Tissue culture of isolated glomeruli in experimental crescentic glomerulonephritis.

As a means of studying mechanisms of response to injury in glomerulonephritis, glomeruli from normal sheep and rabbits and from sheep and rabbits with experimental crescentic glomerulonephritis have been isolated and grown in tissue culture. The cellular outgrowths from the normal and diseased glomeruli have been compared. The outgrowth of glomeruli from normal animals contained only two cell populations whose microscopic and ultrastructural appearances were of epithelial and mesangial cells. The same cells were also observed in the outgrowths of glomeruli from animals with crescenti nephritis but in addition a third population of cells was present in large numbers. These cells were identified as macrophages by their mobility, ultrastructure, phagocytic capacity, and presence of Fc receptors. Glomerular outgrowth from sheep with crescentic glomerulonephritis contained 170 +/- 20 (SEM) macrophages and outgrowths from rabbits with crescentic nephritis contained 64 +/- 6 (SEM) macrophages per glomerulus. We have previously observed large numbers of macrophages in the outgrowth of isolated glomeruli from humans with rapidly progressive crescentic glomerulonephritis. The predominance of the macrophage in cultures of glomeruli from both human and animal crescentic glomerulonephritis suggests that this is an important cell in the inflammatory reaction occurring in crescentic glomerulonephritis and may comprise a substantial proportion of the cells forming the crescent.

Interactions between methylating and pyridyloxobutylating agents in A/J mouse lungs: implications for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis.

The tobacco-specific nitrosamine, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone, is activated to lung DNA methylating and pyridyloxobutylating intermediates. It is likely that both pathways play a role in lung tumor initiation by this nitrosamine. Previous studies indicated that O(6)-methylguanine (O(6)-mG) persistence is critical for lung tumor formation in A/J mice. The model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc), enhanced the tumorigenic activity of a model methylating agent, acetoxymethylmethylnitrosamine (AMMN), presumably by increasing O(6)-mG persistence in lung DNA. We have been testing the hypothesis that the pyridyloxobutylation pathway increases the mutagenic activity of the DNA methylation pathway by preventing the repair of O(6)-mG by O(6)-alkylguanine-DNA alkyltransferase (AGT). In this study, we report that NNKOAc depletes AGT in lungs but not livers of A/J mice. The consequences of AGT depletion by NNKOAc were then compared with those observed with a known AGT inhibitor, O(6)-benzylguanine (O(6)-bG). NNKOAc and O(6)-bG had similar effects on the levels of AMMN-derived O(6)-mG at 4 and 96 h postinjection. This increase in O(6)-mG levels correlated to increased lung tumor multiplicity in animals simultaneously treated with AMMN (0.75 or 1 micromol) and NNKOAc or O(6)-bG. Only NNKOAc significantly increased lung tumor multiplicity at doses of 0.25 or 0.5 micromol AMMN. The results from these studies indicate that the pyridyloxobutylating agent, NNKOAc, can influence the tumorigenic activity of methylating agents in two ways. At low AMMN doses, the increase in tumor multiplicity is dominated by the additive tumorigenic properties of AMMN and NNKOAc. At higher AMMN doses, NNKOAc appears to enhance the tumorigenic activity of AMMN through enhanced depletion of the repair protein, AGT, leading to increased O(6)-mG persistence. It is likely that similar interactions are important for the organospecific effects of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone.

Apoptosis and the expression of genes of the Bcl-2 family and TGF-beta1 in rat renal allografts transplanted after donor-specific blood transfusion.

