Contrasting effects of pyrazinoylguanidine and hydrochlorothiazide in patients with renal insufficiency.

A single blind crossover study with washout phases showed that pyrazinoylguanidine (PZG) reduced elevated serum concentrations of urea, triglycerides, and cholesterol in patients with renal insufficiency. Pyrazinoylguanidine was saluretic, without affecting serum potassium or glucose concentrations. The onset of PZG's antihypertensive effect occurred within 4 hours. In contrast, hydrochlorothiazide (HCTZ) increased serum concentrations of urea, triglycerides, and glucose, without affecting cholesterol.

Calcium transients during Fc receptor-mediated and nonspecific phagocytosis by murine peritoneal macrophages.

Studies with populations of macrophages have produced conflicting results concerning the possibility that the concentration of intracellular ionized calcium [( Ca2+]i) may act as an important mediator for phagocytosis. Since asynchronous changes in [Ca2+]i in individual cells undergoing phagocytosis may be averaged to undetectability in population studies, we studied single adhering murine macrophages using fura-2 and our previously described digital imaging system. The proportion of macrophages phagocytosing IgG-coated latex beads was greater than for uncoated beads (percent phagocytosing cells: 71 +/- 7 vs. 27 +/- 7, P less than 0.01). Phagocytosis of IgG-coated and uncoated beads was always associated with a calcium transient that preceded the initiation of phagocytosis. No calcium transients were detected in cells that bound but did not phagocytose beads. Four major differences between Fc receptor-mediated and nonspecific phagocytosis were detected: (a) the duration of calcium transients was longer for nonspecific phagocytosis compared with Fc receptor-mediated phagocytosis (69.9 +/- 10.2 vs. 48.7 +/- 4.7 s, P less than 0.05) and the magnitude of calcium transients was less for nonspecific phagocytosis (178 +/- 43 vs. 349 +/- 53 nM, P less than 0.05); (b) removal of extracellular calcium abolished the calcium transients associated with nonspecific phagocytosis but had no effect on those associated with receptor-mediated phagocytosis; (c) in the absence of extracellular calcium, buffering intracellular calcium with a chelator reduced Fc receptor-mediated phagocytosis but had no additive inhibitory effect on nonspecific phagocytosis; and (d) inhibition of protein kinase C (PKC) with staurosporine inhibited nonspecific phagocytosis but had no effect on receptor-mediated phagocytosis. Our observations suggest that despite both types of phagocytosis being associated with intracellular calcium transients, the role played by intracellular calcium in the signaling pathways may differ for Fc receptor-mediated and nonspecific phagocytosis by elicited murine macrophages.

Specific identification of urinary fibrinogen, fibrinogen degradation products, and cross-linked fibrin degradation products in renal diseases and after renal allotransplantation.

We have applied a sensitive and discriminating electrophoretic technique that distinguishes between fibrinogen, fibrin polymers, fibrinogen degradation products (FDP), and cross-linked fibrin degradation products (XLDP) to evaluate urinary fibrinogen antigen in control subjects and in patients with a variety of renal diseases and after renal allograft transplantation. Although only one of 11 controls showed the trace presence of urinary fibrinogen, 14 of 28 patients with renal disease had urinary fibrinogen antigen, mostly as fibrinogen or fibrin monomer. Thrombin treatment failed to remove fibrinogen from urine, indicating that methods using this step to eliminate clottable protein will overestimate the quantity of urinary fibrinogen and fibrin degradation products. No association was found between the amount or type of antigen and the specific clinical diagnosis or the presence of proteinuria or hematuria, although urinary FDP and XLDP were found only with greater degrees of renal impairment. Fifteen patients were evaluated for 3 weeks after renal transplantation surgery by serial urine and serum electrophoretic analysis. Urinary FDP and XLDP were found significantly more often in the first week after surgery and in association with episodes of acute renal transplant rejection (ARTR) than at other times. This suggests that fibrin deposition and degradation is involved in the pathologic process of ARTR and that identification of specific XLDP and FDP could have diagnostic and prognostic application in such patients.

Binding and catabolism of aggregated immunoglobulins containing C3b by U937 cells.

