-B-3/GAG ratio were found in OA, compared with RA or normal knees, with higher 3-B-3 and 3-B-3/GAG in LJOA and NGOA than in CPA. SF 7-D-4 and 7-D-4/GAG were reduced in RA, compared with normal and OA; SF 5-D-4 was reduced in OA compared with normal. GAG and HA concentrations were decreased in both OA and RA. No correlations with radiographic scores were observed, but SF 7-D-4 was lower in `inflamed' compared with `non-inflamed' RA and OA knees. In patients with bilateral samples there were strong correlations between right and left knees for all SF variables.
CONCLUSIONS—Changed concentrations of SF CS and KS can be detected in OA with a profile that differs from that seen in RA. Clinical subgrouping and local joint inflammation may influence these measures, supporting different pathogenesis within OA subgroups and requirement for careful patient characterisation in SF studies.

     

Chondroitin and keratan sulphate epitopes, glycosaminoglycans, and hyaluronan in progressive versus non-progressive osteoarthritis

OBJECTIVE—To determine if a single time point estimation of chondroitin sulphate (CS) or keratan sulphate (KS) epitopes, hyaluronan (HA), or total glycosaminoglycans (GAG) in knee synovial fluid at time of hospital referral can predict subsequent radiographic progression of knee osteoarthritis.
METHODS—Two groups of hospital referred patients with knee osteoarthritis were compared: (1) a "progressive" group (n = 45), showing further reduction in radiographic joint space of at least one grade (0-3) in at least one compartment; and (2) a "non-progressive" group (n = 25) in whom radiographs showed no change during the mean follow up period of 2.3 years (median 2, range 1 to 5 years). Knee synovial fluid obtained at the first visit was examined by ELISA for: CS epitopes, using monoclonal antibodies 3B3 and 7D4; KS epitope, using monoclonal antibody 5D4; and HA, using biotinylated HA binding region of cartilage proteoglycan. Total sulphated GAG were measured by dye binding with 1:9 dimethylmethylene blue.
RESULTS—In patients with bilateral synovial fluid data right and left knee values were closely correlated for all variables. There were no significant differences between CS and KS epitopes, HA, total sulphated GAG, or ratios of individual CS or KS epitopes to total GAG, between progressive and non-progressive groups.
CONCLUSIONS—Single time point estimation of CS, KS, HA, or total GAG in synovial fluid does not distinguish radiographically progressive and non-progressive knee osteoarthritis patients followed for two years.

     

Sex hormones and metabolism of glycosaminoglycans. I. Effect of orchidectomy and administration of testosterone in rabbits

The effect of orchidectomy in male rabbits and administration of testosterone to orchidectomized animals on the metabolism of glycosaminoglycans (GAG) has been studied. The response of the different GAG fractions in the aorta varies with the nature of the GAG, and in some cases is different in different segments of the aorta. Orchidectomy produced an increase in hyaluronic acid fraction, decrease in heparin sulphate fraction, and no response in the chondroitin sulphate A fraction in the aortic arch, thoracic aorta, and abdominal aorta. Chondroitin sulphate C and chondroitin sulphate B fractions decreased only in the abdominal aorta and were not significantly altered in the other two segments, while heparin fraction decreased only in the thoracic aorta and was not affected in the other segments. Administration of testosterone to the orchidectomized animals counteracted these changes in the aortic GAG fractions. The enzymes concerned with the synthesis of precursors of GAG—L-glutamine: D-fructose-6-phosphate aminotransferase, UDPG dehydrogenase, and UDPG pyrophosphorylase—all decreased in the orchidectomized animals; testosterone administration increased their activity in the orchidectomized animals. Enzymes concerned with degradation of GAG—β-glucuronidase, β-hexosaminidase, aryl sulphatase, cathepsin, and hyaluronidase—increased in the orchidectomized animals and decreased on administration of testosterone. Concentration of PAPS and activity of sulphate-activating system and sulphotransferase also decreased in the orchidectomized animals, and testosterone administration tended to restore this decrease to normal levels.     

