17-ß-estradiol affects BLyS serum levels and the nephritogenic autoantibody network accelerating glomerulonephritis in NZB/WF1 mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease that predominantly affects fertile women, suggesting sex hormones are involved in disease pathogenesis. B lymphocyte stimulator (BLyS) has been found to be elevated in SLE patients and to drive a lupus-like syndrome in transgenic mice. Our aim was to evaluate the effects of estrogen administration on BLyS and nephritogenic anti-C1q and anti-dsDNA antibodies in lupus-prone NZB/WF1 mice. We implanted pellets releasing 17-ß-estradiol (18.8 µg/day) on the back side the ear of 10 NZB/WF1 mice (group 1), and compared them with 10 mice intraperitoneally injected with PBS 200 µl twice a week (group 2), as controls. We evaluated BLyS, anti-dsDNA and anti-C1q serum levels starting one week after pellet implantation. We also analyzed time to proteinuria onset, proteinuria-free survival and overall survival. Kidneys, spleen, liver and lungs were harvested for histological analysis. Mice were bred until natural death. BLyS serum levels were higher in group 1 than in group 2 mice at each evaluation. Group 1 mice developed nephritogenic antibodies and proteinuria significantly earlier and at higher levels than controls. Direct correlation between BLyS and anti-C1q (R (2 )= 0.6962, p < 0.0001) or anti-dsDNA (R (2 )= 0.5953, p < 0.0001), and between anti-C1q and anti-dsDNA autoantibodies (R (2 )= 0.5615, p < 0.0001) were found. Proteinuria-free and global survival rates were significantly lower in group 1 than in controls. Histological analyses showed more severe abnormalities in group 1 mice. Estrogen administration is associated with increased levels of BLyS as well as of anti-C1q and anti-dsDNA antibodies, leading to accelerated glomerulonephritis and disease progression in NZB/WF1 mice.

Glomerular c4d immunoreactivity in acute rejection biopsies of renal transplant patients.

In renal transplant patients, glomerulitis may be present in all types of acute rejection, often accompanied by diffuse C4d staining of peritubular capillaries: C4d3 positivity in more than 50% of peritubular capillaries. It may progress to chronic transplant glomerulopathy, characterized by capillary basement membrane multilayering, proteinuria, and progressive loss of renal function. While C4d3 is a recognized marker of an antibody-mediated reaction, the significance of glomerular C4d (GlC4d) staining is unknown. The aim of this study was to evaluate GlC4d immunoreactivity and its correlation with C4d3 in acute rejection biopsies. Paraffin-embedded acute rejection biopsies from 90 renal transplant patients were evaluated according to the Banff classification. Biopsies showing C4d-positive endothelial cells in more than 50% of glomeruli were considered GlC4d-positive. C4d3-positive staining prevalence was 23%. GlC4d-positive staining showed an 89% concordance rate (r = 0.81, P < .0001; Cohen's k = 0.80, P < .0001). GlC4d detection sensitivity was 0.80 and specificity 0.97. C4d3 and GlC4d immunoreactivity was significantly associated with glomerulitis (P < .006 and P < .03, respectively) and with proteinuria at the time of biopsy (P < .03 and P < .01, respectively). Interestingly, GlC4d positivity correlated better than C4d3 positivity with the presence of posttransplant circulating anti-human leukocyte antigen alloantibodies (P < .04 and P = .7, respectively). Patients with C4d3- or GlC4d-positive acute rejections underwent graft loss due to interstitial fibrosis and tubular atrophy more frequently than those with C4d0- or GlC4d-negative rejections (P < .0001 and P < .005, respectively), whereas no differences were observed in graft loss due to death. In conclusion, C4d3 and GlC4d stains showed a high correlation rate. Compared with C4d3, GlC4d staining demonstrated good sensitivity and excellent specificity. Our results suggested that GlC4d staining may indicate glomerular endothelial damage and be of prognostic value.

Histomorphometric evaluation of bone grafts harvested by different methods.

AIM: Many studies proposing the use of autologous bone to correct bony defects of the oral district have been published, and numerous protocols have been proposed to simplify the harvesting of particulate bone. However, no qualitative evaluation of the harvested bone has been reported. The study provides a qualitative evaluation of autologous bone harvested by 9 methods: the harvested bone was analysed through microphotography and histomorphometric analysis, measuring the surface area of bone fragments and the percentages of vital and necrotic bone. METHODS: Nine harvesting methods were employed: round bur on low-speed hand-piece (40000 rpm), bur on high-speed hand-piece, spiral implant bur on low-speed hand-piece (1000 rpm), safe scraper, Rhodes' back-action chisel, rongeur pliers, gouge shaped bone chisel, mectron piezosurgery. Ten bone harvests were taken from the retromolar bone using the 9 methods, during extraction of embedded wisdom teeth (indication to extraction was for orthodontic reasons). The histocytological preparations were analysed with microphotography and histomorphometric analysis, evaluating particle size, percentage of vital bone and number of osteocytes per unit of surface area. RESULTS: Histomorphometric analysis showed that non-vital bone accounted for 100% of harvested bone, with a complete absence of osteocytes, in specimens harvested with burs and safe scraper. Percentage of non-vital bone was intermediate, with a low number of cells, in specimens harvested with back-action, gouge shaped bone chisel, spiral bur and piezosurgery. The best results were achieved with rongeur pliers and by en bloc harvesting. CONCLUSIONS: The results show that the best methods to harvest vital bone are: gouge shaped bone chisel, back-action, en bloc harvesting, rongeur pliers and piezosurgery, although the latter method leaves some empty gaps in the tissue. The bone harvested with round bur on low-speed hand-piece, bur on high-speed hand-piece, spiral implant bur, or safe scraper are not suitable for grafting as indicated by the absence of osteocytes and the predominance of non-vital bone.

Immunolocalisation of dystrophin in the immature human neurons and muscles.

Dystrophin, the product of the Duchenne muscular dystrophy gene, has been shown to be developmentally regulated in both human muscle and brain tissues. We consequently performed an immunocytochemical study using electron microscopy to localise the protein in the immature human fetal muscle and neurons. Results demonstrated that, even if dystrophin was partially associated to the plasma membrane in both tissues, some product was also linked to the neurofilaments network in neurons and to microfilaments in muscle. An intense staining was also found in satellite cells.