Synovial fluid proteins are required for the induction of interleukin-1ß production by monosodium urate crystals.

OBJECTIVES: Monosodium urate (MSU) crystal deposition in gouty joints promotes the release of inflammatory mediators, in particular interleukin (IL)-1ß. The induction of IL-1ß production by MSU crystals requires a co-stimulus. The objective of this study was to determine which part of the synovial fluid (SF) provides co-stimulation to MSU crystals to induce IL-1ß in macrophages. METHOD: The lipidic fraction (LF) and the protein fraction (PF) were isolated from the SF of patients with arthropathies. The PF was subfractionated according to different molecular weight (MW) ranges. THP-1 cells or human primary monocytes were stimulated with MSU crystals in the presence or absence of SF or SF fractions. IL-1ß and IL-8 production and IL-1ß mRNA expression were assessed by an enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qPCR). RESULTS: Exposure of monocytes/macrophages to MSU crystals alone induced the moderate release of IL-8 but not of IL-1ß. The production of IL-1ß required the presence of both SF from patients with inflammatory arthritis (SFi) and MSU crystals. SF from patients with non-inflammatory arthritis, that is patients with osteoarthritis (OA), did not affect the IL-1ß production but slightly enhanced the secretion of IL-8. Both MSU crystals and SFi were required for the induction of the IL-1ß transcript, which was not expressed in the presence of either stimulus alone. SFi fractionation demonstrated that the MSU crystal co-stimulus was contained in the PF of SFi with MW > 50 kDa but not in the LF. CONCLUSIONS: This study shows that the SF of inflammatory arthritis patients, including gout patients, contains proteins required for the induction of IL-1ß by MSU crystals in macrophages whereas lipids are not involved.

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.

Cytoarchitectureal changes in hippocampal subregions of the NZB/W F1 mouse model of lupus.

Over 50% of clinical patients affected by the systemic lupus erythematosus disease display impaired neurological cognitive functions and psychiatric disorders, a form called neuropsychiatric systemic lupus erythematosus. Hippocampus is one of the brain structures most sensitive to the cognitive deficits and psychiatric disorders related to neuropsychiatric lupus. The purpose of this study was to compare, layer by layer, neuron morphology in lupus mice model NZB/W F1 versus Wild Type mice. By a morphometric of cells identified on Nissl-stained sections, we evaluated structural alterations between NZB/W F1 and Wild Type mice in seven hippocampal subregions: Molecular dentate gyrus, Granular dentate gyrus, Polymorph dentate gyrus, Oriens layer, Pyramidal layer, Radiatum layer and Lacunosum molecular layer. By principal component analysis we distinguished healthy Wild Type from NZB/W F1 mice. In NZB/W F1 mice hippocampal cytoarchitecture, the neuronal cells resulted larger in size and more regular than those of Wild Type. In NZB/W F1, neurons were usually denser than in WT. The Pyramidal layer neurons were much denser in Wild Type than in NZB/W F1. Application of principal component analysis, allowed to distinguish NZB/W F1 lupus mice from healthy, showing as NZBW subjects presented a scattered distribution and intrasubject variability. Our results show a hypertrophy of the NZB/W F1 hippocampal neurons associated with an increase in perikaryal size within the CA1, CA2, CA3 region and the DG. These results help advance our understanding on hippocampal organization and structure in the NZB/W F1 lupus model, suggesting the hypothesis that the different subregions could be differentially affected in neuropsychiatric systemic lupus erythematosus disease. Leveraging an in-depth analysis of the morphology of neural cells in the hippocampal subregions and applying dimensionality reduction using PCA, we propose an efficient methodology to distinguish pathological NZBW mice from WT mice."

Induction of the 'ASIA' syndrome in NZB/NZWF1 mice after injection of complete Freund's adjuvant (CFA).

Adjuvants, commonly used in vaccines, may be responsible for inducing autoimmunity and autoimmune diseases, both in humans and mice. The so-called 'ASIA' (Autoimmune/inflammatory Syndrome Induced by Adjuvants) syndrome has been recently described, which is caused by the exposure to a component reproducing the effect of adjuvants. The aim of our study was to evaluate the effect of injection of complete Freund's adjuvant (CFA) in NZB/NZWF1 mice, a lupus-prone murine model. We injected 10 NZB/NZWF1 mice with CFA/PBS and 10 with PBS, three times, 3 weeks apart, and followed-up until natural death. CFA-injected mice developed both anti-double-stranded DNA and proteinuria earlier and at higher levels than the control group. Proteinuria-free survival rate and survival rate were significantly lower in CFA-treated mice than in the control mice (p = 0.002 and p = 0.001, respectively). Histological analyses showed a more severe glomerulonephritis in CFA-injected mice compared with the control mice. In addition, lymphoid hyperplasia in spleen and lungs, myocarditis, and vasculitis were observed in the former, but not in the latter group. In conclusion, the injection of CFA in NZB/NZWF1 mice accelerated autoimmune manifestations resembling 'ASIA' syndrome in humans.

