Serology and tissue lesions in rabbits immunized with Streptococcus mutans

Rabbits were immunized i.v. or i.d. with sterile suspensions of disrupted Streptococcus mutans strain MT703 or K1R. Indirect immunofluorescence assays indicated that sera from four of 10 rabbits immunized i.d. contained antibodies reactive with monkey and human heart and kidney components; 19 of 24 rabbits immunized i.v. had antibodies reactive with these tissues. Heart-reactive antibodies were also detected by immunoelectrophoresis and indirect radioimmunoassay. These antibodies were absorbed well by cytoplasmic membranes, a whole cell extract, and an alkali extract of S. mutans but only weakly by intact bacteria. Between 6 and 8 weeks after the first i.v. administration of S. mutans vaccines, rabbits developed proteinuria and hematuria with subsequent weight loss and lethargy. Approximately 25% of the animals died from illness between the fifth and sixth month of immunization. In 13 of 15 rabbits, immune deposits of C3 and IgG, IgM, or IgA and fibrinogen were seen in kidneys within the glomeruli, basement membranes of the peritubular capillaries, and in the interstitium. In the heart, deposits were seen along the capillaries of the myocardium. In 8 of 14 rabbits, focal deposits of S. mutans antigen were detected in glomeruli and in the kidney interstitium. The kidneys showed gross pathologic and histopathologic changes. Most kidneys were pale and enlarged. Microscopic examination revealed hypercellularity of the glomeruli, presence of neutrophils, thickening of glomerular and tubular basement membranes, tubular atrophy, edema, and fibrosis of the interstitium. The kidney disease presented features of poststreptococcal glomerulonephritis. Microscopic examination of heart sections revealed mild perivascular infiltration by polymorphonuclear leukocytes and plasma cells in some of the rabbits.     

C4d immunohistochemical staining is a sensitive method to confirm immunoreactant deposition in formalin-fixed paraffin-embedded tissue in membranous glomerulonephritis

Although the diagnosis of membranous glomerulonephritis (MGN) may be suspected on routine histology of formalin-fixed paraffin-embedded tissue, fresh-frozen tissue must be used to show the immunologic nature of the process by direct immunofluorescence (IF). The efficiency of IF or immunoperoxidase (IP) detection of IgG and C3 using paraffin sections is controversial. This study was designed to evaluate whether glomerular C4d deposition using an IP method in formalin-fixed paraffin-embedded tissue may be a useful marker for MGN. We showed characteristic glomerular, granular basement membrane deposition of C4d in 31 (100%) cases of idiopathic MGN and in 5 cases (100%) of pure class V membranous lupus nephritis, in which we had a positive diagnosis of the lesions for conventional IF study. Control cases were negative. Nineteen cases of different glomerulopathies, including IgA nephropathy, primary type I membranoproliferative glomerulonephritis, focal segmental glomerulosclerosis and minimal change disease showed diverse reproducible patterns of C4d deposition, without intrinsic background. Our results indicate that staining of formalin-fixed paraffin-embedded tissue for C4d can be used for confirmation of granular basement membrane immunoreactant deposition in cases of MGN. This proved to be a reliable method that could potentially obviate the need for rebiopsy in cases with absence of glomeruli in renal frozen sections or when other adjunct IF or IP methods on paraffin sections are negative. C4d immunostaining, using an IP method, deserves a place as an adjunct method in the biopsy diagnosis of MGN.     

Spontaneous glomerular immunoglobulin deposition in young Sprague-Dawley rats

The frequency, age-onset and distribution of spontaneously deposited immunoglobulins (Igs) in glomeruli of Sprague-Dawley rats has been investigated. Groups of rats (n = 10) were examined at 4-7 day intervals from birth (presuckling) until 30 days of age. Findings were compared with circulating immunoglobulin concentrations in each age group. Immunoglobulins were undetectable in immature kidneys of newborn rats. However, as early as 5 days, scanty IgA and IgM deposits were observed predominantly in mesangial areas of mature glomeruli, corresponding to low circulating concentrations of these immunoglobulins. By contrast, glomerular IgG deposits were not observed until 21 days, despite relatively high concentrations of circulating maternal IgG from birth. Mesangial deposition of immunoglobulins increased with age. Absence of complement C3c or electron dense deposits associated with this mesangial localization suggests that immunoglobulins were not deposited as immune complexes. Accumulation of non-phlogogenic immunoglobulins in the mesangium of normal rats supports the concept that the mesangium is constantly perfused by circulating macromolecules and filtration residues. The results indicate problems of interpretation of the significance of endogenous immunoglobulin deposition in models of experimental glomerulonephritis, even in studies involving weanling rats.     