Use of avidin-biotin-glucose oxidase complex to detect antimalarial antibody in serum by light microscopy

An immunohistochemical assay was developed combining an avidin-biotin-glucose oxidase complex procedure (ABC-GO) with light microscopy to detect specific antibody against Plasmodium falciparum. Thin blood films were prepared from culture material of P. falciparum and fixed with acetone. Antibody was detected by successive incubations with test serum, biotinylated goat antihuman antibody, avidin-biotin-glucose oxidase complex, and glucose oxidase substrate. In the presence of reactive serum, a blue precipitate formed on the parasites and could be visually observed with a 40x objective. Sera from patients with single infections for P. vivax or P. ovale were unreactive. No cross-reactivity was observed with sera from patients with rheumatoid arthritis, filariasis, amebiasis, schistosomiasis, dengue, scrub typhus, leptospirosis, or toxoplasmosis. The sensitivity of ABC-GO is comparable to that of the indirect fluorescent antibody test.     

A simple monoclonal antibody based ELISA for free protein S. Comparison with PEG precipitation

A simple monoclonal antibody based ELISA for free Protein S, compatible with out existing ELISA for total Protein S has been developed, and its performance compared with the conventional PEG precipitation method of free Protein S assay. The normal range (mean±2 SD ) was 0.19–0.54 iu/ml free Protein S. The mean intra assay variation was 5.24% and the mean inter assay variation was 5.50%. A total of 102 routine diagnostic samples from patients referred for prothrombotic investigation (six assays for each method), were assayed by PEG precipitation (mean 0.32 iu/ml, SD 10.60), and the monoclonal ELISA (mean 0.34, SD 0.9). Paired t-test analysis of the two data sets indicated no significant difference between them (P < 0.001). In this sample population, there was no significant difference in free Protein S values when assayed by monoclonal based ELISA or by PEG precipitation. The monoclonal assay has proved to be reliable, accurate and precise. The monoclonal ELISA is simpler, quicker and easier to perform in routine use. Data generated is directly comparable to that generated by PEG precipitation. This methodology would be suitable for laboratories currently measuring Protein S by ELISA.     

Glucose buffer is suitable for blood group conversion with α-N acetylgalactosaminidase and α-galactosidase

Background

It is well known that the buffer plays a key role in the enzymatic reaction involved in blood group conversion. In previous study, we showed that a glycine buffer is suitable for A to O or B to O blood group conversion. In this study, we investigated the use of 5% glucose and other buffers for A to O or B to O blood group conversion by α-N-acetylgalactosaminidase or α-galactosidase.

Materials and methods

We compared the binding ability of α-N-acetylgalactosaminidase/α-galactosidase with red blood cells (RBC) in different reaction buffers, such as normal saline, phosphate-buffered saline (PBS), a disodium hydrogen phosphate-based buffer (PCS), and 5% commercial glucose solution. The doses of enzymes necessary for the A/B to O conversion in different reaction buffers were determined and compared. The enzymes’ ability to bind to RBC was evaluated by western blotting, and routine blood typing and fluorescence activated cell sorting was used to evaluate B/A to O conversion efficiency.

Results

The A to O conversion efficiency in glucose buffer was similar to that in glycine buffer with the same dose (>0.06 mg/mL pRBC). B to O conversion efficiency in glucose buffer was also similar to that in glycine buffer with the same dose (>0.005 mg/mL pRBC). Most enzymes could bind with RBC in glycine or glucose buffer, but few enzymes could bind with RBC in PBS, PCS, or normal saline.

Conclusion

These results indicate that 5% glucose solution provides a suitable condition for enzymolysis, especially for enzymes combining with RBC. Meanwhile, the conversion efficiency of A/B to O was similar in glucose buffer and glycine buffer. Moreover, 5% glucose solution has been used for years in venous transfusion, it is safe for humans and its cost is lower. Our results do, therefore, suggest that 5% glucose solution could become a novel suitable buffer for A/B to O blood group conversion.     