UNLABELLED: Factors involved in "operational" tolerance in animal models induced by recipient pre-treatment with donor-specific blood transfusion (DSBT) need elucidation. This study examined apoptosis, expression of genes of the Bcl-2 family and of TGF-beta(1) in isografts, rejecting and tolerant allografts. METHODS: Adult inbred Dark Agouti (DA) kidneys were transplanted, with immediate nephrectomy of recipient kidneys, to (1) ALLO, inbred Albino Surgery (AS) rats; (2) DSBT ALLO, AS rats who received two DA blood transfusions under cover of cyclosporine prior to transplantation; or (3) ISO, DA rats. Grafts were retrieved on day 1, 3, or 5. Apoptosis was assessed by TUNEL. RNA was extracted and reverse transcribed to cDNA for quantification by real-time PCR, relative to the 18s housekeeping gene. RESULTS: Apoptosis was negligible in ISO while it increased in allograft groups from day 1. On day 5, apoptosis in ALLO (114.0 +/- 30.6), involved renal tubular cells and leukocytes compared to DSBT ALLO (9.7 +/- 4.0) and ISO (0.9 +/- 0.3) involving leukocytes only. On day 1, DSBT ALLO had higher expression of Bax than ALLO or ISO. On day 3, DSBT ALLO and ALLO had higher TGF-beta(1) mRNA than ISO. On day 5, Bcl-2 expression was significantly decreased (P < .001) in ALLO compared to DSBT ALLO and ISO. Bad and Bid were higher in DSBT ALLO than in ALLO. TGF-beta(1) was higher in DSBT ALLO compared to ISO. CONCLUSIONS: Decreased expression of anti-apoptotic Bcl-2 gene may be implicated in increased apoptosis in rejecting allograft while expression of pro-apoptotic genes may be involved in the establishment of operational tolerance.

Modulation of inhibition of the human mitogen response by methylprednisolone.

The in vitro response of human lymphocytes to phytohaemagglutinin was inhibited in a dose-related fashion by methylprednisolone added to the cell culture. The inhibition by steroid was 100-fold less than expected from published values of human lymphocyte receptor affinity. The dose response curve for inhibition was complex, frequently featuring a plateau region suggestive of a heterogeneous system, possibly caused by the presence of more than one subpopulation of cells. When lymphocytes were separated by unit velocity sedimentation, slow sedimenting lymphocytes were found to be highly steroid sensitive whereas rapidly sedimenting lymphocytes were steroid resistant. However, the addition of cultured macrophages rendered the steroid-sensitive fractions relatively steroid resistant. Similarly, unseparated peripheral blood lymphocytes at low concentrations were found to be highly steroid sensitive despite the addition of 2-mercaptoethanol. This sensitivity was lost by the addition of culture macrophages. Thus, macrophages play a vital role in the relative steroid resistance of human lymphocytes. No evidence was found that macrophages actually reduced steroid concentration in culture, and it is suggested that a cellular interaction is required to increase resistance to steroids.

Composition of interstitial cellular infiltrate identified by monoclonal antibodies in renal biopsies of rejecting human renal allografts.

Monoclonal antibodies and a four-layer immunoperoxidase technique were used to analyze and quantitate the various infiltrating cell types found in percutaneous renal biopsies of 25 patients undergoing acute renal allograft rejection. Cell markers included monoclonal antibodies to the human leukocyte-common antigen (PHM 1), mononuclear phagocytes (PHM 2, FMC 32), T cells and T cell subsets (OKT 3, OKT 4, and OKT 8), B cells (7.2), polymorphs (FMC 10, FMC 13), and plasma cells (OKT 10). The proportion of labelled interstitial cells was expressed as a percentage of the total number of infiltrating leukocytes identified with PHM 1, and correlated with the histologically graded intensity of rejection. In mild rejection 32% of the infiltrating cells were T lymphocytes, of which 90% were OKT-8-positive cytotoxic-suppressor cells, and 52% were macrophages. Similarly, in moderate rejection T cells composed 42% of the infiltrate (with 67% of T cells expressing OKT 8 antigen), and macrophages formed 38% of the total cells. By contrast, in severe rejection, the T cell component was decreased to 15% of the cells, of which 78% were OKT-8-positive; these were preponderantly macrophages (60%) and polymorphs (22%). These studies demonstrate that cytotoxic-suppressor T cells and macrophages are the major cells mediating acute interstitial graft rejection.

Transforming growth factor-beta1 and tumor growth factor-beta-inducible gene-H3 in nonrenal transplant cyclosporine nephropathy.

