Effect of a novel adsorbent on cytokine responsiveness to uremic plasma

BACKGROUND: Middle molecules such as beta2-microglobulin (beta2M) and advanced glycation end products (AGE)-modified proteins contribute to inflammation in uremia. The BetaSorb column is a new adsorptive device, which contains copolymeric beads, suitable for removal of beta2M and other middle molecules. We assessed the effect of this column on the bioreactivity of uremic plasma, as measured by cytokine responsiveness. METHODS: Uremic plasma was perfused in vitro through the column (10 mL/min) and samples were collected after 10 to 30 passes. Endotoxin-stimulated tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) production by THP-1-derived monocytes was measured following brief exposure to uremic plasma. beta2M levels were measured. The contribution of AGE-modified proteins to the bioreactivity of uremic plasma was explored. RESULTS: TNF-alpha and IL-10 production markedly decreased after 30 passes (629 +/- 78 vs. 144 +/- 62 pg/mL; 207 +/- 25 vs. 117 +/- 23 pg/mL; P=0.04). The column removed beta2M efficiently with a marked decline in plasma levels by 99% after 30 passes. Neutralization of AGE receptor (RAGE) resulted in a further reduction in the bioreactivity of uremic plasma. This was observed with nonperfused, as well as perfused, uremic plasma, suggesting that AGE-modified proteins were biologically active and still present after perfusion. CONCLUSION: The sorbent beads removed uremic solute(s) that prime monocytes to enhanced cytokine production. Removal of beta2M was efficient, and of native and AGE-modified middle molecules likely.     

Selective removal of circulating immune complexes from patient plasma

The principle of a patient-specific immunoadsorber (PsIA) is demonstrated. Studies with model systems (HSA/anti-HSA) on immobilization, stability, and leakage form the basis for the presented fast-performance liquid chromatography (FPLC) and batch experiments, which were conducted using two different protein A adsorbers and autologous and heterologous PsIA systems. Experiments to determine the binding capacity of protein A adsorbers and PsIAs are described. In all experiments, the adsorption of plasma IgG, total protein, and C1q and C3d circulating immune complexes were measured. Plasma of patients with autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus) was investigated. Analysis was performed in both the initial plasma and the flow-through or supernatant. Results of the investigations using FPLC and batch experiments were compared. Autologous PsIA systems are suitable for the selective removal of elevated levels of circulating immune complexes in the plasma.     

Restricted entry of an anti-rat epididymal protein IgG into the rat epididymis

A rat epididymal protein (MW 32 000) was isolated and purified from the rat caudal epididymal fluid. Mono-specific antiserum against this protein was raised in rabbits, purified and labelled with ¹²⁵I. The labelled IgG was infused intravenously into anaesthetized male rats, and the transfer of the labelled IgG across the luminally perfused cauda epididymidis was studied. It was found that during the 2 h infusion period, radioactivity in blood rose, but no radioactivity could be detected in the perfusates, irrespective of whether the epididymis was perfused with Krebs bicarbonate solution or a solution which resembled the rat caudal fluid in ionic composition. In some experiments, rats were given a single intravenous injection of labelled IgG and radioactivity in the epididymal content was measured 10 days later. It was found that despite a high IgG level in blood and liver, no radioactivity could be detected in epididymal fluid and sperm. It is concluded that the blood-epididymis barrier restricts the passage from blood to lumen, an immunologlobulin directed against an epididymal protein.     

Immunosorbent Chemistry: A Study of Agarose-Based Column Sorbents for the Removal of Low-Density Lipoprotein (LDL) from Blood

Anti-LDL antibody was covalently attached to agarose beads in order to prepare an immunosorbent for the removal of LDL from plasma or blood. Both the conditions of antibody coupling and the type of agarose matrix used were critical to the optimization of LDL binding capacity. Sorbents binding 6-8 mg lipoprotein cholesterol/ml column volume were obtained using cyanogen bromide or glutaraldehyde coupling procedures and crosslinked 2% agarose beads. The sorbent could be regenerated by washing with 1 M acetic acid, a reagent that was also an effective disinfectant. In vitro perfusions of whole blood over small columns of 212-300 mu beads showed excellent flow rates (2 ml/min/cm2 under 50-100 cm saline pressure); recovery of leukocytes and platelets exceeded 90%, and complement was not activated. Leakage of antibody and bead matrix was negligible. The antibody-agarose beads could not be sterilized by conventional techniques, but withstood treatment with 0.34% phosphoric acid in 80% ethanol at 37 degrees C, a novel method of chemical sterilization.     

