Clinical outcomes in chronic intervillositis of unknown etiology.

INTRODUCTION: Chronic intervillositis of unknown etiology (CIUE) is a histopathological lesion of the placenta that is frequently accompanied by unfavourable pregnancy outcomes, e.g. miscarriage, fetal growth restriction (FGR) and intrauterine fetal death. Earlier described case series and cohorts have been based on diverse diagnostic criteria of CIUE. To improve our understanding of clinical outcomes associated with CIUE, we report the obstetric and perinatal outcomes in a cohort based on the recently described diagnostic criteria. METHODS: CIUE is defined as an infiltrate occupying 5% or more of the intervillous space with approximately 80% of mononuclear cells positive for CD68 in the absence of an infection. Thirty-eight cases were included. Also previous and subsequent pregnancies were described. RESULTS: Pregnancies accompanied by CIUE frequently resulted in FGR (51.6%) and pre-term birth (55.3%). Twenty-nine out of 38 pregnancies (76.3%) with CIUE resulted in a living baby. Women with CIUE frequently have had a miscarriage (16/38; 42%). Four-teen subsequent pregnancies in 8 women resulted in 2 miscarriages, 2 terminations of pregnancy for FGR, 1 early neonatal death and 9 living babies (9/14; 64.3%). Histopathologically confirmed CIUE recurred in 5 out of 10 subsequent pregnancies. Two pregnancies with recurrent CIUE were terminated, one pregnancy ended in a late miscarriage and another resulted in term birth complicated by FGR. Recurrent CIUE can also be accompanied by an uncomplicated pregnancy (1/5; 20%). CONCLUSION: This study provides additional insight into the clinical phenotype of CIUE and emphasises the need for further research to understand the pathophysiology behind different pregnancy outcomes in CIUE.

Myxoid tumours of soft tissue: the so-called myxoid extracellular matrix is heterogeneous in composition.

AIM: Myxoid tumours of soft tissue are characterized by their so-called 'myxoid' extracellular matrix. The aim was to investigate the composition and possible function of this matrix which is poorly understood. METHODS AND RESULTS: Using Alcian Blue staining with and without pretreatment with hyaluronidase and application of the critical electrolyte concentration method followed by densitometry, the glycosaminoglycan composition of three different myxoid tumours was studied. The composition of glycosaminoglycans varied with tumour type and grade, despite their general characterization as myxoid tumours. Intramuscular myxoma contained similar amounts of the various glycosaminoglycans as grade I myxofibrosarcoma; grade III myxofibrosarcoma contained less hyaluronic acid and more heparan sulphate, whereas extraskeletal myxoid chondrosarcoma contained predominantly chondroitin-4 and -6 sulphates. Western blot identified albumin as a major protein in tumour lysates, and its presence in the extracellular matrix and cytoplasm of the majority of tumours was demonstrated by immunohistochemistry; production of albumin by the tumour cells was confirmed by quantitative polymerase chain reaction. CONCLUSIONS: The extracellular matrix of myxoid tumours of soft tissue has a heterogeneous composition consisting of, amongst others, glycosaminoglycans and albumin, which appear to play an active role in their morphogenesis.

ECM homeostasis in renal diseases: a genomic approach.

Chronic renal disease is in general histologically accompanied by a vast amount of scar tissue, ie glomerulosclerosis and interstitial fibrosis. Scarring results from excessive accumulation of extracellular matrix (ECM) components, a process driven by a plethora of cytokines and growth factors. Studies in experimental renal disease which target these regulators using gene therapy limit or prevent the development of scarring. This review focuses specifically on the role of transforming growth factor-beta, platelet-derived growth factor, connective tissue growth factor, hepatocyte growth factor, and epidermal growth factor. The results obtained in animal models hold promise for molecular intervention strategies in human renal disease. Microarray technology allows large-scale gene expression profiling in kidney tissue to identify common molecular pathways in a step towards discovery of new drug targets. Molecular techniques are expected to be used for diagnostic and prognostic purposes in nephrological practice to supplement renal biopsy. Several studies already show that molecular techniques might be of use in routine diagnostic practice. Improvement of diagnosis and prediction of outcome in renal patients might lead to more efficient and earlier therapeutic intervention.

Cytokine profile of cervical cancer cells.