Investigations on glucose uptake in isolated human leucocytes from normal and diabetic subjects

Summary 1. Leucocyte preparations have been obtained from the blood of 135 healthy control persons and 10 poorly-controlled insulin-dependent diabetics according to a procedure described earlier. Cells were suspended in a Krebs-Ringer-Bicarbonate-Buffer. Glucose uptake, lactate production and cell glucose space were determined after incubating the cells for 1 h at 37° C in an atmosphere of O₂/CO₂ (95/5). Extracellular water-space of the cell sediment was corrected by measuring U-¹⁴C-sucrose levels in the medium before and after addition to the cell sediment. — 2. The cells showed intact structures and amoeboid motility under the light-microscope as well as under the phase-contrast-microscope. During an incubation lasting 1 h, the ATP/ADP quotient diminished by about 20%; the following metabolites: glucose-6-phosphate, fructose-1,6-diphosphate, 1,3-phosphoglycerate, pyruvate and lactate increased slightly up to markedly, especially glucose-6-phosphate and lactate. — 3. Sources of methodological errors were investigated in preliminary experiments. Disregarding corrections for the extracellular space of the cell sediments led to a dilution effect which imitates an apparent glucose uptake. For measurements of intracellular glucose and cell glucose space the procedure of Crofford and Renold provided the best results. Falsely high values for cell glucose have been found using the glucose oxidase reaction. — 4. Glucose uptake of healthy leucocytes increases at first steeply, later on less with rising medium glucose concentrations. Addition of insulin (50–500 mU/ml) gave no consistent effects. At medium glucose concentrations of 400 and 600 mg/100 ml intracellular glucose could be demonstrated with statistical significance. Intracellular glucose and cell glucose space were not affected by insulin to any marked degree. Determinations of lactate production indicated that human leucocytes utilize the largest portion of glucose via glycolysis. — 5. At medium glucose concentrations of 600 mg/100 ml leucocytes from diabetics who had received no insulin for 14 h prior to incubation showed a significant diminution of glucose uptake. — 6. Theoretical curves for inward transport and phosphorylation have been calculated from the data for glucose uptake and intracellular glucose concentration.The authors are greatly indepted to Prof. Wilbrandt, Department of Pharmacology, University Bern, for advice and discussion.     

A serum inhibitor of insulin action on muscle. II. Quantitative assessement of inhibitory activity

Summary 1. The intraperitoneal assay of insulin has been used to compare the effectiveness of insulin injected in serum with that of insulin injected in 5% albumin. The difference was interpreted as being due to a serum inhibitor of insulin action on muscle. — 2. To obtain a quantitative assessment of the inhibitory activity, the insulin effectiveness was estimated by the arithmetic subtraction of the effect of the serum alone from that of the insulin added to the serum. Since the effect of insulin is related to the logarithm of the dose, such a subtraction requires that the two forms of insulin-like activity are essentially different. — 3. Dose-response curves for insulin injected in albumin and in serum were parallel, despite the considerable ILA of the serum; and analysis of the phenomenon clearly shows that the effects of the serum ILA and the added insulin were arithmetically additive. — 4. The ILA of the serum was distinguishable from that of the added insulin in not being suppressed by potent anti-insulin serum in the intraperitoneal assay system used. — 5. The consistency in the results obtained by the application of this procedure to the data of a variety of experiments provides effective support for the procedure.Supported by United States Public Health Service, Grant No. F2, AM-23, 661.     

The synaptic proteins neurexins and neuroligins are widely expressed in the vascular system and contribute to its functions

Unlike other neuronal counterparts, primary synaptic proteins are not known to be involved in vascular physiology. Here, we demonstrate that neurexins and neuroligins, which constitute large and complex families of fundamental players in synaptic activity, are produced and processed by endothelial and vascular smooth muscle cells throughout the vasculature. Moreover, they are dynamically regulated during vessel remodeling and form endogenous complexes in large vessels as well as in the brain. We used the chicken chorioallantoic membrane as a system to pursue functional studies and demonstrate that a monoclonal recombinant antibody against β-neurexin inhibits angiogenesis, whereas exogenous neuroligin has a role in promoting angiogenesis. Finally, as an insight into the mechanism of action of β-neurexin, we show that the anti-β-neurexin antibody influences vessel tone in isolated chicken arteries. Our finding strongly supports the idea that even the most complex and plastic events taking place in the nervous system (i.e., synaptic activity) share molecular cues with the vascular system.     