We studied Fc receptor and C3b receptor (CR1) function on U937 cells, a human monocyte cell line. C3b was incorporated into stable soluble heat aggregates of 125I-IgM (A-IgM) and 125I-IgG (A-IgG) by using functionally pure classical pathway components. C3b incorporation was verified by the ability of aggregates to bind to human red cells and by cosedimentation of 125I and 131I during ultracentrifugation. Cell uptake and degradation of A-IgG X C3b was increased up to twofold compared with A-IgG not containing C3b molecules. However, A-IgG X C3b bound to CR1 after Fc receptors were blocked with nonradiolabeled A-IgG were also not endocytosed and catabolized. Moreover, A-IgM X C3b was bound but not degraded by U937 cells. As expected, uptake of A-IgM without C3b was negligible. CR1-mediated binding of A-IgM X C3b was specifically inhibited both by a murine monoclonal antibody against the human CR1 that blocks C3b binding and by C3b oligomers generated by trypsin activation of C3, but not by monoclonal antibodies against the iC3b receptor (CR3). We conclude that CR1 on U937 cells cause increased binding of A-IgG, and this increased binding leads to increased Fc-mediated endocytosis and catabolism of model immune complexes. However, binding of soluble ligands by CR1 alone, even when binding is multivalent, does not lead to endocytosis and degradation of soluble ligands bearing C3b.

Multiple sclerosis: free light chains in cerebrospinal fluid.

We found free light chains in the CSF of 18 MS patients, but not in any of 14 patients with other neurologic disease. CSF from all MS patients contained kappa and lambda chain dimers, less frequently contained light chain monomers, and never contained free gamma heavy chains. Light chains were not detected in matched serum samples. CSF from MS patients did not release free light chains from whole IgG in CSF of controls. The findings suggest that these free light chains originate in plasma cells, not from degradation of whole IgG.

Multiple sclerosis. Cerebrospinal fluid immune complexes that bind C1q.

We used a sensitive C1q-binding assay to measure levels of soluble immune complexes in 182 samples of cerebrospinal fluid (CSF) from control patients and patients with multiple sclerosis (MS). Soluble immune complexes in CSF were detected in 16% of patients with progressive MS, 38% of patients with exacerbating-remitting MS, 55% of patients with infectious or inflammatory diseases, 3% of patients with noninflammatory neurologic disorders, and in 0% of control patients with back pain. No correlations were found between the results of the C1q-binding assay and abnormalities of other CSF parameters. These included an elevated level of myelin basic protein, pleocytosis, oligoclonal bands, or an increased IgG level. Because of the lack of correlation to laboratory indexes of disease activity and the nonspecificity of a positive test, the C1q-binding assay seems to have little clinical usefulness in the diagnosis or management of patients with MS.

Blood clearance and tissue localization of soluble aggregates of IgG in NZB/W and NZB mice.

We studied the capacity of the mononuclear phagocytic system (MPS) of NZB/W and NZB mice to clear trace and saturating doses of soluble heat-aggregates of IgG (A-IgG) from the blood. Mature female NZB/W mice (aged 5-7 months) with early glomerulonephritis showed no differences in MPS clearance of A-IgG compared with younger NZB/W mice without glomerulonephritis. In contrast, mature NZB mice had a more rapid clearance of A-IgG and greater MPS localization of A-IgG than their younger counterparts. Further studies showed that older NZB/W mice (greater than 10 months) had a slightly more rapid clearance of A-IgG than 2-5-month-old mice (t 1/2 = 3.34 +/- 0.27 SEM vs 3.76 +/- 0.34 SEM, P less than 0.01), whereas NZB mice mice older than 10 months of age had a markedly more rapid clearance than 2-5-month-old NZB mice (t 1/2 = 2.84 +/- 0.15 SEM vs 3.76 +/- 0.32, P less than 0.005). The more rapid clearance seen in NZB mice was partly explained by greater splenic localization of A-IgG and appeared to be restricted to Fc- and/or C3b-receptor mediated clearance, in that clearance of aggregated albumin was not changed. We conclude that NZB/W mice have no impairment in MPS clearance capacity at the onset of their glomerulonephritis, and slightly increased clearance capacity late in the course of their disease. Thus, the presence of circulating immune complexes and the development of glomerulonephritis in NZB/W mice is unlikely to be due to a diminished MPS clearance capacity. NZB mice have an increase in MPS capacity to clear A-IgG as a function of age.

Isolation of model soluble immune complexes in the fluid phase by monoclonal anti-IgG-ferritin antibodies.

Ferritin conjugates of monoclonal IgG anti-human gamma chain (anti-IgG-F) were reacted with soluble heat aggregates of IgG (A-IgG) and with soluble DNA-anti-DNA complexes to increase the S rate of the model soluble immune complexes (ICx) and thus facilitate isolation of ICx in the fluid phase and provide an immunochemical marker for subsequent ultrastructural analysis. A-IgG appeared as globular or curvilinear structures with individual IgG molecules arranged in a random fashion. The technique appears promising for characterization of other soluble ICx.

IgG aggregates of different sizes stimulate or suppress Ig secretion by human lymphocytes in vitro.