OBJECTIVE: In patients with cervical carcinoma, the presence of cytokines produced by T(H)2 cells, and the presence of an eosinophilic inflammatory infiltrate, has been associated with a less effective immune response and tumor progression. In the present study, we have investigated the cytokine profile of cervical carcinoma cells. In addition, we have measured whether differences in cytokine profiles between normal and malignant cervical epithelial cells are present. METHODS: For this purpose we have determined the mRNA expression patterns of 20 relevant cytokines by RT-PCR and Southern blotting in 3 normal primary cervical epithelial cell cultures (NPE) and 10 cervical cancer cell lines (CCCL). RESULTS: TGF-beta(1), IL-4, IL-12p35, and IL-15 were produced by all CCCL and NPE. TNF-alpha, IL-10, IL-5, and RANTES were present in most NPE, but not in any of the CCCL. MCP-1 was expressed in all CCCL but in only one NPE. The presence of the anti-inflammatory cytokine TGF-beta(1) in cervical carcinomas was confirmed by RNA in situ hybridization on tissue sections of carcinomas from which the CCCL originated. CONCLUSIONS: Our results suggest that cervical carcinoma cells produce immunomodulatory cytokines and that cytokine expression patterns change after malignant transformation. The implications of locally produced cytokines by cervical cancer cells are further discussed.

Malignant melanoma is genetically distinct from clear cell sarcoma of tendons and aponeurosis (malignant melanoma of soft parts).

Clear cell sarcoma of tendons and aponeuroses (malignant melanoma of soft parts) and conventional malignant melanoma may demonstrate significant morphologic overlap at the light microscopic and ultrastructural level. Consequently, the clinically relevant distinction between primary clear cell sarcoma and metastatic melanoma in the absence of a known primary cutaneous, mucosal or ocular tumour may occasionally cause diagnostic problems. A balanced translocation, t(12;22)(q13;q13), which can be detected, amongst others, using the reverse transcriptase polymerase chain reaction (RT-PCR) or fluorescent in situ hybridization (FISH), has been identified in a high percentage (50-75%) of clear cell sarcomas and is presumed to be tumour specific. Whether this chromosomal rearrangement is present in malignant melanoma has, to date, not as yet been studied by molecular genetic or molecular cytogenetic techniques. Using RT-PCR and FISH, a series of metastases from 25 known cutaneous melanomas and 8 melanoma cell lines (5 uveal and 3 cutaneous) were screened for the t(12;22)(q13;q13) translocation. Primers for RT-PCR were chosen based upon published breakpoint sequences. The Cosmids G9 and CCS2.2, corresponding to the 5' region of EWS and 3' region of ATF-1 respectively, were used as probes. The translocation was not identified in any of the melanomas or melanoma cell lines analysed in this study; in contrast this translocation was identified in 3 out of 5 clear cell sarcomas using these techniques. This allows distinction between translocation positive cases of clear cell sarcoma and malignant melanoma at a molecular genetic level. Consequently, in diagnostically challenging cases, this represents a valuable tool for the clinicopathologic differentiation between these two entities, with an important impact on patient management and prognosis.

Idiotype usage by polyclonally activated B cells in experimental autoimmunity and infection.

Both in animal models and in human systemic lupus erythematosus (SLE) the occurrence of nephritogenic autoantibodies bearing dominant idiotypes has been described. In this study we investigate the relation between the induction pathway of polyclonal B cell activation and the production and glomerular deposition of nephritogenic antibodies with shared dominant idiotype(s). Polyclonal B cell activation was induced in several experimental models characterized by glomerular immune deposit formation. We monitored the occurrence of dominant idiotypes among immunoglobulins deposited in the glomeruli. In addition, we studied the species specificity of the dominant idiotypes, by monitoring their presence in kidney sections of patients with an immunologically mediated kidney disease. Anti-idiotype antisera against two monoclonal anti-DNA autoantibodies were used, derived from MRL-lpr/lpr mice, i. e. clone H241 and clone H130. Autoantibodies with the H241 idiotype were present in immune complex depositions in all experimental models but not in humans. We therefore conclude that the presence of this dominant idiotype is independent of the induction pathway of polyclonal B cell activation. However, autoantibodies bearing the H130 idiotype were only detected in kidney sections of mice with spontaneous lupus.

Cisplatin effects on F-actin and matrix proteins precede renal tubular cell detachment and apoptosis in vitro.

In primary cultures of porcine proximal tubular kidney cells and LLC-PK1 cells cisplatin (5 - 50 microM) caused apoptosis and cell detachment; in both systems cell detachment occurred, preceded by a loss of cytoskeletal F-actin stress fibers within 4 - 6 h, and a reduction of mRNA encoding for fibronectin, collagen a2 type (IV) and laminin B2 within 17 - 41 h. Prevention of F-actin damage by phalloidin prevented nuclear fragmentation, suggesting a relation between F-actin damage and apoptosis. Overexpression of Bcl-2 also prevented apoptosis, but did not prevent damage to the F-actin skeleton or the reduction of mRNA expression of the matrix proteins. These results suggest that Bcl-2 overexpression interferes with apoptotic signals downstream of F-actin. The relevance of these results for cell detachment in kidney toxicity is discussed.