The pattern of basal and stimulated insulin responses to intravenous glucose in first degree relatives of Type 1 (insulin-dependent) diabetic children and unrelated adults aged 5 to 50 years

Summary The pattern of insulin secretion was studied in 107 normal individuals aged 5 to 50 years. Intravenous glucose tolerance tests were performed on 64 islet-cell antibody negative siblings of diabetic children and on 43 normal adults. Puberty was staged using Tanner's criteria and subjects were grouped as follows: I — stage 1 (n=22), II — stages 2 and 3 (n=18), III — stages 4 and 5 (n=20), IV — adults >17 years (n=47). Basal and stimulated (incremental 0–10 and 10–60 min areas) insulin responses rose throughout puberty (Groups I–III), declined following puberty until the third decade (Groups III and IV) and then appeared constant thereafter. Insulin levels in the 17.6–22.5 year group were lower than in the 12.6–15 year group (p<0.01). Fasting insulin to glucose ratios and incremental 0–60 min insulin to glucose area ratios produced a similar age-related pattern indicating that changes in insulin levels were independent of glucose concentrations. Gender did not affect these changes and multiple regression analysis showed that HLA haplotype sharing did not influence insulin responses in siblings of diabetic patients. Age and pubertal status must be carefully considered when interpreting intravenous glucose tolerance tests from patients suspected of having early abnormalities of carbohydrate metabolism.     

Therapie bösartiger Hämoblastosen mit monoklonalen Antikörpern

The success of rituximab, the first monoclonal antibody ever licensed for the treatment of a human malignancy, has not only increased survival and cure rates in many non-Hodgkin lymphomas of the B-cell type, but has prompted an explosion in the development of novel antibodies and biologically active substances with specific cellular targets (“targeted therapy”) both in the field of hematological malignancies and solid tumors. The chimeric anti-CD20 monoclonal antibody rituximab is the first drug that was shown to increase overall survival in follicular lymphomas and to cut lymphoma-associated deaths in patients with CD20⁺ aggressive lymphomas into half. In this review the current role of monoclonal antibodies in the treatment of different hematological neoplasms will be discussed and perspectives for their future use in leukemia and lymphomas will be shown.     

Monoclonal Antibodies to Human Lysyl Oxidase

Hybridoma antibodies against human lysyl oxidase were produced by fusing Sp. 2.0-Ag 14 myeloma cells with spleen cells from mice hyperimmunized with lysyl oxidase isolated from umbilical cords. Hybridomas positive by enzyme-linked immunosorbent assay (ELISA) for human lysyl oxidase were cloned by the dilution method. Eight hybridomas producing antibodies were isolated, three of which also recognized purified bovine aortic lysyl oxidase in an ELISA. One of these antibodies, monoclonal antibody I, was purified and attached to Sepharose CL-4B. The immobilized antibody was effective in binding an enzymatically active 30,000 dalton species. Immunoblot analysis of four of these antibodies showed reactivity against dic 30,000 dalton catalytically active enzyme and the 24,000 dalton fragment of lysyl oxidase. These monoclonal antibodies should be useful tools for studying the localization and biosynthesis of lysyl oxidase.     

The Evolving Role of Monoclonal Antibodies and Dendritic Cell Therapy in Hematologic Malignancies

The approach for treatment of hematological cancers had changed in the last decade from non-specific eradication of tumor cells by chemotherapy to more specific strategies by activation of immune system.

There are number of potential targets of immune responses in patients with hematological malignancies. Some have been developed like monoclonal antibody therapy and others that have yet to be define like dendritic cell infusion.

In this review, we will discuss the evolving role of monoclonal antibody therapy and donor dendritic cell infusion in mounting on immune response in hematological malignancies.     