The regulatory effects of IgG aggregates on Ig production in vitro by human peripheral blood lymphocytes were shown to be highly dependent on the aggregate size and the degree of mitogenic stimulation. Covalently linked oligometers of IgG were prepared with dimethylsuberimidate cross-linking and chromatographic separation; larger aggregates were prepared by heating (63 degrees C) and preparative zonal ultracentrifugation. The storage and culture conditions used were shown to preserve the stability of aggregate sizes. Although both positive and negative regulatory effects were seen with cells isolated directly from blood, more predictable dose-related effects were seen if cells were vigorously washed, possibly due to the removal of IgG or natural immune complexes bound by the cells in vivo. Some preparations of small IgG oligomers produced marked stimulation of Ig production, especially in cells cultured without mitogen or with suboptimal pokeweed mitogen doses. Aggregates containing six or more IgGs suppressed Ig production, especially when cells were stimulated by mitogen at optimal concentrations.

Preparation of monoclonal antibody-ferritin conjugates of high specific activity.

125I-monoclonal IgG anti-gamma chain antibodies were conjugated to ferritin using glutaraldehyde as a bifunctional reagent. The molar ratio of IgG:ferritin:glutaraldehyde resulting in the highest yield was determined. Free IgG was separated from IgG bound to ferritin by sucrose density gradient ultracentrifugation; free ferritin was separated from antibody-ferritin conjugates by differential salt precipitation. The IgF:ferritin molar ratio of the resulting product was 1:1.4, containing over 90% ferritin-IgG "monomers"; 70-90% of the 125I activity bound immunospecifically to sepharose-IgG or aggregated human globulin (AHG). The product was used as an immunologic EM marker for AHG. Monoclonal antibody-ferritin conjugates prepared by this method should prove useful for quantitative ultrastructural analysis of surface antigens.

Immunologic abnormalities in hemodialysis patients: improvement after pyridoxine therapy.

8 male patients undergoing maintenance hemodialysis were studied to determine the effect of administering supplements of pyridoxine hydrochloride, 50 mg/day for 3-5 weeks, on tests of immune function. In the 3 patients who initially had abnormal nitroblue tetrazolium reduction tests, the values returned to normal with therapy (p less than 0.05). The generation of chemotactic factors from plasma was defective in all evaluated patients and improved after pyridoxine therapy in 4 of 5 patients (p less than 0.01). The lymphocyte subpopulations changed with a rise in the populations of null cells after supplementation with pyridoxine. In addition, lymphocyte transformation in response to mitogens improved in the 3 patients who initially showed low values in these assays. The improvements occurred with pyridoxine therapy even though some patients who responded had no evidence for vitamin B6 deficiency before therapy, as indicated by a normal erythrocyte glumatic-pyruvic transaminase index. We conclude that several parameters of immune function are improved with pyridoxine supplementation. Studies are necessary to establish the minimum daily intake of pyridoxine which will maintain improved values of these tests of immune function in hemodialysis patients.

Decreased Fc receptor avidity and degradative function of monocytes from patients with systemic lupus erythematosus.

We studied the binding and degradation of stable, soluble heat aggregates of 125I-IgG (A-IgG) by monocytes from 30 patients with systemic lupus erythematosus (SLE) and 30 normals. Relative avidities (KE) for Fc receptor (FcR) binding of A-IgG and maximal binding of A-IgG by monocytes were determined from Scatchard plots of binding data obtained at 4 degrees C. Rates of degradation (Vmax) of A-IgG at 37 degrees C were calculated from Lineweaver-Burke plots of the Michaelis-Menton equation. KE were decreased in SLE monocytes (15.5 X 10(-9) L/M) as compared with normals (20.1 X 10(-9) L/M, p less than 0.005) and Vmax were decreased for SLE (0.89 ng/hr) as compared with normals (1.11 ng/hr, p less than 0.005). The maximal FcR binding by SLE monocytes was not statistically different in SLE patients and normals, but monocytes from SLE patients with active disease showed a lower maximal binding capacity for A-IgG (4.9 ng/10(5) cells) than normals (5.4 ng/10(5) cells, p less than 0.05). KE and Vmax in SLE were also lower for patients with active disease than for normal subjects. KE in patients whose anti-ssDNA binding was greater than 20% were lower than for those with DNA binding of less than 20% (p less than 0.005). These data suggest that patients with active SLE have diminished numbers of available FcR on their circulating monocytes, possibly due to interiorization of FcR during endocytosis of endogenous circulating immune complexes.

Increased Fc receptor activity in monocytes from patients with rheumatoid arthritis: a study of monocyte binding and catabolism of soluble aggregates of IgG in vitro.

