Anti-proteinase 3 antibodies in diffuse systemic sclerosis (SSc) with normotensive renal impairment: is it suggestive for an overlapping between SSc and idiopathic vasculitis?

OBJECTIVE: To test the prevalence of anti-neutrophil cytoplasmic antibodies (ANCA) in systemic sclerosis (SSc) and to verify a possible association of ANCA with normotensive renal involvement in SSc. PATIENTS AND METHODS: 51 patients affected by SSc, 35 with diffuse scleroderma (dSSc) and 16 with limited scleroderma (lSSc), were tested for ANCA by indirect immunofluorescence (IIF) on human ethanol and formalin-acetone-fixed granulocytes (before and after DNase treatment), by conventional enzyme linked immuno-sorbent assay (ELISA) and by capture-ELISA. RESULTS: Six out of 51 selected SSc patients had ANCA by IIF (11.7%) and five presented a perinuclear/nuclear atypical ANCA pattern. In all cases we only found anti-proteinase3 (aPR3) antibodies. All ANCA positive patients had diffuse form of SSc (17.1%), all were anti-Scl70 positive (aScl70), five patients had proteinuria, three had microscopic haematuria. All ANCA positive patients were normotensive with normal renin plasma levels, the mean erythrocyte sedimentation rate (ESR) was higher in this group compared to the other SSc patients. CONCLUSIONS: Our study shows that aPR3 is not rare in dSSc. According to the clinical and serological findings and to the recent literature, we can hypothesise that when ANCA are found in SSc, an overlapping of scleroderma with systemic necrotizing vasculitis should be suspected.     

Glomerular Autoimmune Multicomponents of Human Lupus Nephritis In Vivo (2): Planted Antigens

Glomerular planted antigens (histones, DNA, and C1q) are potential targets of autoimmunity in lupus nephritis (LN). However, the characterization of these antigens in human glomeruli in vivo remains inconsistent. We eluted glomerular autoantibodies recognizing planted antigens from laser-microdissected renal biopsy samples of 20 patients with LN. Prevalent antibody isotypes were defined, levels were determined, and glomerular colocalization was investigated. Renal and circulating antibodies were matched, and serum levels were compared in 104 patients with LN, 84 patients with SLE without LN, and 50 patients with rheumatoid arthritis (RA). Autoantibodies against podocyte antigens (anti–α-enolase/antiannexin AI) were also investigated. IgG2 autoantibodies against DNA, histones (H2A, H3, and H4), and C1q were detected in 50%, 55%, and 70% of biopsy samples, respectively. Anti-DNA IgG3 was the unique non-IgG2 anti-DNA deposit, and anti-C1q IgG4 was mainly detected in subepithelial membranous deposits. Anti-H3, anti-DNA, and anti-C1q IgG2 autoantibodies were also prevalent in LN serum, which also contained IgG3 against the antigen panel and anti-C1q IgG4. Serum and glomerular levels of autoantibodies were not strictly associated. High serum levels of all autoantibodies detected, including anti–α-enolase and antiannexin AI, identified LN versus SLE and RA. Anti-H3 and anti–α-enolase IgG2 levels had the most remarkable increase in LN serum and represented a discriminating feature of LN in principal component analysis. The highest levels of these two autoantibodies were also associated with proteinuria>3.5 g/24 hours and creatinine>1.2 mg/dl. Our findings suggest that timely autoantibody characterization might allow outcome prediction and targeted therapies for patients with nephritis.     

Identification of glomerular immune deposits in cryoglobulinemia glomerulonephritis

To provide further evidence of the nature of intraglomerular immune deposits in essential mixed cryoglobulinemia (EMC), we used two mouse monoclonal antibodies against cross-reactive idiotypes present on monoclonal rheumatoid factors (MoRFs) from patients with type II-EMC. MoAb Cc1 reacted with 9 of 16 circulating IgMk MoRFs tested, and MOAb Lc1 with four of the remaining. Using indirect immunofluorescence and immunoperoxidase techniques, we could identify the same cross-reactive idiotype of the serum MoRF in the renal biopsy specimens from 11 of 13 patients with EMC glomerulonephritis. Kidney specimens from the three patients, whose MoRF was not recognized by MoAbs Cc1 and Lc1, were negative. Two out of 30 control renal biopsies from patients with other forms of glomerulonephritis were shown to contain idiotype (Cc1 and Lc1) positive material. Both patients had serum polyclonal RF which could account for this finding. In conclusion, our results provide direct evidence that serum cryo-MoRF participate in the formation of glomerular immune deposits and, presumably, in the pathogenesis of renal damage in EMC glomerulonephritis.     