A sensitive double antibody radioimmunoassay for Human Growth hormone (HGH): levels of serum HGH following rapid tolbutamide infusion

Summary A double antibody radio-immunoassay for human growth hormone is described. — The assay can detect 0.0625 mg HGH/ml serum and has good reproducibility. It was found that: 1. a highly pure labelled hormone; 2. a specific and very potent guinea pig antihuman growth hormone antibody; and 3. at least five days of incubation for the first reaction were necessary to achieve this accuracy and sensitivity. -Porcine and rat growth hormone, sera from cow, guinea pig, rabbit, mouse, and toad fish did not react with the guinea pig anti-HGH serum used in the assay. — In four patients after hypophysectomy, HGH concentrations disappeared almost completely, and in another patient no rise of the hormone was seen during an IV insulin tolerance test.-Undiluted human serum appears to produce falsely high levels of HGH. — Normal males exhibited fasting HGH levels from 0 –2.2 mg/ml (mean 0.8 mg/ml). Females ranged from 0.6–15.0 mg/ml (mean 5.1 mg/ml) and 15 acromegalics from 8.0–103.0mg/ml (mean 31.2 mg/ml). — During a rapid tolbutamide tolerance test, serum HGH rose between 2.5- and 82-fold over the fasting levels within 10 to 70 minutes following the glucose nadir.Performed in part during a postdoctoral fellowship Stiftung Volkswagenwerk, Germany.     

Crystal structure of human prion protein bound to a therapeutic antibody

Prion infection is characterized by the conversion of host cellular prion protein (PrP^C) into disease-related conformers (PrP^Sc) and can be arrested in vivo by passive immunization with anti-PrP monoclonal antibodies. Here, we show that the ability of an antibody to cure prion-infected cells correlates with its binding affinity for PrP^C rather than PrP^Sc. We have visualized this interaction at the molecular level by determining the crystal structure of human PrP bound to the Fab fragment of monoclonal antibody ICSM 18, which has the highest affinity for PrP^C and the highest therapeutic potency in vitro and in vivo. In this crystal structure, human PrP is observed in its native PrP^C conformation. Interactions between neighboring PrP molecules in the crystal structure are mediated by close homotypic contacts between residues at position 129 that lead to the formation of a 4-strand intermolecular β-sheet. The importance of this residue in mediating protein–protein contact could explain the genetic susceptibility and prion strain selection determined by polymorphic residue 129 in human prion disease, one of the strongest common susceptibility polymorphisms known in any human disease.     

Serum insulin in acromegaly

Summary The response of serum insulin to 100 g of glucose by mouth was determined in 23 acromegalics simultaneously by radioïmmunoassay and by assay on adipose tissue and muscle. The values obtained were compared with those of a normal group. 8 patients suffered from active acromegaly, and 15 were in an inactive phase of the disease. The method used by us for the determination of insulin-like activity on mouse-diaphragm in vivo is described. 1. After an oral glucose load, the changes in the concentrations of serum insulin, determined by radioimmunoassay, and of insulin-like activities, estimated by assay on adipose tissue and muscle, were largely parallel. — 2. At all interval serum insulin levels were higher in the acromegalic group than in the normal one. — 3. Patients with active acromegaly showed significantly higher 1 and 2 h values than the group with inactive acromegaly. — 4. No significant differences of insulin concentrations were found between the acromegalic groups with and without impairment of carbohydrate tolerance. — 5. In four patients investigated before and after irradiation or operation of the pituitary, there was a significant drop of serum insulin 1 and 2 h after oral glucose. — 6. Comparing the various insulin levels in the subgroups with different degrees of activity of acromegaly and in the patients seen before and after therapy, we found a parallel change of the Insulinogenic Index (Seltzer). — There is good agreement between the response of serum insulin as determined by radioimmunoassay and that of the insulin-like activity measured on fat and muscle tissue, in acromegaly. Each of the three methods used permits the assessment of the degree of activity of an acromegaly and the success of an intervention.We wish to express our gratitude to the Deutsche Forschungsgemeinschaft and the Landesamt für Forschung of Nordrhein-Westfalen for grants which made this work possible.     