Novel Composite Sorbent Beads for Paraquat Removal by Hemoperfusion

The present report describes the development and performance of a column that is to be used for removal of paraquat from circulating blood in a hemoperfusion-type set-up. The key element of the system is a newly developed sorbent material containing fuller's earth entrapped in cross-linked agarose beads (Talosit). The technique produces a sorbent material exhibiting a very large active surface area while allowing for high mobility of paraquat molecules within the beads and favorable flow characteristics of packed column. Cross-linking of the agarose (by epichlorohydrin) also has a most beneficial effect on the mechanical strength of the beads as well as on their stability to sterilization in an autoclave. The composite beads exhibit good blood compatibility. A scanning electron microscope analysis of the beads showed no adherence of cellular blood components after contact with blood. Moreover, no significant changes in plasma composition had taken place when the beads were properly conditioned prior to contact with fresh human blood. A comparative study of paraquat removal from saline solution by the new beads and by cellulose-coated activated charcoal (Adsorba-300C) indicates a higher removal rate with the former. The results obtained so far with this new sorbent are very promising and extension of these studies to in vivo hemoperfusion is under way.     

Development of cellulose-DNA immunoadsorbent

The aim of this study was to prepare a DNA immunoadsorbent for the specific, extracorporeal removal of anti-DNA antibodies from the blood of patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Two kinds of cellulose beads were applied as a carrier. Calf thymus DNA was covalently coupled to the carrier using the epichlorohydrin method. Efforts were focused on optimization of conditions for activation and coupling, trying to couple as much DNA as possible to a certain amount of carrier. It was found that the activation level increased with the increase of NaOH concentration and the amount of epichlorohydrin used. The mole of epichlorohydrin must be in excess of that of NaOH because excess NaOH could react further with the epoxy groups in the beads resulting in a decrease of activation level. High activation level could be obtained in a medium of 3.0 M NaOH. The DNA coupling was found to be mainly temperature and pH dependent. Using 0.1 M Tris-HCl buffer, pH 8 at a temperature of 50-90 degrees C, more than 3 mg of DNA could be coupled to 1 ml of wet beads. Prolonging the coupling reaction under 50 degrees C to 72 h resulted in the same coupling capacity as that obtained under 90 degrees C. To evaluate the adsorption ability for anti-DNA of this immunoadsorbent, batch and circulation tests were applied using SLE patient plasma. The immunoadsorbents showed excellent adsorption capacity, especially the cellulose with smaller size (200-300 microm). The incubation of 20 ml of patient's plasma with 1 ml of adsorbent resulted in an 80% decline in the anti-DNA antibody level. In the circulation tests, 30 ml of plasma was circulated through a column containing 3 ml of adsorbent. The maximum decline in anti-DNA level, 80%, was obtained after 60 min. Such high adsorption capacity and high adsorption rate suggest this immunoadsorbent may be used for treatment. For comparison, 1,4-butanediol diglycidyl ether activation method and other DNA sources were tested with the same protocol.     

Osteoclast-specific monoclonal antibodies coupled to magnetic beads provide a rapid and efficient method of purifying avian osteoclasts.