Bortezomib alleviates antibody-mediated rejection in kidney transplantation by facilitating Atg5 expression

Antibody-mediated rejection (AMR) is one of the most dominant mechanisms responsible for the loss of kidney grafts. Previous researches have shown that donor-specific antibodies (DSAs) are the major mediators of AMR. In order to prolong the survival time of grafts, it is vital to reduce the incidence of AMR and inhibit the generation of DSAs. We established an animal model of AMR by performing kidney transplantation in pre-sensitized rats. Then, we investigated the effect of bortezomib (BTZ) on AMR. We found that BTZ could reduce the serum level of DSAs and alleviate post-transplantation inflammation in peritubular capillaries (PTCs) and glomeruli, which was demonstrated by the reduction of C4d and IgG deposition in PTCs, and the reduced number of B cell and plasma cell in peripheral blood and the transplanted kidney (p < 0.05). Our results also suggested that BTZ increased the number of regulatory T cell (Treg) and significantly reduced the proportion of T helper (Th17) cell (p < 0.05). Besides, BTZ induced the significant upregulation of anti-inflammatory cytokines but downregulated pro-inflammatory cytokines (p < 0.05). After dealing with Atg5 siRNA-lentivirus, the effect of BTZ alleviating AMR was reversed and Th17/Treg proportions were also significantly modulated. Collectively, these findings show that BTZ slows down the process of AMR and Atg5 may be the key mechanism. Furthermore, Atg5 silencing results may be demonstrated that Atg5 alleviated AMR by modulating the ratio of Th17/Treg.     

Pathogenic significance of IgA receptor interactions in IgA nephropathy

IgA nephropathy (IgAN), the most common primary glomerulonephritis worldwide, frequently progresses to renal failure. The pathogenesis of this disease involves the deposition of undergalactosylated IgA1 complexes in the glomerular mesangium. How the IgA1 complexes are generated and why they are deposited in the mesangium remains unclear. We propose a model wherein two types of IgA receptors participate in sequential steps to promote the development of IgAN, with FcalphaRI (CD89) being initially involved in the formation of circulating IgA-containing complexes and, subsequently, transferrin receptor (CD71) in mediating mesangial deposition of IgA1 complexes.     

Urinary Thrombin: A Novel Marker of Glomerular Inflammation for the Diagnosis of Crescentic Glomerulonephritis (Prospective Observational Study)

Background

Crescentic glomerulonephritis (CresGN), an uncommon rapidly progressive disease, is characterized by severe glomerular inflammation with fibrin deposition. The lack of specific CresGN biomarkers delays diagnosis and threatens life. Because fibrin deposits in CresGN glomeruli indicate thrombin generation, we hypothesized that thrombin is excreted in urine and is a specific CresGN biomarker.

Methods

We measured urinary thrombin activity in 200 untreated patients (17 with CresGN, 183 with primary glomerulonephritis) and controls (8 patients with healed CresGN, 11 with nephrosclerosis, and 10 with tubulointerstitial nephritis, and 66 healthy volunteers). CresGN types included 15 pauci-immune and 2 immune complex. We assessed the diagnostic accuracy of thrombinuria in 169 patients with hematuria and proteinuria. Renal biopsy tissues were immunostained for tissue factor and fibrin. We analyzed the relationship of thrombinuria to plasma thrombin-antithrombin complex, hematuria, proteinuria, glomerular filtration rate, glomerular fibrin deposition, antineutrophil cytoplasmic antibodies (ANCAs), and C-reactive protein (CRP). We studied changes in thrombin activities after glucocorticoid treatment in 12 patients with thrombinuria.

Results

The highest thrombinuria occurrence was in CresGN (70.6%), followed by membranoproliferative glomerulonephritis (41.7%), IgA nephropathy (9.2%), and acute glomerulonephritis (0%). More than 75% of patients with nonproliferative glomerulonephritis manifested no thrombinuria. No controls had thrombinuria. Thrombinuria showed high CresGN specificity (90.1%) and moderate sensitivity (70.6%) and was detected in 4 of 7 patients with ANCA-negative CresGN. In CresGN, thrombinuria was associated with fibrin deposition in glomerular extracapillary tissue, where monocytes/macrophages expressed tissue factor. Thrombinuria in CresGN was unrelated to plasma thrombin-antithrombin complex, hematuria, proteinuria, glomerular filtration rate, and CRP. After glucocorticoid treatment, thrombinuria in patients with CresGN rapidly disappeared but proteinuria and hematuria persisted.

