Alfred F. Michael

Polyantigenic Expansion of Basement Membrane Constituents in Diabetic Nephropathy

The immunohistopathology of the intrinsic basement membrane-associated antigens were examined in diabetic nephropathy. In early and moderate stages of disease there was polyantigenic expansion of all the intrinsic components of mesangium, glomerular basement membrane (GBM), and tubular basement membrane (TBM) assessed by polyclonal antisera to collagen types IV and V, laminin, and by monoclonal antibodies to type IV collagen and fibronectin and to four other intrinsic components of normal renal extracellular matrices (MBM10, 11, 12, and 15). In the mesangium the first intrinsic antigens to increase were fibronectin and type V collagen. In late stages of disease, there was a diminution in the mesangium of all of these antigens with the exception of type V collagen, which persisted. Additionally, antigens appeared in the mesangium, recognized by MBM11 and MBM15, which are normally present in fetal but not adult mesangial regions. Similarly, in the GBM in late stages of disease, there was a decrease in all of the antigens, except for a persistence of the antigen recognized by MBM15. However, in TBM all of the antigens assessed increased in early, moderate, and severe disease. These studies document the complexity of polyantigenic alterations in the development of diabetic nephropathy.

Studies of the Rate of Regression of the Glomerular Lesions in Diabetic Rats Treated with Pancreatic Islet Transplantation

Diabetes was induced in Lewis rats with streptozotocin. Six to eight months later glomeruli showed mesangial thickening: IgG, IgM and C3 were seen in large quantities in the mesangium by immunofluorescent microscopy. Ten animals then had successful pancreatic transplantation resulting in normal glucose and insulin levels within one to three weeks. Biopsies obtained within the first two weeks following transplantation demonstrated a significant reduction in mesangial thickening and in mesangial staining for IgG, IgM and C3. Three to four weeks after transplantation C3 staining was no longer detected. A gradual reduction in mesangial IgG and IgM localization continued so that by nine weeks following islet transplantation only minimal staining for immunoglobulins was present. Although mesangial thickening was reduced, this abnormality could still be detected in most animals six to nine weeks after transplantation. Three rats showed improvement in glomerular morphology within two weeks despite persistent hyperglycemia. These rats had normal insulin levels at this time. Islet transplantation in inbred diabetic rats effectively returns glucose and insulin levels to normal and results in rapid regression of the light microscopic and immunopathologic glomerular lesions. These studies support the concept of reversible mesangial dysfunction in diabetic rats.

Lung T Cells in Hypersensitivity Pneumonitis

Monoclonal antibodies OKT3 (all T cells), OKT4 (T-helper/inducer), and OKT8 (T-suppressor/cytotoxic) were used to determine surface phenotypes of bronchoalveolar lavage and peripheral blood lymphocytes in patients with chronic hypersensitivity pneumonitis. Similar studies were done in asymptomatic pigeon breeders, patients with sarcoidosis, and nonsmoking controls. Increased numbers of lavage T cells were found in patients with hypersensitivity pneumonitis and sarcoidosis and in asymptomatic pigeon breeders. The predominant T-cell subset in patients with hypersensitivity pneumonitis and in asymptomatic pigeon breeders was T8 +; in contrast, the predominant subset in those with sarcoidosis was T4 +. Peripheral blood T-cell subsets were normal in all groups. Thus, most lung T lymphocytes in chronic hypersensitivity pneumonitis belong to the T8 + subset; the local cellular immune response in hypersensitivity pneumonitis and sarcoidosis are different; and the pattern of alveolitis, as determined by bronchoalveolar lavage, is not the sole determinant of lung impairment after exposure to hypersensitivity pneumonitis antigens.

Neoantigen of the polymerized ninth component of complement. Characterization of a monoclonal antibody and immunohistochemical localization in renal disease.