Osteoclasts are the major cell type responsible for normal and pathologic bone resorption. Obtaining highly purified populations of these multinucleated cells has been problematic, although such populations would greatly facilitate investigations of osteoclast regulation and activity. A new immunomagnetic protocol has been devised to surmount these difficulties, employing avian osteoclast-directed monoclonal antibodies (designated 121F, 35L, and 75B) surface coupled to uniformly small, magnetic polystyrene beads covalently conjugated with sheep antimouse IgG. Presentation of these antiosteoclast antibody-coated beads to mixed cell preparations derived from marrow-depleted, collagenase- and/or trypsin-treated chick tibiae and wing bones, followed by magnetic separation and washing, results in efficient and selective binding of osteoclasts to the immunomagnetic beads within minutes. The specific nature of this bead-cell interaction is further demonstrated by the progressive decline in antiosteoclast antibody-coated bead binding to osteoclasts by uncoated beads or beads coated with an irrelevant antibody. Under optimal conditions, these isolations typically yield more than a 100-fold enrichment and greater than a 90% purification of osteoclasts from subpopulations of either predominantly nonviable or viable osteoclasts. Although scanning electron microscopy reveals that immunomagnetically purified and cultured osteoclasts internalize large numbers of the antibody-coated beads, such cells appear unimpaired in their ability to attach to tissue culture plastic or devitalized cortical bone slices and to produce resorption pits characteristic for osteoclasts. Additional studies to ascertain the most effective method for removal (desorption) of antibody-coated beads from magnetically isolated osteoclasts demonstrate that moderate physical agitation is at present the most effective protocol to dislodge antibody-coated beads from the cell surface while maintaining osteoclast viability and function. This immunomagnetic technique therefore provides a gentle method for the isolation of highly purified populations of osteoclasts from heterogeneous bone cell populations in a rapid, efficient, and selective manner.     

Protein A Immunoadsorption Therapy in HIV-Related Immune Thrombocytopenia: A Preliminary Report

Nine homosexual patients with immune thrombocytopenia were treated with autologous plasma that had been perfused over silica-immobilized Staphylococcus aureus protein A (SpA). Pretreatment platelet counts ranged from 10,000 to 98,000 cells/mm3 (mean: 54,000 cells/mm3). Six patients responded to therapy. Platelets increased by a mean of 95,000 cells/mm3 (p less than 0.007) and reached normal levels (greater than 150,000 cells/mm3) in four patients. Increased platelet counts are presently sustained in these four individuals after 5 months of follow-up. Increases in platelet counts significantly correlated with decreases in platelet-associated IgG (PAIgG), platelet-directed IgG (PDIgG), and immune complexes (CIC). PAIgG and PDIgG declined by a mean of 67% (p less than 0.003) and 58% (p less than 0.007), respectively. CIC decreased by a mean of 37% (p = 0.02). Complement was concomitantly activated in all four examined patients. C3a and C5a increased 23-fold and 2.6-fold, respectively, while total hemolytic complement decreased by 50%. Activated complement components and removal of CIC and IgG thus may contribute to the platelet-enhancing activity of SpA immunoadsorption therapy.     

Platelet aggregating factor in the epidemic form of hemolytic-uremic syndrome in childhood

Plasma from six out of eleven children with the epidemic forms of hemolytic-uremic syndrome caused the aggregation of homologous platelets as has been described in thrombotic thrombocytopenic purpura. IgG purified from normal adults inhibited the platelet aggregation induced by plasma collected from three children during the acute phase of the disease. This inhibition by IgG may contribute to the reported successful management by infusions of plasma or plasma exchanges.     

Selective Removal of Anti-Acetylcholine Receptor Antibodies and IgG In Vitro with an Immunoadsorbent Containing Immobilized Sulfathiazole

Adsorption therapy, which treats the autoimmune disease by the selective removal of pathogenic substances from the blood, is an active subject. In this therapy, bioactive molecules such as antigen, antibodies and protein A are usually used as affinity-ligands. The disadvantages of using these substances as affinity-ligands are the availability, sterilizability, and ability to cause antigenic reactions. After surveying hundreds of synthetic compounds, it was found that polyacrylic beads [Eupergit C (EC)] immobilized covalently with sulfathiazole (ST) are an ideal biomimetic ligand for the selective adsorption of IgG (24.3 +/- 0.4 mg IgG/g wet beads). In an in vitro study, this adsorbent selectively removed the antiacetylcholine (AChR) receptor antibody of myasthenia gravis (MG) patients. Further experiments demonstrated that the Fab fragment was adsorbed three times more efficiently than the Fc fragment of IgG.     

Circulating antibodies to lung protein(s) in patients with cryptogenic fibrosing alveolitis.

