Luigi M. Sena

The Fate of Aggregated Immunoglobulin A Injected in IgA Nephropathy Patients and Healthy Controls

Organ uptake of IgA-containing immunologically active material was studied in humans by intravenous (IV) injection of 131I-labeled heat-aggregated human secretory IgA (HAS-IgA) in nine patients affected by primary IgA nephropathy and 10 normal volunteers. Aggregated secretory IgA was found to be removed almost exclusively by the liver. The peak activity in liver was reached at 21.1 minutes (range, 18 to 26 minutes) in patients and 19 minutes (range, 14 to 22 minutes) in controls. The rate of increase of liver radioactivity was found to be significantly slower in patients (with a mean slope of 5.0; range, 3.4 to 7.1 v 7.6, 5.6 to 11.4; P less than 0.02). The mean liver to precordium ratio at the peak time was significantly lower in patients (mean value, 2.3; range, 1.9 to 3.1) compared with controls (mean value, 3.3; range, 2.4 to 4.0) (P less than 0.02). These data confirm the pivotal role of the liver in the removal of aggregated IgA in humans and the defective clearance capacity of this test probe in IgA nephropathy patients.

Met overexpression confers HGF-dependent invasive phenotype to human thyroid carcinoma cells in vitro.

The proto‐oncogene c‐MET encodes the tyrosine kinase receptor for hepatocyte growth factor (HGF), a pleiotropic cytokine controlling growth, survival, motility, invasive migration, and differentiation of epithelial cells. Like several other epithelial neoplasms, thyroid carcinomas have been found to overexpress c‐MET at both the mRNA and protein level. The biological relevance of Met overexpression to thyroid carcinoma natural history, however, remains to be elucidated. Therefore, we analyzed Met expression and response to HGF in two cell lines established from human thyroid carcinomas. In both lines we observed that the overexpressed and constitutively tyrosine phosphorylated HGF receptor maintained biochemical responsiveness to the ligand. Both cell lines were also found to respond to HGF by consistently increasing their motility and invading in vitro reconstituted basal membranes. Conversely, no effect of HGF could be observed in proliferation and survival assays. These data show that overexpression of Met specifically confers to transformed thyroid cells a motile‐invasive phenotype that is dependent on exogenous HGF stimulation. J. Cell. Physiol. 180:365–371, 1999.

Polymorphism of the Uteroglobin Gene in Systemic Lupus Erythematosus and IgA Nephropathy

Uteroglobin (UG) is a multifunctional protein with anti-inflammatory/immunomodulatory properties. The UG gene is located on the long arm of chromosome 11 (11q12.3–q13.1) in a region linked to some immune disorders. A guanine-adenine substitution at position 38 (A38G) has been found in the noncoding region of exon 1 that is significantly correlated with an increased risk of developing immune-mediated diseases. Recently an experimental model of UG knockout mice showed that in mice, UG deficiency causes severe glomerulopathy with mesangial deposition of IgA-fibronectin complexes. To detect the presence of polymorphisms in the UG coding sequence, the DNA of 109 patients with IgA nephropathy (IgAN), and 32 patients with systemic lupus erythematosus (SLE) were tested for the nucleotide sequence of all three UG exons by heteroduplex analysis. We detected heterozygous DNA only for exon 1 due to the A38G substitution, as confirmed by sequencing. We tested for A38G polymorphism, by restriction endonuclease digestion (Sau96I), both in SLE patients and in IgAN patients. Twenty patients with either membranous nephropathy (12) or focal and segmental glomerular sclerosis and 120 healthy subjects served as controls. Compared with both healthy controls and non-IgA control patients, the frequency of the 38A allele was significantly higher in SLE patients (38 of 64 alleles versus 89 of 240 alleles, p = 0.002, and versus 7 of 40 alleles, p < 0.001). IgAN patients showed an allelic distribution similar to both control groups. A subgroup of 18 IgAN patients undergoing renal replacement therapy because of end-stage renal disease showed a significant increase in 38A allele frequency (5 of 36 38G alleles versus 31 of 36 38A alleles, p < 0.001). UG is an immunomodulatory agent that is able to (a) inhibit the activity of several phospholipase A2 (PLA2s), (b) interfere with the function of both neutrophils and monocytes, and (c) prevent immune recognition, perhaps by masking surface antigens. This could account for the role this molecule plays in SLE. The A38G polymorphism is located within a region corresponding to the rat minimal promoter that proved to be important in the transcriptional regulation of UG. Although the significance of any alterations in the UG exon 1 noncoding region in humans has yet to be clarified, initial evidence suggests that it may alter the control of immune response and of inflammation.