Cyclosporine nephropathy (CyAN) is a major limiting factor in the otherwise successful widespread use of cyclosporine in solid organ transplant. Transforming growth factor-beta1 (TGF-beta1) has been implicated as an important fibrogenic cytokine in the development of this disease. TGF-beta-inducible gene-H3 (beta(ig)-H3) is a TGF-beta1- induced gene product, which acts as a marker for biologically active TGF-beta1. This study reports TGF-beta1 gene expression and beta(ig)-H3 tissue distribution in non-renal allograft CyAN. Renal tissue from nine patients who had developed CyAN after successful heart or heart-lung transplantation and from four kidneys removed for tumour were analyzed for TGF-beta1 gene expression beta(ig)-H3 protein with reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. TGF-beta1 gene expression was increased in CyAN compared to nephrectomy (P<0.0001). Beta(ig)-H3 protein expression was identified in distal convoluted tubular epithelium and parietal glomerular epithelium in CyAN, and not in nephrectomy samples. Expression of TGF-beta1 mRNA was significantly higher in renal tissue from patients not receiving angiotensin converting enzyme inhibitor (ACEI) therapy for hypertension (P<0.05). These findings support the hypothesis that TGF-beta1 is an important cytokine in the development of CyAN, independent of its role in chronic rejection in renal allografts.

Phenotypic definition of primed T cells in human renal allografts. Use of the CD45R marker.

Immunohistological studies indicate that T cells and macrophages are the major components of human kidney allograft infiltrates. Recent work has demonstrated a division of T lymphocytes into 2 subpopulations with distinct functions on the basis of their expression of the CD45R antigen (CD45R+ "naive" and CD45R- "memory" T cells). This study analyzes CD45R expression on circulating T cells and T cells infiltrating renal allografts in patients undergoing rejection and/or cyclosporine nephrotoxicity. The percentage of circulating T cells that expressed CD45R in patients with rejecting (63 +/- 4) or stable grafts (66 +/- 3) was not different from values obtained for normal donors (62 +/- 3). In contrast, the percentage of T cells expressing CD45R infiltrating rejecting grafts was 21 +/- 2 and was not affected by the stage of rejection; in patients with CsA toxicity the value was 22 +/- 6. The reduced proportion of T cells that expressed CD45R in the allograft may reflect a change in status from the naive state due to alloantigenic stimulation (which can be demonstrated in vitro) and/or a propensity of memory T cells to enter or be retained in an allograft.

Development of chronic injury and nature of interstitial infiltrate in a model of chronic renal allograft rejection.

A model of chronic renal rejection in the Dark-Agouti to Albino-Surgery rat combination is described. In a number of cases, the original allograft was replaced by a second Dark-Agouti allograft. Seventy-five percent of rats experienced early episodes of rejection that subsided spontaneously. Second allografts had better initial renal function. Variable degrees of tubular atrophy, interstitial fibrosis, vascular damage, glomerulosclerosis, deposition of humoral mediators, and mononuclear leukocyte infiltrate were observed in all long-term allografts. Chronic damage increased with time, and was less severe in second allografts. At 5 days, total interstitial infiltrate was similar to that seen in unmodified rejection, but there was a significant increase in CD4+ cells and a decrease in ED2 and IL-2R expression. Subsequently, the total interstitial infiltrate decreased with time, although it remained significantly higher than in isografts and residual kidneys from uninephrectomized rats. No significant decrease over time was seen in numbers of CD4+ and CD45RC+ cells. The latter had a marked focal distribution after 100 days. Total leukocyte infiltrate was similar in original and second allografts, but there were changes in the proportions of leukocyte subpopulations, including significantly lower numbers of CD45RC+ cells in the latter. The persistence of CD45RC+ cells throughout the course of chronic rejection and their lower numbers in the second allografts favors a role for these cells in the development of chronic injury. The model of chronic renal allograft rejection characterized in this study will be valuable in further studies of the mechanisms of injury in this pathology.

Cytomegalovirus infection complicating renal transplantation and its relationship to acute transplant glomerulopathy.