Immunoglobulin heavy chain switch region gene polymorphisms in glomerulonephritis

Much evidence suggests that primary IgA nephropathy (IgAN) and idiopathic membranous nephropathy (MN) are immune complex mediated diseases. Moreover, genetic factors may play an important role in their pathogenesis. Recently, restriction fragment length polymorphisms (RFLPs) of the immunoglobulin heavy chain genes have been described which appear to associate with glomerulonephritis. We have studied RFLPs of the switch region of the IgM (S mu) and IgA1 (S alpha 1) heavy chain in MN and IgAN. DNA obtained from British Caucasoids with IgAN (N = 75), MN (N = 43), and normal controls (N = 73), was digested with the restriction enzyme Sac1, and studied using Southern blot techniques and hybridization with a 32P labelled DNA probe homologous to S mu. This probe detects RFLPs at the S mu and S alpha 1 loci. The genotypic and allelic frequencies of the S mu and S alpha 1 alleles in IgAN and MN was similar to normal controls. Caucasoid subjects with IgAN from Northern and Southern Europe (Finland and Italy, respectively) were also studied to determine whether an ethnic variation in genetic susceptibility to IgAN exists. The frequency of the S mu and S alpha 1 alleles was similar between the patient groups and their respective local healthy controls. These results do not support the recent findings of an association with RFLPs of the S mu and S alpha 1 loci in IgAN and MN, and suggest that the immunoglobulin heavy chain switch region genes are not important in conferring disease susceptibility to IgAN or MN.     

Fenofibrate-induced acute hepatitis with pseudo-cholangitis]

We report the case of acute hepatitis probably due to fenofibrate. Clinical features included abdominal pain and fever. Serum aminotransferases and alkaline phosphatase activities were moderately increased. Rechallenge induced a relapse of the symptoms and liver test abnormalities. High levels of eosinophils were present in the blood and in the liver suggesting an immunoallergic mechanism. Fenofibrate withdrawal was rapidly followed by favorable outcome.     

Anti-glomerular basement membrane antibodies in the diagnosis of Goodpasture syndrome: a comparison of different assays.

BACKGROUND: The role of anti-glomerular basement membrane (GBM) antibodies in the pathogenesis of Goodpasture syndrome (GPS) is firmly established. Untreated, the disease may follow a fulminating course. Early identification of patients has important implications in terms of management and prognosis. Therefore, a diagnostic test for the determination of circulating anti-GBM antibodies, of very high sensitivity and specificity, is necessary. A number of assays, using different antigenic substrates, are available, but studies comparing the 'performances' of the different tests are scarce. METHODS: The aim of our work was to evaluate the sensitivity and specificity of four immunoassay-based anti-GBM antibodies kits. Thirty-four serum samples from 19 GPS patients, 41 pathological and 28 normal controls were studied retrospectively (the follow-up samples were not included in the analysis of performance data). Cut-off limits were derived from receiver operating characteristics curve analysis. RESULTS: All the assays showed a comparable good sensitivity (between 94.7 and 100.0%), whereas specificity varied considerably (from 90.9 to 100.0%). The better performance in terms of sensitivity/specificity was achieved by a fluorescence immunoassay which utilizes a recombinant antigen. CONCLUSION: All the assays have a good performance, with high sensitivity; however, the specificity may vary considerably.     

Phospholipid vesicles as carriers in aquaculture: preparation and stability study of thiamine hydrochloride-loaded liposomes

The aim of this work is to study liposomes as carriers of nutrients and therapeutic agents in aquaculture with Venerupis decussatus and Venerupis pullastra larvae. Multilamellar (MLVs) and large unilamellar (LUVs) vesicles were prepared from a commercial mixture of soy phosphatidylcholine, rich in unsaturated and polyunsaturated fatty acids, cholesterol, and hydrated with a solution of vitamin B1 both in distilled and sea water. Carboxyfluorescein-loaded liposomes were also prepared in order to test the uptake of vesicles by larvae. The stability of formulations was checked by monitoring the size of vesicles and their drug leakage. In order to limit the vitamin loss, liposome freeze-drying was studied. Dried formulations were also prepared by using different amounts of trehalose as cryoprotectant. We found that freeze-dried vesicles, rehydrated after two weeks, had a vitamin retention (R%) equal to 95%, while their diameter significantly increased. By contrast, liposomes freeze-dried in the presence of trehalose displayed a lower R%, but higher bilayer stability. Finally, when CF-loaded vesicles were added to Venerupis decussatus and Venerupis pullastra larvae incubated in filtered sea water, a bright and diffused fluorescence was present in most of the larvae, a fact which can be regarded as evidence of liposome uptake by Venerupis larvae.     