Vaccination of mice for research purpose: alum is as effective as and safer than complete Freund adjuvant.

Systemic lupus erythematosus (SLE) is an autoimmune disease involving many organ systems. Glomerulonephritis (GLN) is one of the major causes of morbidity and mortality in SLE. It has recently been demonstrated that adjuvants of vaccines could cause the so called ASIA syndrome. The study aimed to assess the effects of Complete Freund's Adjuvant (CFA) vs alum injections in NZB/NZWF1 mice. Mice (n=10 each group) were injected with a total volume of 200 µL of: CFA in PBS (group 1), alum in PBS (group 2), PBS (group 3) as controls, PTX3/CFA (group 4), PTX3/alum (group 5), 3 times, 3 weeks apart /given in each injection, three weeks apart from ten weeks of age. Urine samples were collected weekly to evaluate proteinuria. Blood samples were collected before every injection, at 21 weeks of age, and at death to evaluate levels of anti-PTX3 and anti-dsDNA. Proteinuria free survival and survival rates were analyzed by the Kaplan-Meier method using Mantel-Cox's test for comparisons. CFA-treated mice developed both anti-dsDNA antibodies and proteinuria earlier and at higher levels than alumtreated and PBS-injected mice, starting from 13 weeks of age. Proteinuria free survival rates (proteinuria ≥ 300 mg/dL) and survival rates were lower in CFA-treated mice than those treated with alum or injected with PBS (P<0.001 for all). No difference was observed between the alum-treated group and PBS-injected mice. Notably, groups 4 and 5, immunized with PTX3, developed anti-PTX3 antibodies and no significant difference was observed. Alum seems to be as effective as and safer than CFA as adjuvant, since it did not affect disease progression in immunized NZB/NZWF1 mice.

[HDL inhibit cytokine production in a mouse model of urate crystal-induced inflammation]. / Le HDL inibiscono la produzione di citochine nel modello murino di infiammazione da microcristalli di urato monosodico.

OBJECTIVES: To evaluate whether high density lipoproteins (HDL) affect monosodium urate (MSU) crystal-induced inflammation in the murine air pouch model. METHODS: MSU crystals were prepared by Denko's method and sterilized by heating at 180°C for 2 h before each experiment. Human HDL were isolated from peripheral blood of healthy volunteers. MSU crystals (2 mg in 1 ml of PBS) were injected into subcutaneous air pouches in mice in the presence or absence of HDL (0.1 mg). Negative control pouches received 1 ml of PBS. To recover pouch fluid, the pouches were washed with 2 ml of PBS after the animals were sacrificed. The leukocyte count in the lavage fluids was obtained using a hemocytometer and differential leukocyte count was determined by May-Grunwald-Giemsa staining. IL-6, KC, CCL2 and TNF-α levels were measured in exudates by ELISA. RESULTS: MSU crystals increased the number of leukocytes and the neutrophil migration, as well as the concentrations of IL-6, KC and CCL2 in pouch fluids, while the TNF-α levels were not detectable. The treatment with HDL led to a reduction in all inflammatory parameters: the leukocyte count decreased by 73%; the neutrophil density decreased by 35%; the IL-6, KC and CCL2 concentration decreased by 4-, 6- and 5-fold respectively. CONCLUSIONS: This study shows that HDL may limit the inflammatory process by inhibiting leukocyte recruitment and cytokine release. HDL are likely to represent a mechanism of control of crystal-induced inflammation.

Galectin-3 orchestrates the histology of mesentery and protects liver during lupus-like syndrome induced by pristane.

Galectin-3 (Gal-3) controls intercellular and cell-extracellular matrix interactions during immunological responses. In chronic inflammation, Gal-3 is associated with fibrotic events, regulates B cell differentiation and delays lupus progression. Gal-3 deficient mice (Lgals3-/-) have intense germinal center formation and atypical plasma cell generation correlated to high levels IgG, IgE, and IgA. Here, we used pristane (2,6,10,14-tetramethylpentadecane) to induce lupus-like syndrome in Lgals3-/- and Lgals3+/+ BALB/c mice. Mesentery and peritoneal cells were monitored because promptly react to pristane injected in the peritoneal cavity. For the first time, mesenteric tissues have been associated to the pathogenesis of experimental lupus-like syndrome. In Lgals3+/+ pristane-induced mice, mesentery was hallmarked by intense fibrogranulomatous reaction restricted to submesothelial regions and organized niches containing macrophages and B lymphocytes and plasma cells. In contrast, Lgals3-/- pristane-treated mice had diffuse mesenteric fibrosis affecting submesothelium and peripheral tissues, atypical M1/M2 macrophage polarization and significant DLL1+ cells expansion, suggesting possible involvement of Notch/Delta pathways in the disease. Early inflammatory reaction to pristane was characterized by significant disturbances on monocyte recruitment, macrophage differentiation and dendritic cell (DC) responses in the peritoneal cavity of pristane-induced Lgals3-/- mice. A correlative analysis showed that mesenteric damages in the absence of Gal-3 were directly associated with severe portal inflammation and hepatitis. In conclusion, it has suggested that Gal-3 orchestrates histological organization in the mesentery and prevents lupoid hepatitis in experimental lupus-like syndrome by controlling macrophage polarization, Notch signaling pathways and DC differentiation in mesenteric structures.