BACKGROUND--It has been hypothesised that cryptogenic fibrosing alveolitis has an immunological pathogenesis mediated by T lymphocytes. It is, however, recognised that patients may show dysregulation of the humoral immune system and that the presence of large numbers of B lymphocytes in open lung biopsies may be associated with a poor prognosis. Evidence of a role for the humoral immune system in the pathogenesis of cryptogenic fibrosing alveolitis has been suggested, but attempts to demonstrate circulating immunoglobulin to antigen within the lung have been inconclusive. METHODS--Plasma samples from 22 patients with cryptogenic fibrosing alveolitis, 22 patients with sarcoidosis, and 17 healthy controls were screened by SDS-PAGE and Western blotting for the presence of autoantibodies to lung proteins derived from cryptogenic fibrosing alveolitis, sarcoid and control lung tissue, as well as four normal non-pulmonary tissues. Possible site(s) of target protein(s) within the lung tissue were identified by immunohistochemical examination using IgG purified from the plasma of six patients and two controls. RESULTS--Eighteen of the plasma samples from patients with cryptogenic fibrosing alveolitis had reactive IgG to lung protein(s) in the 70-90 kDa molecular weight range compared with five of 18 plasma samples from patients with sarcoidosis and one of 17 controls. Plasma from patients with cryptogenic fibrosing alveolitis recognised antigen(s) of the same molecular weight in control and sarcoid lung tissue, but not non-pulmonary tissues, with a similar frequency. Immunohistochemical staining of cryptogenic fibrosing alveolitis biopsy material using IgG purified from plasma samples from patients with cryptogenic fibrosing alveolitis, but not control samples, revealed fine linear positivity in the lung parenchyma in a pattern suggestive of reaction with alveolar lining cells. The pattern was cytoplasmic/membranous and not nuclear. CONCLUSIONS--Patients with cryptogenic fibrosing alveolitis have a high frequency of plasma IgG autoantibodies to protein(s) within lung tissue associated with alveolar lining cells. This is believed to be the site where immunological injury occurs in cryptogenic fibrosing alveolitis, but the significance of these antibodies to the aetiology and pathogenesis is as yet unclear.     

Purification and characterization of a boar sperm protein interacting with immunoglobulins from an infertile woman.

Immunoglobulin G (IgG) fraction was prepared from a serum obtained from an infertile woman (IS) that induced sperm agglutination of human and boar sperm. The antisperm antibodies interacted with a 50-kD boar sperm protein, determined by immunoblot. The 50-kD protein was extracted with deoxycholate (DOC) and purified by affinity chromatography on concanavalin A column, ion exchange chromatography on CM52 column, affinity chromatography on IS IgG-protein G-agarose, and by preparative electrophoresis with electroelution. The purified 50-kD protein migrated as a single homogeneous band when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by Western blot. The production of antibodies against specific sperm proteins may be a cause of immunological infertility.     

Chimeric pig hearts resist hyperacute rejection in ex vivo perfusion model

With surrogate tolerogenesis. the recipient immune system is engrafted within the donor pig before organ transplant. Chimeric pig hearts may resist hyperacute rejection by inducing accommodation. This hypothesis was tested using an ex vivo isolated piglet heart perfusion model. Processed sheep marrow was infused into fetal pigs at 45 days gestation. Heart explants from chimeric or nonchimeric pigs were suspended in a Langendorff apparatus and perfused with plasma from unsensitized sheep or sensitized sheep. Nonchimeric hearts perfused with plasma from unsensitized functioned for 240 min (N = 3). Nonchimeric hearts perfused with sensitized plasma deteriorated rapidly, functioning at 19+/-12 min (N = 6); Immunohistochemistry of heart graft revealed extensive deposition of IgG, IgM in the microvascular. In contrast, chimeric hearts perfused with sensitized plasma functioned for 183+/-46 min (N = 3)(p <.001); Deposition of IgG, IgM had substantially less. Heart grafts procured from chimeric pigs survived in the presence of antidonor IgG, IgM, and complement, demonstrating that chimeric pig hearts resist hyperacute rejection.     