The incidence of cytomegalovirus (CMV) infection was established, using laboratory criteria, in 298 patients receiving 362 renal allografts (164/298 = 55%). The incidence of CMV infection did not differ between azathioprine/prednisolone-treated and cyclosporine-treated patients (55% vs. 57% NS). The use of antithymocyte globulin (ATG) increased the incidence of CMV infection (78% vs. 51%: P less than 0.01). Donor and recipient CMV status, known for 116 allografts, did not correlate with the incidence of CMV infection (recipient CMV-positive = 50%; recipient CMV-negative = 54%: NS). CMV infection was responsible for 8 patients' deaths (2.7% mortality). Thirty-three patients with acute transplant glomerulopathy were identified (11%). There was no correlation between acute transplant glomerulopathy and CMV infection. Glomerulopathy was associated with poor graft survival (22/33 patients with a graft survival of less than 6 months). Thus CMV infection, although a common complication of renal transplantation with significant morbidity and mortality, is not closely associated with acute transplant glomerulopathy. Further, the lack of correlation of donor-recipient CMV serologic status with graft outcome limits the usefulness of pretransplantation donor screening.

Severe visceral disease in subacute cutaneous lupus erythematosus.

Subacute cutaneous lupus erythematosus has been heralded as a marker of relatively benign subset of systemic lupus erythematosus. In this article, we describe two cases with severe visceral disease and suggest that it may be less reliable as a predictor of benign disease than was previously accepted.

Histochemistry of glomerular cells in animal models of crescentic glomerulonephritis.

A histochemical enzyme profile has been used to determine the origin of the cells of glomerular crescents in rabbit and sheep models of rapidly progressive crescentic glomerulonephritis. Crescentic glomeruli were examined for beta-glucuronidase, esterase and acid phosphatase, the characteristic phagolysosomal enzymes of the monocyte-macrophage series and its transformed tissue counterpart, the epithelioid cell. More than 50% of the cells of the glomerular crescents of animals with experimental crescentic glomerulonephritis contained large amounts of all the enzymes, whereas the cells of normal glomeruli contained only trace amounts of acid phosphatase and esterase and no glucuronidase. These findings support the hypothesis that the major proportion of the cells of glomerular crescents are monocytes which have accumulated in Bowman's space rather than intrinsic glomerular cells which have proliferated.

Tissue culture of isolated human glomeruli.

Glomerular cells have been grown in a reproducible manner from 5 normal human kidneys. A technique is described which combined mechanical disruption of renal cortex and microdissection, and provides large numbers of pure glomeruli within 30--45 mintues. Histological examination shows this technique produces intact glomeruli without cell disturbance. During tissue culture, glomeruli attach to the flask and the intrinsic cells migrate onto the flask and divide. Variations of culture conditions have shown that glomeruli are robust without fastidious culture requirements. Intact kidney tissue can be left at 4 degrees C for perios up to 24 hours prior to isolated of individual glomeruli without affecting subsequent cellular growth in culture. They grow in most commonly used media although the cells require 20% foetal calf serum for optimum growth. Their pH optimum is between 7.0 and 7.4 with temperature optimum of 37 degrees C. as glomeruli must attach prior to cell growth, minimum movement is critical to promote optimum growth. Under these optimum conditions a regular and predictable growth of cells of two distinct types, has been observed over 14 days; one of these types is probably epithelial.

Primary T-cell-rich B-cell lymphoma in the kidney presenting with acute renal failure and a second malignancy.

Infiltration of the kidney is commonly found in lymphoma, but acute renal failure arising from bilateral renal infiltration is uncommon. Primary renal lymphoma may occur and is usually of B-cell lineage. It is rare for patients with lymphoma to develop acute renal failure as their initial clinical presentation. Recently, an association between primary renal lymphoma and a second primary malignancy has been reported. We describe the first case of a renal T-cell-rich B-cell lymphoma presenting as acute renal failure, which was associated with a second primary pulmonary malignancy.

A robust route to enzymatically functional, hierarchically self-assembled peptide frameworks.

The addition of enzyme biofunctionality to self-assembling peptide nanofibers is challenging since such additions can inhibit functionality or self-assembly. We introduce a method for peptide nanofiber enzyme functionalization, demonstrated by the attachment of a polymerization synthase to peptide nanofibers. The enzyme generates a biocompatible, biodegradable biopolyester coat on the fibers with applicablity in medical engineering. This approach provides a template for generation of functional bionanomaterials.