Conclusions

Thrombinuria was specific for glomerular inflammation, was unaffected by systemic inflammation or coagulation, and demonstrated good diagnostic accuracy for CresGN including ANCA-negative cases. Thrombinuria measurement may provide risk-free diagnosis and screening for CresGN.     

Plasma C3 and C4 Concentrations in Management of Glomerulonephritis

As part of a larger study of serial complement profiles in glomerulonephritis plasma C3 and C4 concentrations were measured using commercially available immunodiffusion plates. A total of 303 samples were obtained from 128 patients suffering from forms of nephritis associated with hypocomplementaemia—namely, lupus nephritis, mesangiocapillary glomerulonephritis (M.C.G.N.), and acute glomerulonephritis.These simple measurements of C3 and C4 gave clinically useful information. In lupus nephritis C3 and C4 generally correlated and C4 concentrations were more often and more profoundly depressed than C3 concentrations. Neither C3 nor C4 concentrations alone correlated well with the antinuclear factor titre.In both acute glomerulonephritis and M.C.G.N. the C3 concentrations were frequently lower than 20% of normal (which was never the case in patients with lupus), while the C4 concentration was usually normal and was almost never depressed in the absence of C3 depression. This suggests activation of complement at the C3 level by the “bypass” pathway in acute nephritis as well as in M.C.G.N., though both may be operating in some patients. In acute glomerulonephritis but not in M.C.G.N. C3 concentrations returned to normal within eight to 12 weeks.The two varieties of M.C.G.N. identified by the site of the deposits in the capillary glomerular walls differed in their C3 levels. In 10 patients with intramembranous dense linear deposits the C3 was always low over very long periods of time, rising in three out of four patients only after transplantation and immunosuppression. Other patients with M.C.G.N., in contrast, often showed normal C3 concentrations. Concentrations of C4 did not differ in either group, being normal in 80% of samples from all types.     

La glomérulonéphrite de shunt: manifestations cliniques et histopathologiques.

In patients with cerebrospinal fluid internal shunts, immune complex glomerulonephritis sometimes develops. Of two new cases the first was classic, while the second was in an adult who had had a ventriculoatril shunt for 8 years; furthermore, the patient had acute renal failure and is the first to have been reported to have Peptococcus septicemia. Shunt glomerulonephritis is characterized by the following: (a) its occurrence following, most often, Staphylococcus albus infection in a patient who usually has a ventriculoatrial shunt; (b) transitory improvement of the symptoms by antibiotherapy only; and (c) full recovery if the prosthesis is removed. Laboratory studies show a low serum concentration of the C3 component of complement, the presence of cryoglobulins and a positive rheumatoid factor test. These abnormalities are reversible with removal of the prosthesis. Optical microscopy of a renal biopsy specimen in the two cases showed cellular proliferation of the glomerular tuft, electron microscopy demonstrated subepithelial deposits and immunofluorescent studies revealed intramembranous and intramesangial immune complexes. These features are similar to those observed in experimental nephritis induced in animals by foreign protein.     

Dermatitis herpetiformis sera or goat anti-transglutaminase-3 transferred to human skin-grafted mice mimics dermatitis herpetiformis immunopathology

Dermatitis herpetiformis (DH) is characterized by deposition of IgA in the papillary dermis. However, indirect immunofluorescence is routinely negative, raising the question of the mechanism of formation of these immune deposits. Sárdy et al. (2002. J. Exp. Med. 195: 747-757) reported that transglutaminase-3 (TG3) colocalizes with the IgA. We sought to create such deposits using passive transfer of Ab to SCID mice bearing human skin grafts. IgG fraction of goat anti-TG3 or control IgG were administered i.p. to 20 mice. Separately, sera from seven DH patients and seven controls were injected intradermally. Biopsies were removed and processed for routine histology as well as direct immunofluorescence. All mice that received goat anti-TG3 produced papillary dermal immune deposits, and these deposits reacted with both rabbit anti-TG3 and DH patient sera. Three DH sera high in IgA anti-TG3 also produced deposits of granular IgA and TG3. We hypothesize that the IgA class anti-TG3 Abs are directly responsible for the immune deposits and that the TG3 is from human epidermis, as this is its only source in our model. These deposits seem to form over weeks in a process similar to an Ouchterlony immunodiffusion precipitate. This process of deposition explains the negative indirect immunofluorescence results with DH serum.     