Renal membrane-bound carbonic anhydrase. Purification and properties

Microsomes from perfused human donor kidneys were separated by differential centrifugation in sucrose, and thoroughly washed before solubilization by the nonionic detergent nonyl-beta-D-glucoside. The solubilized material was first applied onto an affinity chromatographic column of acetazolamide-oxirane-SepharoseR-CL-4B to remove contaminating cytoplasmic carbonic anhydrase isozymes CA I and CA II. It was then added onto an affinity column of p-aminomethylbenzene sulfonamide coupled to CM Bio-gel AR to purify the membrane-bound carbonic anhydrase activity. This resulted in a 50% pure enzyme. It was then concentrated and fractionated on an anion-exchange column, and desalted and purified to homogeneity (SDS-PAGE and isoelectric focusing) by gel filtration. The enzyme was now purified 411-fold from extractable membrane protein. Its molecular weight was 34.4 kDa from gel filtration and SDS-PAGE, and 36.7 kDa from amino acid analysis. The amino acid composition differed from that of the cytoplasmic isozymes CA I, II, and III. Antisera, produced in rabbits against the purified SDS-treated enzyme, reacted with native nondenatured membrane enzyme protein but only weakly with CA II. Kinetically the enzyme was similar to CA II with respect to hydrase and esterase activities and to inhibition by various sulfonamides. Considered together, the data suggest that the human kidney contains a membrane-bound carbonic anhydrase protein that differs from the cytoplasmic isozymes CA I, II, and III and the secretory form (CA VI) in the saliva.     

Identification of a monocyte phagocytic defect in a subpopulation of patients with nephritis

Experimental studies have demonstrated the cardinal role played by the mononuclear phagocyte system in the removal of antigen-antibody complexes. To assess the functional capacity of phagocytes in patients with renal disease, 33 normal subjects, 10 patients with mesangial proliferative glomerulonephritis, 8 patients with membranous nephropathy, and 8 patients with moderately severe chronic renal failure were studied by an in vitro assay, measuring the ability of isolated monocytes to ingest sheep erythrocytes coated with IgG antibody and to phagocytize latex beads. Monocytes from four patients with mesangial proliferative glomerulonephritis and one patient with membranous nephropathy exhibited a subnormal capacity to ingest the antibody-coated erythrocytes. Additionally, monocytes from two of the four patients with mesangial proliferative glomerulonephritis and a defect in ingesting sensitized erythrocytes had a subnormal capacity to phagocytize latex beads. The results are interpreted in the context of a hypothesis which suggests that patients with immune nephritis show various forms of immune deficit.     

Plasma and dialysate immunoglobulin G in continuous ambulatory peritoneal dialysis patients: a multicenter study

Peritoneal dialysate immunoglobulin (Ig)G concentrations were measured in 120 continuous ambulatory peritoneal dialysis (CAPD) patients evaluated at four dialysis centers in different countries to assess the normal range for dialysate IgG and to investigate the relationships of this protein levels with peritoneal episodes, For 65 of these patients, plasma IgG levels were determined, and IgG clearances were calculated. The mean dialysate concentration of IgG was 6.9 +/- 4.2 mg/dl, and there was no difference between men and women or between patients who had or had not previously undergone hemodialysis. Dialysate IgG concentrations were significantly related to residual renal creatinine clearance and negatively correlated with dialysate volume, plasma albumin and total protein. There were no significant correlations between IgG levels in the dialysate and age, protein losses in the dialysate, time on CAPD or time from the last peritonitis episode. Plasma and dialysate IgG were unrelated to the incidence of peritonitis, statistical analysis being performed with different methods. These results suggest that IgG levels in the dialysate or plasma are not a major factor in the prevention of CAPD peritonitis.     