A monoclonal antibody to a neoantigen of the C9 portion of the membrane attack complex (MAC) of human complement has been developed and characterized. The distribution of this neoantigen was assessed by indirect immunofluorescence microscopy in nephritic and nonnephritic renal diseases. The antibody (Poly C9-MA) reacted on enzyme-linked immunosorbent assay (ELISA) with a determinant in complement-activated serum that was undetectable in normal human serum (NHS). Zymosan particles incubated in NHS had positive immunofluorescent staining with Poly C9-MA; however, binding of Poly C9-MA was not observed with zymosan particles incubated in sera deficient in individual complement components C3, C5, C6, C7, C8, or C9. Reconstitution of C9-deficient sera with purified C9 restored the fluorescence with Poly C9-MA. Poly C9-MA reacted positively by ELISA in a dose-dependent manner with purified MC5b-9 solubilized from membranes of antibody-coated sheep erythrocytes treated with NHS but not with intermediate complement complexes. Poly C9-MA also reacted in a dose-dependent manner on ELISA and in a radioimmunoassay with polymerized C9 (37 degrees C, 64 h) (poly C9) but not with monomeric C9. Increasing amounts of either unlabeled poly C9 or purified MC5b-9 inhibited the 125I-poly C9 RIA in an identical manner. These studies demonstrate that Poly C9-MA recognizes a neoantigen of C9 common to both the MAC and to poly C9. By immunofluorescence, Poly C9-MA reacted minimally with normal kidney tissue in juxtaglomerular loci, the mesangial stalk, and vessel walls. Poly C9-MA stained kidney tissue from patients with glomerulonephritis in a pattern similar to that seen with polyclonal anti-human C3. In tissue from patients with nonnephritic renal disease--diabetes, hypertension, and obstructive uropathy--Poly C9-MA was strongly reactive in the mesangial stalk and juxtaglomerular regions, tubular basement membranes, and vascular walls. Poly C9-MA binding was especially prominent in areas of advanced tissue injury. Poly C9-MA frequently stained loci where C3 was either minimally present or absent. These studies provide strong evidence for complement activation not only in nephritic but also in nonnephritic renal diseases.

The glomerular mesangium: I. Kinetic studies of macromolecular uptake in normal and nephrotic rats

This study was designed to define quantitatively the function of the rat glomerular mesangium in the uptake and processing of intravenously administered protein macromolecules (radiolabeled aggregated human IgG, AHIgG-(125)I), to relate this function to that of the general reticuloendothelial system, and to examine the effects of increased glomerular permeability to protein on the mesangial cell system.Mesangial localization of human IgG as demonstrated by immunofluorescent microscopy showed good correlation with concentrations of AHIgG-(125)I in preparations of isolated glomeruli. In normal rats the concentrations of AHIgG-(125)I in glomeruli were similar to those of lung, liver, and spleen and demonstrated a rapid decrease with increasing time intervals after aggregate administration. In rats given aminonucleoside of puromycin a marked increase in mesangial uptake of aggregates was found while studies of nephrotic lungs, liver, spleen, and blood showed no such differences. Glomerular levels of AHIgG-(125)I in aminonucleoside animals could not be correlated with the quantity of proteinuria. Nephrotic and control animals given unaggregated human IgG showed little glomerular localization by immunofluorescent microscopy; no difference in the concentration of this protein in nephrotic as compared to control glomerular isolates was found.Thus, the mesangium in normal animals functions in a manner analogous to that of the general reticuloendothelial system. In nephrotic rats the mesangial uptake of macromolecules is makedly increased, a finding not observed in other tissues.

Pancreatic Islet Transplantation: Effects on the Glomerular Lesions of Experimental Diabetes in the Rat

Diabetes was induced in Lewis rats with streptozotocin. Six to nine months later glomeruli showed significant mesangial matrix thickening; by immunofluorescent microscopy large quantities of IgG, (β1C and in some instances, IgM were seen in a mesangial distribution. Sustained normoglycemia was then achieved in nine of these animals by successful pancreatic islet isotransplantation. Three months following transplantation five of these animals had decreased mesangial matrix while four had no further progression of glomerular lesions. All had a marked decrease in IgG, IgM and β iC in the mesangium. In contrast, untreated diabetic rats, over the same time period, demonstrated progressive mesangial thickening and focal tuft sclerosis. Immunoglobulins and complement were in the mesangium in quantities equal to or greater than seen in earlier biopsies. Thus, successful pancreatic islet transplantation in the rat results in regression or arrest of the diabetic glomerular lesion.

Nephritogenic antigen determinants in epidermal and renal basement membranes of kindreds with Alport-type familial nephritis.