Modulation of renal disease in MRL/lpr mice genetically deficient in the alternative complement pathway factor B

In systemic lupus erythematosus, the renal deposition of complement-containing immune complexes initiates an inflammatory cascade resulting in glomerulonephritis. Activation of the classical complement pathway with deposition of C3 is pathogenic in lupus nephritis. Although the alternative complement pathway is activated in lupus nephritis, its role in disease pathogenesis is unknown. To determine the role of the alternative pathway in lupus nephritis, complement factor B-deficient mice were backcrossed to MRL/lpr mice. MRL/lpr mice develop a spontaneous lupus-like disease characterized by immune complex glomerulonephritis. We derived complement factor B wild-type (B+/+), homozygous knockout (B-/-), and heterozygous (B+/-) MRL/lpr mice. Compared with B+/- or B+/+ mice, MRL/lpr B-/- mice developed significantly less proteinuria, less glomerular IgG deposition, and decreased renal scores as well as lower IgG3 cryoglobulin production and vasculitis. Serum C3 levels were normal in the B-/- mice compared with significantly decreased levels in the other two groups. These results suggest that: 1) factor B plays an important role in the pathogenesis of glomerulonephritis and vasculitis in MRL/lpr mice; and 2) activation of the alternative pathway, either by the amplification loop or by IgA immune complexes, has a prominent effect on serum C3 levels in this lupus model.     

Immunocytochemical studies of desmin and vimentin in pericapillary cells of chicken

The composition of intermediate filaments in pericytes was examined by immunofluorescent and immunoelectron microscopic labeling of frozen sections of various chicken microvascular beds in situ. Pericytes in capillaries of cardiac muscle, exocrine pancreas, and kidney (peritubular capillary) were found to contain both desmin and vimentin. In some capillaries where pericytes do not exist, cells apposed to endothelial cells--the Ito cell in the hepatic sinusoid and the reticular cell in the splenic sinusoid--were shown to contain both of the intermediate filament proteins. In contrast, podocytes and mesangial cells around renal glomerular capillaries contained only vimentin. The presence of desmin supports the hypothesis that pericytes may have a contractile apparatus similar to that of vascular smooth muscle cells. Our results also revealed that even in microvascular beds where pericytes are not found, cells having both desmin and vimentin exist next to endothelial cells and may assume similar functions to pericytes.     

Reduced hnRNPA3 increases C9orf72 repeat RNA levels and dipeptide‐repeat protein deposition

Intronic hexanucleotide (G₄C₂) repeat expansions in C9orf72 are genetically associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat RNA accumulates within RNA foci but is also translated into disease characterizing dipeptide repeat proteins (DPR). Repeat‐dependent toxicity may affect nuclear import. hnRNPA3 is a heterogeneous nuclear ribonucleoprotein, which specifically binds to the G₄C₂ repeat RNA. We now report that a reduction of nuclear hnRNPA3 leads to an increase of the repeat RNA as well as DPR production and deposition in primary neurons and a novel tissue culture model that reproduces features of the C9orf72 pathology. In fibroblasts derived from patients carrying extended C9orf72 repeats, nuclear RNA foci accumulated upon reduction of hnRNPA3. Neurons in the hippocampus of C9orf72 patients are frequently devoid of hnRNPA3. Reduced nuclear hnRNPA3 in the hippocampus of patients with extended C9orf72 repeats correlates with increased DPR deposition. Thus, reduced hnRNPA3 expression in C9orf72 cases leads to increased levels of the repeat RNA as well as enhanced production and deposition of DPR proteins and RNA foci.     

Pathogenesis of Berger's disease: recent advances on the involvement of immunoglobulin A and their receptors

Immunoglobulin A (IgA) nephropathy or Berger's disease is the most common form of primary glomerulonephritis in the world and one of the first cause of end-stage renal failure. IgA nephropathy is characterized by the accumulation in mesangial areas of immune complexes containing polymeric IgA1. While epidemiology and clinical studies of IgA nephropathy are well established, the mechanism(s) underlying disease development is poorly understood. The pathogenesis of this disease involves the deposition of polymeric and undergalactosylated IgA1 in the mesangium. Quantitative and structural changes of IgA1 play a key role in the development of the disease due to functional abnormalities of two IgA receptors: The FcalphaR (CD89) expressed by blood myeloid cells and the transferrin receptor (CD71) on mesangial cells. Abnormal IgA induce the release of soluble CD89 which is responsible for the formation of circulating IgA complexes. These complexes may be trapped by CD71 that is overexpressed on mesangial cells in IgA nephropathy patients allowing pathogenic IgA complex formation.     