The glomerular deposition of PAS positive material correlates with renal function in human kidney diseases.

General agreement exists on the correlation of renal insufficiency with the severity of tubulointerstitial abnormalities in the biopsy. This could not be shown for the severity of glomerular pathology by semiquantitative methods. The relation between renal function and glomerular pathology was therefore evaluated in patients with various kidney diseases using quantitative measurements. Fifty-five patients and ten controls were studied. Histomorphometric measurements of renal biopsies were obtained for both frozen and paraffin sections and were correlated with serum creatinine values. The frozen sections were stained with an indirect immunoperoxidase technique using antibodies against collagen types I, IV, V, VI, laminin, fibronectin, decorin and heparansulphate proteoglycan core protein. The contribution of each component to the composition of the mesangial extracellular matrix was scored with a semiquantitative technique. All glomerular histomorphometric indices correlated with the severity of renal insufficiency expressed as serum creatinine at the time of biopsy. However, quantitative estimates of the glomerular deposition of periodic acid-Schiff positive extracellular matrix seemed to be the most important structural correlate of renal function (r = 0.524, p = 0.0001). Semiquantitative estimates of interstitial extracellular matrix accumulation were less correlated with renal function (r = 0.370, p = 0.01). The composition of the mesangial extracellular matrix did not differ in patients and controls, with the exception of the extent of laminin staining, which was significantly higher in patients. The extent of fibronectin staining in patients correlated with the severity of glomerular structural abnormalities. This study demonstrates that the severity of renal insufficiency in a variety of renal diseases correlates with the severity of glomerular pathology, when quantitative scoring is applied.

Differential effects of sex hormones on autoantibody production and proteinuria in chronic graft-versus-host disease-induced experimental lupus nephritis.

In patients with systemic lupus erythematosus, the female-to-male ratio is as high as 10:1. Sex hormones are thought to play a role in this difference in susceptibility. In a previous study, we demonstrated a high susceptibility of female mice to the development of glomerulonephritis after induction of chronic graft-versus-host disease (GVHD), compared with male mice. In order to unravel further this gender-related difference (C57B1/10*DBA/2)F1 hybrid mice were either castrated or ovariectomized and treated with 17beta-ethinyloestradiol or testosterone-decanoate preceding the induction of chronic GVHD. Testosterone-decanoate reduced significantly the development of albuminuria in females. In contrast, proteinuria of 17beta-ethinyloestradiol-treated female mice was in the same range as that of sham-operated mice. Autoantibody levels against glomerular basement membrane, renal tubular epithelium, dsDNA and ssDNA, as determined by ELISA, were higher in 17beta-ethinyloestradiol-treated female mice than in all other groups. Immunofluorescence studies showed the presence of immunoglobulin and complement deposits in glomeruli of all animals, without significant differences between the experimental groups. Our findings confirm earlier observations, in that testosterone-decanoate is shown to be an inhibitory compound, whereas 17beta-ethinyloestradiol has stimulating properties in autoimmunity. Moreover, our results show for the first time differential hormonal effects on autoantibody levels and proteinuria in experimental lupus nephritis.

Characterization of reactivity of monoclonal autoantibodies with renal antigens in experimental lupus nephritis.

Mice with chronic graft-versus-host disease (GvHD), induced by injection of DBA/2 lymphocytes into (C57BL10*DBA/2)F1 hybrids, develop a lupus-like syndrome with immune complex glomerulonephritis. Circulating autoantibodies are reactive with various self-antigens, including DNA, renal tubular epithelium (RTE), and laminin-1. To elucidate the reactivity of autoantibodies with renal antigens in experimental lupus nephritis further, the reactivity of the autoantibodies was studied in more detail by generating hybridomas from GvHD spleen cells. Hybridomas were selected for reactivity with RTE and laminin-1 coated on nitrocellulose sheets. Four stable clones were obtained (GV1-GV4). Monoclonal antibody (mAb) GV1 showed no reactivity on kidney sections, while GV2 stained the brush border of proximal tubular epithelial cells. Both GV1 and GV2 reacted only with RTE in ELISA. GV3 showed a nuclear staining pattern, while GV4 stained matrix structures on F1 kidney sections. GV3 and GV4 both reacted with RTE, laminin-1, ssDNA, and dsDNA in ELISA. Growth of hybridomas in mice, but not passive transfer of the mAbs, led to glomerular Ig binding for mAbs GV3 and GV4 without development of proteinuria. Our results show that in addition to anti-nuclear autoantibodies cross-reactive with renal antigens, autoantibodies reactive with renal antigens and not with DNA are generated during chronic GvHD. Based on these results, combined with those of earlier experiments, we conclude that a combination of autoantibodies against multiple epitopes is necessary for the induction of glomerular damage in this model for lupus nephritis.