[Synoviocyte cultures from synovial fluid]. / Realizzazione di colture di sinoviociti da liquido sinoviale.

UNLABELLED: The study of the pathogenetic mechanisms of rheumatic diseases is in general carried out through "in vitro" systems based on cellular cultures models. The difficulties to achieve fresh human tissue prompted us to develop a simpler method to obtain fibroblast-like synovial cells from synovial fluid (SF). METHODS: SF was collected from the knees of 5 patients with rheumatoid arthritis (RA), 4 with osteoarthritis (OA) and 5 with psoriatic arthritis (PsA). The pellet obtained after centrifugation was resuspended in DMEM/HamF12 containing 10% foetal calf serum, 1% peni-streptomycin, 4 ng/ml of fibroblast grow factor and incubated at 37 degrees C in T25 culture flasks. Synoviocytes were also obtained from fresh synovial membranes (SM) by explants technique. Both types of cells were characterized by immunocytochemistry and their inflammatory response to synthetic monosodium urate crystals was studied through the measurement of nitric oxide (NO). RESULTS: Adherent synoviocytes were obtained from the culture of 2/5 SF from RA, 4/4 SF from OA and 5/5 SF from PsA. Synoviocytes isolated from both SF and SM expressed surface antigens CD90, CD55, and the intracellular prolyl-4-hydroxylase. Morphologically, the cells showed the typical spindle-shape fibroblast-like appearance. NO levels induced by UMS crystals in SF synoviocytes were similar to those obtained in SM synoviocytes. CONCLUSION: Adherent cells obtained from SF showed the phenotype and the reactivity of tissue synoviocytes. Due to the easy accessibility of SF, this method may represents an useful alternative when synovial tissues is not promptly available.

Subnormothermic machine perfusion for non-heart-beating donor liver grafts preservation in a Swine model: a new strategy to increase the donor pool?

We previously reported that subnormothermic machine perfusion (sMP; 20°C) is able to improve the preservation of livers obtained from non-heart-beating donors (NHBDs) in rats. We have compared sMP and standard cold storage (CS) to preserve pig livers after 60 minutes of cardiac arrest. In the sMP group livers were perfused for 6 hours with Celsior at 20°C. In the CS group they were stored in Celsior at 4°C for 6 hours as usual. To simulate liver transplantation, both sMP- and CS-preserved livers were reperfused using a mechanical continuous perfusion system with autologus blood for 2 hours at 37°C. At 120 min after reperfusion aspartate aminotransferase levels in sMP versus CS were 499 ± 198 versus 7648 ± 2806 U/L (P < .01); lactate dehydrogenase 1685 ± 418 versus 12998 ± 3039 U/L (P < .01); and lactic acid 4.78 ± 3.02 versus 10.46 ± 1.79 mmol/L (P < .01) respectively. The sMP group showed better histopathologic results with significantly less hepatic damage. This study confirmed that sMP was able to resuscitate liver grafts from large NHBD animals.

A new liver autotransplantation technique using subnormothermic machine perfusion for organ preservation in a porcine model.

BACKGROUND: Hepatic resection is the gold standard of therapy for primary and secondary liver tumors, but few patients are eligible for this procedure because of the extent of their neoplasms. Improvements in surgical experience of liver transplantation (OLT), hepatic resection and preservation with sub-normothermic machine perfusion (MP) have prompted the development of a new model of large animal autotransplantation. METHODS: Landrace pigs were used in this experiment. After intubation, hepatectomy was performed according to the classic technique. The intrahepatic caval vein was replaced with a homologous tract of porcine thoracic aorta. The liver was perfused with hypothermic Celsior solution followed by MP at 20 °C with oxygenated Krebs solution. An hepatectomy was performed during the period of preservation, which lasted 120 minutes, then the liver was reimplanted into the same animal in a 90° counterclockwise rotated position. The anastomoses were performed in the classic sequence. Samples of intravascular fluid, blood and liver biopsies were obtained at the end of the period of preservation in MP and again at 1 and 3 hours after liver reperfusion to evaluate graft function and microscopic damage. RESULTS: All animals survived the procedure. The peak of aspartate aminotransferase was recorded 60 minutes after reperfusion and the peak of alanine aminotransferase and lactate dehydrogenase after 180 minutes. Histopathologic examination under the light microscope identified no necrosis or congestion. Intraoperative echo-color Doppler documented good patency of the anastomosis and normal venous drainage. CONCLUSION: This system made it possible to perform hepatic resections and vascular reconstructions ex situ while preserving the organ with mechanical perfusion (ex vivo, ex situ surgery). Improving surgical techniques regarding autotransplantation and our understanding of ischemia-reperfusion damage may enable the development of interesting scenarios for aggressive surgical treatment or radiochemotherapy options to treat primary and secondary liver tumors unsuitable for conventional in situ surgery.