Inhibition of B cell death causes the development of an IgA nephropathy in (New Zealand white x C57BL/6)F(1)-bcl-2 transgenic mice

Little is known about the pathogenic mechanisms of IgA nephropathy, despite being the most prevalent form of glomerulonephritis in humans. We report in this study that in (New Zealand White (NZW) x C57BL/6)F(1) mice predisposed to autoimmune diseases, the expression of a human bcl-2 (hbcl-2) transgene in B cells promotes a CD4-dependent lupus-like syndrome characterized by IgG and IgA hypergammaglobulinemia, autoantibody production, and the development of a fatal glomerulonephritis. Histopathological analysis of glomerular lesions reveals that the glomerulonephritis observed in these animals resembles that of human IgA nephropathy. The overexpression of Bcl-2 in B cells selectively enhances systemic IgA immune responses to T-dependent Ags. Significantly, serum IgA purified from (NZW x C57BL/6)F(1)-hbcl-2 transgenic mice, but not from nontransgenic littermates, shows reduced levels of galactosylation and sialylation and an increased ability to deposit in the glomeruli, as observed in human patients with IgA nephropathy. Our results indicate that defects in the regulation of B lymphocyte survival associated with aberrant IgA glycosylation may be critically involved in the pathogenesis of IgA nephropathy, and that (NZW x C57BL/6)F(1)-hbcl-2 Tg mice provide a new experimental model for this form of glomerulonephritis.     

IgA nephropathy in adults: immunohistologic findings and clinical course

Laboratory examination of specimens from 123 consecutive renal biopsies performed at Victoria General Hospital, Halifax revealed six cases of mesangial deposition, predominantly of IgA, unassociated with systemic disorders. Immunohistologic examination showed deposits of only IgA in one specimen, IgA and IgG in two and IgA, IgG and IgM in three. Glomerular deposits of C3 were seen in five of the specimens, and properdin was seen in three. Glomeruli in all the specimens showed increased matrix and increased numbers of cells in the mesangium. Electron microscopy revealed deposits in the mesangium or capillary wall in all five of the specimens so studied. All six patients had proteinuria, four had microscopic hematuria, and three had hypertension; in one patient the disease progressed to renal failure.     

Defective oral tolerance promotes nephritogenesis in experimental IgA nephropathy induced by oral immunization.

Oral tolerance, an important feature of the mucosal immune system, appears to protect against immune-mediated disease by blunting production of systemic IgG and IgM antibody directed toward immunogens chronically present at mucosal surfaces. In this study, we explored the role of oral tolerance and mucosal immunoregulation in an experimental model of IgA nephropathy (IgAN), an important form of nephritis in humans. Cyclophosphamide and estradiol were used to inhibit the expression of oral tolerance, which otherwise develops after chronic oral presentation of Ag. BALB/c mice given drinking water containing 0.1% bovine gamma globulin (BGG) continuously for 14 wk were randomly assigned to groups given either 2 mg of cyclophosphamide i.p., 2 mg of estradiol s.c. or both drugs. Groups of control mice received neither BGG nor drugs. In three separate experiments, a low percentage of saline-treated orally immunized mice had microscopic hematuria (0 to 20%), as did nonimmunized controls (0 to 20%). However, 58 to 83% of mice given estradiol and/or cyclophosphamide at appropriate times developed significant hematuria. If drugs were given at suboptimal times, only 25 to 56% of mice developed hematuria. Drug-treated immunized mice also had more serum IgG and IgM anti-BGG antibodies than control and saline groups. Immunofluorescence showed significantly more glomerular deposits of IgG, IgM, and C3 in drug-treated immunized mice compared to saline-treated immunized and normal untreated control mice. Hematuria and glomerular deposits of IgG, IgM, and C3 paralleled serum IgG and IgM antibody. All immunized mice showed significant mesangial IgA and BGG deposits and there were no differences in such deposits between saline- and drug-treated immunized mice. We suggest that blunting of oral tolerance with promotion of systemic IgG and IgM antibody production leads to nephritis in chronically orally immunized mice and that glomerular immune complexes containing IgG and/or IgM promote complement deposition and hematuria in IgAN. Analogous defects in oral (or more generally mucosal) tolerance could play a role in the genesis of symptomatic human IgAN.     

Nephrotic Syndrome in Monkey Infected with Human Quartan Malaria

A splenectomized aotus monkey infected with human quartan malaria (Plasmodium malariae) developed oedema and proteinuria. Histological examination revealed a generalized diffuse glomerulonephritis and immunofluorescent staining showed granular deposits of IgM in the glomeruli. The pathological picture resembled that shown by human patients with the quartan malaria nephrotic syndrome.