Progression of chronic renal disease in humans is associated with the deposition of basement membrane components and decorin in the interstitial extracellular matrix.

The degree of impairment of renal function in patients with chronic renal failure correlates closely with the extent of fibrosis in the tubulointerstitium, i.e. of interstitial extracellular matrix (ECM) accumulation. The composition of this pathological extracellular matrix and the relation between the ECM composition on the one hand and the severity of histological changes and renal function on the other has not been investigated. This prompted us to perform the present study. The severity of histological abnormalities and the composition of the interstitial ECM were assessed using a semiquantitative scoring technique in 57 biopsies from patients with kidney disease of diverse etiology and variable degrees of renal failure and were contrasted with the results of 9 control biopsies. Tissue sections were stained with an indirect immunoperoxidase technique using antibodies against collagen I, III, IV, V, VI, laminin, fibronectin, decorin and heparansulphate proteoglycan core protein (HSPG). Collagen IV, laminin and HSPG were virtually absent from the interstitium in controls. These components were more widely distributed in patients and the extent of their deposition correlated with the severity of interstitial histological abnormalities. In patients with severe interstitial damage the deposition became diffuse. Collagen type I and III were already diffusely distributed in the interstitium of controls and did not increase significantly as interstitial damage became more severe. However, the extent of collagen III staining in patients was significantly higher than in controls. Decorin staining showed a patchy distribution both in patients and controls. The overall distribution was significantly increased in patients. The extent of the deposition of both collagen V and VI was significantly increased in patients when compared with controls. Only the distribution of collagen V correlated with the severity of histological abnormalities. Our findings suggest that an increased interstitial deposition of extracellular matrix substances which are generally regarded as basement membrane components contributes more to the development of interstitial fibrosis and renal failure than the deposition of the fiber forming interstitial collagens type I and III, which are prominent in controls and in patients irrespective of the severity of histological abnormalities. Decorin staining was significantly enhanced in patients and was found to be the best predictor both of the severity of interstitial fibrosis and of renal failure. This could mean that decorin is important in human renal pathology. Collagen V and VI staining was significantly increased in patients when compared with controls. To our knowledge this is the first study in which this is demonstrated.

Optimal method for RNA extraction from mouse glomeruli.

Extraction of RNA has been described for rat and rabbit glomeruli but not for mouse glomeruli. Due to their small size, mouse glomeruli cannot be isolated by relatively simple sieving techniques. Based on recently reported methods for the isolation of mouse glomeruli, we developed an RNA isolation technique by performing comparative methodological studies. Two standard RNA extraction methods were compared. In addition in separate experiments the influence was studied of protease inhibitors and freezing and thawing of whole kidney prior to glomeruli isolation, on the yield and degradation of RNA. Therefore kidneys were perfused with 10 ml 0.01 M PBS containing 1.25% Fe3O4 through the aorta. Kidneys were decapsulated and passed through a 75-microns metal screen. After pelletting and washing, tubes were placed against a magnet and pelleted glomeruli were washed three times. In a second experiment protease inhibitors were added to the PBS. As a third method, kidneys were frozen before the isolation of glomeruli. From isolated glomeruli RNA was extracted using either caesium chloride or lithium chloride method. The yields of RNA (OD 260) were highest using the lithium chloride method. Hybridization of Northern blots of extracted RNA with cDNA probes showed the best results when RNA was extracted using the lithium chloride method, while the caesium chloride method led to considerable degradation of RNA. Freezing of kidney tissue prior to RNA extraction led to the virtual absence of any signal. We then applied this method successfully in an in-vivo model of experimental lupus nephritis. This is the first description of an optimal protocol for the extraction of RNA from mouse glomeruli.(ABSTRACT TRUNCATED AT 250 WORDS)

Gender-related influences on the development of chronic graft-versus-host disease-induced experimental lupus nephritis.