两种病理类型肾病患者尿IgG对人肾小管上皮细胞表达巨噬细胞移动抑制因子的影响

目的 分离纯化表现为肾病综合征(NS)的微小病变型(MCD)及膜性肾病(MN)患者尿IgG，比较它们对人近端小管上皮细胞(HK-2)表达巨噬细胞移动抑制因子(MIF)的影响。方法 采用硫酸铵沉淀&#65380;蛋白G亲和层析纯化尿中IgG，并经SDS-PAGE、Western印迹分析鉴定&#65377;用不同浓度(0&#65380;0.5&#65380;1.0&#65380;2.5&#65380;5.0&#65380;10.0 mg/ml)的上述两种患者的尿IgG分别刺激HK-2细胞6 h，应用RT-PCR检测细胞表达MIF mRNA的变化;应用Western印迹检测细胞中MIF的蛋白水平&#65377; 结果 纯化的尿IgG经SDS-PAGE分析显示其分解为4个片段,以兔抗人IgG抗体进行免疫印迹鉴定，证实这些蛋白条带均为IgG成分&#65377; 两种不同病理类型NS患者的尿IgG均可上调HK-2细胞MIF 的基因及蛋白表达，并呈剂量依赖性&#65377;MN患者的尿IgG 0.1 mg/ml即可明显上调HK-2细胞MIF mRNA和蛋白表达(P < 0.01);而MCD患者的尿IgG需达到2.5 mg/ml才具有显著上调效应&#65377; 结论 呈NS的MCD和MN患者尿IgG可上调HK-2细胞表达MIF&#65377;MN患者尿IgG的作用强于MCD患者,提示这两种不同病理类型患者尿IgG可能存在结构或功能上的差异&#65377;     

Granulocytapheresis in the Treatment of Patients with Rheumatoid Arthritis

Abstract: The G-1 column is an extracorporeal type granulocytapheresis device packed with 220 g cellulose acetate beads to which granulocytes and monocytes specifically adhere. A total of 59 rheumatoid arthritis patients with elevated granulocyte counts from 4 hospitals in Japan received 2 apheresis sessions of 1 h duration/week for a total of 8 times over a period of 4 weeks. About 55 % of the leukocytes which entered the G-1 column were adsorbed onto the beads; 95% were granulocytes, 3.5% monocytes, and 0.4% lymphocytes. Clinical and efficacy assessments showed improvements in swollen joints (p < 0.01), tender joints (p < 0.001), the active joint score (p < 0.001), duration of morning stiffness (p < 0.01), and grip strength (p < 0.001). In good responders, the improvements were observed for up to 12 weeks following the last apheresis. Exacerbation was noted in 2 patients. It is suggested that the efficacy of the G-1 column is attributable to the removal or suppression of hyperactive leukocytes and inflammatory cytokines, inducing a kind of immunomodu-lation.     

Immunodepletion in xenotransplantation

Xenograft transplantation is perhaps the most immunologically difficult problem in transplantation today. An overwhelming hyperacute rejection reaction (HAR) occurs within minutes of organ implantation. Preformed antibodies are thought to initiate this process. We used a pig-to-dog renal xenograft transplant model and investigated methods of decreasing the severity of hyperacute rejection. Female pigs weighing 15-20 kg were used as donors. Recipients were mongrel dogs weighing 15-25 kg. Experimental dogs were all given a number of treatments of IgG depletion using an antibody removal system (Dupont-Excorim). This machine immunoadsorbs plasma against a column containing immobilized staphylococcal protein A, which is known to bind the IgG Fc receptor. An 84% reduction in the IgG levels and a 71% reduction in IgM levels was achieved. Postoperative assessment was made of urine output, time to onset of HAR, and histopathological examination of the rejected kidneys. Although cross-matches between donor lymphocytes and recipient sera remained strongly positive in the treated dogs, there was a two- to fourfold reduction in the titers. The time to onset of HAR was prolonged in the experimental group, and the urine output was increased slightly. The histopathologic changes in the experimental group generally showed signs of HAR, but of less intensity than in the nonimmunodepleted control group.     

Clinical Trials of Immunoadsorbent in Systemic Lupus Erythematosus Therapy

Abstract: Five patients with systemic lupus erythematosus (SLE) were perfused through an extracorporeal shunt filled with DNA-immunoadsorbent (DNA immobilized on carbonized resin beads). High concentrations of anti-DNA antibodies (36.4–67.0%) (binding percentage with ¹²⁵I-DNA) in the serum of SLE patients were reduced to 13.8–53.0%, respectively. The highest removal percentage was 62.1%. Although the decline levels varied, the symptoms of patients, i.e., long-term severe joint pain, severe edema, hydropericardium, and ascites were all relieved considerably. The immunoadsorbent showed satisfactory blood compatibility.