We studied the binding and catabolic function of adherent monocytes from patients with rheumatoid arthritis (RA) and normal subjects using stable, heat aggregates of IgG125I (A-IgG) as a model for soluble immune complexes. Scatchard plots of 4 degrees C binding data showed that RA monocytes had increased binding avidity and higher maximal binding capacity for A-IgG compared with monocytes from normal subjects. These data suggest that RA monocytes have increased numbers of Fc receptors for IgG, although a concomitant increase in the avidity of individual Fc receptors could not be excluded. At 37 degrees C, RA monocytes; kinetic analysis suggested that increases in catabolized A-IgG were due to increased binding of A-IgG with no change in the fractional rates of catabolism. Latex titers of RA patients correlated with the number of Fc receptors detected on RA monocytes. Mononuclear phagocytes from RA patients are often exposed to endogenous immune complexes that may be present in the blood of such patients; immune complexes may stimulate monocytes and possibly other mononuclear phagocytes to increase their capacity to bind and catabolize soluble immune complexes.

Naturally occurring human antibodies to the F(ab')2 portion of IgG.

Antibodies to the F(ab')2 portion of IgG were detected in the sera of patients and normals using a heat-aggregated F(ab')2 fragment in a liquid-phase radioimmunoassay. The antibodies were found in 72% of 82 rheumatoid arthritis patients, in less than 20% of patients with other diseases and in a few normal subjects. Anti-F(ab')2 antibodies were distinct from previously described anti-Fc antibodies and 'serum agglutinators'. They reacted with intact IgG, F(ab')2 and IgM in binding and inhibition assays and they appeared to have specificity for the Fd portion of IgG molecules, possibly to the VH region. The anti-F(ab')2 antibodies were of both 7S IgG and 19S IgM classes and were found in sera as components of intermediate size (between 7S and 19S) and large size (greater than 19S) immune complexes. The possible contribution of autologous anti-F(ab')2 to abnormal immunoregulation is discussed.

Cadmium and the reticuloendothelial system (RES). A specific defect in blood clearance of soluble aggregates of IgG by the liver in mice given cadmium.

The ability of the reticuloendothelial system (RES) to bind and catabolize soluble stable heat aggregates of 125I-IgG (A-IgG) was studied in mice given oral cadmium. Cadmium caused a delay in the circulation clearance of A-IgG in intact animals. The defect was due to impaired liver uptake of A-IgG and correlated with increased liver cadmium. Subsequent catabolism of bound A-IgG by liver slices was not affected. The defect was specific in that clearance of aggregated human serum albumin and colloidal carbon was normal in cadmium mice; this suggests that cadmium may affect either Fc or complement receptors of Kupffer cells in liver.

Poststreptococcal acute glomerulonephritis: fact and controversy.

Poststreptococcal acute glomerulonephritis is prototypic of the immunologic glomerulonephritides. It most commonly follows streptococcal infection of the pharynx or skin. The diagnosis is usually not difficult when a nephritic clinical presentation (with such manifestations as hematuria, edema, and hypertension) is associated with serologic evidence of recent streptococcal infection and a depressed serum complement concentration. Currently, however, the nephritogenic antigen(s) has not been identified and has not been shown to be the same antigen for all nephritogenic streptococci; it may not even be a part of the infecting organism. The development of a vaccine to prevent this illness from occurring is therefore still not possible. Whether poststreptococcal acute glomerulonephritis progresses to chronic renal failure is still uncertain. Painstaking laboratory research together with careful, prospective long-term follow-up studies of patients with poststreptococcal acute glomerulonephritis may provide some of the answers to these critical questions.

Soluble oligovalent antigen--antibody complexes. II. The effect of various selective forces upon relative stability of isolated complexes.

Soluble oligovalent antigen--antibody complexes were isolated and analysed by ultracentrifugation to assess the effect of several forces upon the composition and stability of soluble complexes. Complexes were prepared with fluorescein (F) conjugates of rabbit serum albumin (RSA) or thyroglobulin (RTg) and high affinity rabbit anti-F antibodies. Isolated complexes containing two antigen molecules (Ag2 complexes) tended to dissociate and form an equilibrium with complexes containing one antigen molecule (Ag1 complexes). This equilibrium was thermolabile, concentration dependent and affected by the original combining ratio and the area in the gradient from which complexes were harvested. Small amounts of free antibody dissociated from soluble complexes also to form a dynamic equilibrium; this equilibrium was much less affected by the above parameters. The data support the concept that complexes grow in size by a process analogous to polymerization of simple subunits and that the driving forces for polymerization are of a lower order of magnitude and more affected by physical variables than the primary reaction between antibody and its antigen.