We probed epidermal basement membranes (EBM) of acid-urea denatured skin from members of kindreds with Alport-type familial nephritis (FN) for the presence of antigens reactive with Goodpasture sera (GPS) and serum (FNS) from an Alport patient who developed anti-glomerular basement membrane (GBM) nephritis in a renal allograft. By immunoblotting, GPS reacted primarily with the 28,000 molecular weight (mol wt) monomer but also the 24,000 mol wt and 26,000 mol wt monomers of the noncollagenous globular domain (NC1) of type IV collagen from normal human GBM, while FNS identified only the 26,000-mol wt monomer. FNS reacted with EBM of 12 controls and nine unaffected male kindred members but not EBM of eight affected males. Five affected females exhibited interrupted reactivity of FNS with EBM. GPS showed variable reactivity with EBM and was not discriminating with respect to Alport-type FN. FNS did not stain renal basement members of five affected males. However, the EBM, tubular basement membrane, and Bowmans capsules of affected males contained antigens reactive with GPS. These immunochemical studies suggest that the FNS antigen is distinct from Goodpasture antigen(s). The expression of FNS antigen located on the NC1 domain of type IV collagen is altered in basement membranes of patients with Alport-type FN, and the distribution of this antigenic anomaly within kindreds suggests X-linked dominant transmission of a defective gene.

Tryptophan Metabolism in Man

The amino acid tryptophan is unique because it contains the indole nucleus and because it is metabolized in man through several different biochemical pathways to a number of specific products. It is the precursor of serotonin and 5-hydroxyindoleacetic acid. In addition, after cleavage of the indole ring, it may be metabolized by way of the kynurenine pathway to 3-hydroxyanthranilic acid and ultimately to nicotinamide. In mammalian liver the benzene ring is oxidized and metabolized through a number of intermediate reactions to glutarate, acetate, and carbon dioxide (1, 2). Tryptophan is also the precursor of indolic acids, such as 3-indoleacetic acid. In man this compound is formed both by tissue enzymes and by bacteria in the gut (3). In the intestinal tract, bacteria that contain tryptophanase (4) reductively cleave the side chain of tryptophan and form indole, which is absorbed, conjugated in the liver, and excreted as indican (sulfated potassium ester of indoxyl). In addition to the various reactions involving the indole ring or side chain, tryptophan, like other amino acids, is incorporated into protein. These pathways are schematically shown in Figure 1. The many different enzyme systems and important cofactors, such as pyridoxal phosphate, that take part in these reactions have been ably reviewed (5-7) and will not be discussed here. In the investigation of tryptophan metabolism in man, a number of variables must be considered. These include not only these enzymes and cofactors, but

Systemic lupus erythematosus within the first two decades of life

Forty-nine patients with systemic lupus erythematosus (SLE) during childhood and adolescence presenting over a period of 17 years were followed during treatment with prednisone and azathioprine. The average period of follow-up was 5.7 years. Detailed analyses of clinical parameters of renal function and sequential changes in glomerular abnormalities by percutaneous renal biopsy are reported. Therapy was directed towards normalizing the results of urinalysis and renal function, eliminating proteinuria and maintaining normal serology (normal serum complement and negative antiDNA titers). The 10 year survival of the entire group was 86 per cent. A survival of 73 per cent and 87 per cent over this interval in patients with diffuse and focal proliferative lupus nephritis, respectively, was achieved. The major cause of mortality in this series was infection. It appears that intensive observation and monitoring of serologic parameters in SLE, along with aggressive steroid and immunosuppressive therapy, lead to a prognosis in SLE more favorable than previously reported.

The complement system in chronic glomerulonephritis: Three newly associated aberrations

This report provides a description of 3 patients with chronic glomerulonephritis who present unique serum complement system deficiencies associated with different kidney histopathology and immunopathology. One patient has a severe deficiency of hemolytic C1 predominantly due to diminished C1r function; this abnormality is associated with the end stage of glomerulonephritis. A second patient has a selective deficiency of hemolytic C2 function associated with a membranous glomerulopathy. The third patient has hereditary angioedema with decreased levels of hemolytic C4 and C2; this patients glomeruli show basement membrane thickening, centrolobular and glomerular hyalinization, mesangial proliferation, and interstitial round cell infiltration similar to that seen in patients with the C3-deficient form of chronic glomerulonephritis in children. The possible basis for the deficiencies and the questions that these natural experiments raise concerning the direct or indirect participation of the complement system in immune injury to the kidney are discussed.