Treatment of hematological malignancies with monoclonal antibodies

The success of rituximab, the first monoclonal antibody ever licensed for the treatment of a human malignancy, has not only increased survival and cure rates in many non-Hodgkin lymphomas of the B-cell type, but has prompted an explosion in the development of novel antibodies and biologically active substances with specific cellular targets ("targeted therapy") both in the field of hematological malignancies and solid tumors. The chimeric anti-CD20 monoclonal antibody rituximab is the first drug that was shown to increase overall survival in follicular lymphomas and to cut lymphoma-associated deaths in patients with CD20+ aggressive lymphomas into half. In this review the current role of monoclonal antibodies in the treatment of different hematological neoplasms will be discussed and perspectives for their future use in leukemia and lymphomas will be shown.     

Prenatal diagnosis of molybdenum cofactor deficiency by assay of sulphite oxidase activity in chorionic villus samples

Summary Molybdenum cofactor deficiency is characterized by the absence of sulphite oxidase, xanthine dehydrogenase and aldehyde oxidase, the three known enzymes in man that require the cofactor for their activity. Prenatal diagnosis of the deficiency may be performed by assay of sulphite oxidase activity in cultured amniocytes. However, the activity in amniocytes is low and large numbers of cells are required for reliable assessment. We show that sulphite oxidase is present at high levels in chorionic villi obtained at 10–14 weeks gestation and can be assayed directly in the biopsy sample without cell culture. This assay has been applied to two pregnancies at risk for molybdenum cofactor deficiency with successful diagnoses of an unaffected and an affected fetus.     

夹心免疫-PCR检测旋毛虫循环抗原的研究(英文)

目的建立以多抗为捕获抗体和单抗为检测抗体的夹心免疫-PCR检测旋毛虫循环抗原。方法利用杂交瘤技术制备抗旋毛虫单抗;旋毛虫抗原免疫家兔,分离纯化兔血清,制备兔抗旋毛虫多抗。多抗作为捕获抗体包被酶标板,单抗为检测抗体,选择适宜多抗和单抗浓度,建立夹心ELISA方法,再利用亲和素和生物素的高结合力连接起标记了生物素的抗体和DNA片段,将抗原抗体特异反应的免疫技术同无限扩增DNA片段的基因检测技术结合,利用PCR反应对DNA片段的扩增能力,提高检测灵敏度。结果制备了兔抗旋毛虫多抗,间接ELISA法筛选出与多种寄生虫抗原无交叉反应的单抗F4C6,夹心ELISA检测旋毛虫抗原最低浓度为0.05μg/ml,免疫PCR方法检测最低浓度为0.1ng/ml。结论首次建立夹心免疫-PCR检测旋毛虫抗原的方法,检测旋毛虫抗原的灵敏度高于传统ELISA方法104倍。     

The pathogenesis of tumour hypoglycaemia: Blocks of hepatic glucose release and of adipose tissue lipolysis

Summary Earlier findings on the pathogenesis of tumour hypoglycaemia [15] were confirmed and extended in a second patient with this disease. — A block of hepatic glucose release was found to be the main cause of hypoglycaemia in both patients suffering from large tumours of non-endocrine origin. The free fatty acid level failed to increase upon hypoglycaemia. Low free fatty acid levels correlated with an increased rate of glucose assimilation and glucose oxidation. — Immunoreactive, suppressible and nonsuppressible ILA measuredin vitro andin vivo were normal. — However, the serum of patient Z.B. inhibited lipolysis of adipose tissuein vitro to a greater extent than serum of normal subjects. This difference was no longer present after dialysis of the sera and the antilipolytic activity was now found in the diffusate. — The block of hepatic glucose release may be overcome by a pharmacological dose of intravenous glucagon. The block of hepatic glucose release is of paramount importance for the development of hypoglycaemia since the pharmacological blocking of lipolysis alone does not lead to hypoglycaemia, although it may increase glucose assimilation and glucose oxidation. — An attempt is being made to characterize further the antilipolytic substance which is present in increased amounts in the serum of patients with tumour hypoglycaemia.This work was supported by grants from the Schweizerische Nationalfonds (3336) and from the U.S. Public Health Service (AM 5387).