Mosaic trisomy 9 and lobar holoprosencephaly

The main features of trisomy 9 syndrome in mosaic and non-mosaic forms have been thoroughly described. Characteristic traits are low-set malformed ears, micrognathia, broad nose with bulbous tip, abnormal brain, congenital heart defects, abnormal hands and feet, genital abnormalities, and early death. We report a case of mosaic trisomy 9 with holoprosencephaly (HPE). The propositi was born at 37 weeks, with intra-uterine growth retardation, hypotelorism and single nostril, ventricular septal defect, anterior placement of anus, clenched hands with thumb adduction and ulnar deviation. Facial anomalies characteristic of trisomy 9 included deeply set eyes and short palpebral fissures, flat face with maxillary hypoplasia, small mouth, and low-set posteriorly angulated ears. Cytogenetic analysis showed mosaic trisomy 9 with 17% trisomic cells. Pathology confirmed lobar HPE. In literature, isolated arrhinia, related to the HPE spectrum, was reported in one case of mosaic trisomy 9. Our case raises the question of the causative role of trisomy 9 in full blown HPE.     

Niosomes as carriers for tretinoin. I. Preparation and properties

Tretinoin-loaded niosomes were prepared from polyoxyethylene (4) lauryl ether, sorbitan esters and a commercial mixture of octyl/decyl polyglucosides, in the presence of cholesterol and dicetyl phosphate. Liposomes made of hydrogenated and non-hydrogenated phosphatidylcholine were also prepared as a comparison reference. A study was made of the influence of vesicle composition and preparation method on the vesicle structure (MLV, LUV, SUV), size distribution, entrapment efficiency and in vitro release of incorporated tretinoin. Results showed that in the presence of cholesterol all the amphiphiles used were able to form stable vesicle dispersions with or without tretinoin. Vesicle sizes were dependent on the preparation method, bilayer composition and drug load. Multilamellar (MLV) vesicles were larger than extruded (LUV) and sonicated (SUV) vesicles while drug-loaded vesicles were generally smaller than empty ones. Entrapment efficiencies of tretinoin were always very high especially for multilamellar (91-99%) and extruded (88-98%) vesicles. The in vitro release of tretinoin from the prepared vesicular formulations was studied using the vertical Franz diffusion cells. The rate of drug release through a Silastic membrane from a liposomal and niosomal tretinoin dispersion was generally faster than from a tretinoin solution. Release data showed that tretinoin delivery is mainly affected by the vesicular structure and that tretinoin delivery increased from MLVs to LUVs to SUVs.     

Anti-DNA antibodies: a diagnostic and prognostic tool for systemic lupus erythematosus?

The clinical impact of anti-DNA antibodies lies on their diagnostic power for systemic lupus erythematosus (SLE), being a formal classification criterion. In spite of such a disease association, low-avidity anti-DNA antibodies might also be part of the natural autoantibody repertoire. Their switch to pathogenic high-avidity autoantibodies is the result of the autoimmune process leading to SLE.Anti-DNA antibodies were shown to play a role in SLE pathogenesis and particularly in kidney damage. Accordingly, antibody titres might fluctuate in relation to disease activity, but their prognostic value for flares is still debated.Several methods for anti-DNA detection were described and there is evidence that the assays identify different antibodies with different prognostic value. The results of a multicenter study on four different routine tests for anti-dsDNA antibody detection showed that: (i) Farr assay displays the best diagnostic specificity/sensitivity for SLE, followed by Crithidia luciliae method (CLIFT), (ii) the new generation of solid phase assay (EliA) shows an increased sensibility versus the classical enzyme linked immune assay (ELISA) but a decreased specificity. Antibody titre detected by EliA and Farr assay correlated with disease activity. These findings would suggest that more than one assay should be useful for SLE diagnosis and monitoring.