Autoimmune diseases are far more common in women than in men. In the incidence of systemic lupus erythematosus (SLE), the female-to-male ratio is as high as 10:1. This suggests that sex hormones may play a fundamental role in determining the susceptibility to these diseases. In order to investigate the sex-related differences in the inducibility of chronic graft-versus-host disease-related experimental lupus nephritis, lymphocytes from female DBA/2 donor mice were administered to either male or female (C57BL10 x DBA/2)F1 recipients. An additional group of male recipients received lymphocytes from male DBA/2 donors. After four cell transfers, female recipients developed a significantly higher albuminuria than both male groups. Serum concentrations of autoantibodies against glomerular basement membrane (GBM), collagen IV, and laminin were significantly higher in females 2-4 weeks after induction. Levels of circulating autoantibodies against renal tubular epithelial antigens (RTE) and nuclear antigens were not different between the sexes. In transfer studies, the necessity of the presence of anti-GBM and anti-RTE autoantibodies for the development of glomerulonephritis was confirmed. These findings indicate that: (i) in this model of lupus nephritis, susceptibility to glomerulonephritis is strongly influenced by sex-related genes; and (ii) among the variety of autoantibodies occurring in this model of SLE, both anti-GBM and anti-RTE autoantibodies play a key role in the pathogenesis of glomerulonephritis.

A molecular biologic study of extracellular matrix components during the development of glomerulosclerosis in murine chronic graft-versus-host disease.

BACKGROUND: We studied the development of glomerulosclerosis in murine chronic graft-versus-host disease, a model for human systemic lupus erythematosus. EXPERIMENTAL DESIGN: The disease was induced in (C57BL10 x DBA/2)F1 hybrids by injection of DBA/2 lymphocytes leading to deposition of auto-antibodies in the glomeruli, and a lupus type of nephritis morphologically. We have determined the levels of mRNA coding for laminin (B1 and B2), a 67 kilodalton laminin binding protein, and types I and IV collagen, in control and graft-versus host disease mice at various times after disease induction. RESULTS: Laminin and collagen mRNAs were increased in whole kidneys 4 weeks after induction of the disease. At week 10, all animals displayed dramatic stimulation of alpha 1(I), alpha 1(IV), laminin B1, and B2 mRNAs. The 67 kilodalton laminin binding protein mRNA was also doubled from week 4 to 16. In isolated glomeruli, the mRNA level coding for laminin B2 was already significantly increased from week 8. This enhancement of laminin synthesis corresponds to the mesangial expansion and to the development of laminin-containing spike formations of the glomerular basement membrane at week 8. CONCLUSIONS: The expansion of the mesangial matrix in murine chronic graft-versus-host disease is caused at least in part, by an increased production of extracellular matrix components by glomerular cells. These results demonstrate that the increase of specific extracellular matrix components mRNAs precedes light microscopic changes. Quantitative evaluation of the mRNA levels coding for extracellular matrix proteins may reveal a useful method for the early detection of the development of glomerular sclerosis at the stage preceding the onset of anatomo-clinical changes.

A histologic study of the extracellular matrix during the development of glomerulosclerosis in murine chronic graft-versus-host disease.

The development of glomerulosclerosis was studied in murine chronic graft-versus-host disease (GvHD), which is a model for human systemic lupus erythematosus. The authors investigated the distribution patterns of six components of the extracellular matrix (ECM), i.e., laminin, fibronectin, collagen types I, III, IV, and VI during the course of the disease. All of these ECM components except collagen type I were found in the glomeruli of normal mice, where all of them were intrinsic constituents of the mesangium. Laminin, fibronectin, and collagen type IV were also found in the glomerular capillary walls. Starting 6 weeks after the induction of GvHD and continuing at week 8, the onset of an expansion of the mesangial matrix was observed. At the same time, the amounts of laminin, fibronectin, and collagen types IV and VI increased. Ten weeks after the onset of the disease, glomerulosclerosis developed. Traces of the interstitial collagen type I were found in sclerotic glomeruli. The levels of four ECM components, i.e., collagens III, IV, VI, and laminin were markedly decreased in the sclerotic glomeruli as compared with week 8. In contrast, the amount of fibronectin in the sclerotic glomeruli increased dramatically. Immunoelectron microscopic examination showed fibronectin in the sclerotic lesions, in contrast to laminin, collagen type I, and collagen type IV. It is concluded that the sclerotic lesions in murine chronic GvHD contain fibronectin. The small amounts of the ECM components laminin, as well as collagens III, IV, and VI in the sclerotic glomeruli in GvHD, might represent remnants of mesangial material and collapsed capillary walls. These components are probably replaced by increased production and/or accumulation of collagen type I and fibronectin.