Anti-neuronal antibodies in patients with HCV-related mixed cryoglobulinemia

Mixed cryoglobulinemia (MC) is an immunological disorder characterized by immune-complex-mediated systemic vasculitis involving small vessels, which may present with renal, cutaneous, rheumatologic, and/or neurological manifestations. Until recently, the possible appearance of anti-neuronal autoantibodies in peripheral neuropathy occurring in the context of hepatitis C virus (HCV)-associated IgMk/IgG MC has not been extensively addressed. Therefore, a sample of these patients were evaluated by means of immuno-enzyme methods of anti-neuronal autoantibody detection. A significant increase in plasma titers of both anti-GM1 ganglioside and anti-sulfatide was observed. Abnormal titers were associated with evidence of active neuropathy as assessed by electrophysiologic studies. While peripheral neuropathy was traditionally thought to result from axonal ischemic damage caused by deposits of cryoprecipitable immune complexes in the vasa nervorum, a significant association between anti-GM1 and anti-sulfatide antibodies and involvement of the peripheral nervous system was observed in HCV-associated mixed IgMk/IgG cryoglobulinemia. Anti-neuronal reactivity could be a direct trigger of neurologic injury in this disorder.

Clearance of polymeric IgA aggregates in humans.

Clearance of aggregated human secretory immunoglobulin A (AHS-IgA) was studied in nine patients affected by primary IgA nephropathy (IgAGN) and six normal volunteers from the medical staff. Samples of whole blood, erythrocytes, and serum were taken from 3 to 120 minutes after injection of 0.5 mg of 131iodine-labeled AHS-IgA. No significant radioactivity was recorded on erythrocytes. The clearance curve of AHS-IgA from the circulation, calculated by measuring trichloroacetic acid precipitable radioactivity in serum, was found to be biexponential with an initial fast component significantly prolonged in patients (mean half-time, 19.4 minutes, range, 14 to 26 minutes) compared with controls (mean, 12.2 minutes, range, 7.6 to 16.8 minutes; P less than 0.01). These data indicate that clearance of aggregated polymeric IgA does not involve the erythrocyte transport system and seems to be defective in IgAGN patients.

Mycophenolate mofetil and roscovitine decrease cyclin expression and increase p27kip1 expression in anti Thy1 mesangial proliferative nephritis

The response of mesangial cells to a phlogistic challenge includes cell proliferation and mesangial matrix expansion. Cell proliferation is a highly regulated process which includes enhancing factors such as cyclins, cyclin dependent kinases, and inhibitory proteins, such as p27kip1. The aim of the study was to evaluate the effects of Mycophenolate mofetil (MMF), and roscovitine (R), on the cell cycle regulatory system when administered in the florid phase of the experimental model of mesangial proliferative nephritis induced by the anti Thy‐1 antigen monoclonal antibody. Three days after nephritis induction, different groups were given MMF and R. Rats treated with MMF or R showed a slight decrease in mesangial proliferation and matrix expansion. Samples of cortical tissue were tested by ‘real time’ RT‐PCR in order to study gene expression of cyclins B, D1, D2, D3, E, and the cyclin inhibitor p27kip1. Localization of mRNA was evaluated by in situ hybridization. Real time RT‐PCR analysis showed a significant decrease in cyclins B, D1, D2, and D3 in rats treated with either MMF or R as compared to controls. Both MMF and R treatment induced a significant increase in p27kip1 mRNA expression. In situ hybridization showed a mesangial‐endothelial expression pattern in glomeruli. The number of labelled cells per glomerulus, the number of positive glomeruli in each examined slide as well as cyclin D2 and D3 signal intensity was significantly lower in rats treated with MMF or R as compared to controls, whereas MMF or R treatment up‐regulated p27kip1 mRNA expression. Immunohistochemical evaluation of p27kip1 aimed to examine the influence of MMF or R on protein expression confirmed up‐regulation.

Intradialytic Cytokine Gene Expression

Along with the numerous technological improvements in molecular biology, polymerase chain reaction, which permits analysis of sequences of a very small amount of biological material, enables evaluation of hemodialysis-induced gene transcription of inflammatory cytokines. Blood samples drawn from 22 hemodialysis patients, treated with cellulose- derived or synthetic membranes, were collected at 0 and 15 min of hemodialysis. Total RNA, purified from mononuclear cells, was reverse transcribed and cDNA amplified by polymerase chain reaction primed with specific oligomers in order to determine tumor necrosis factor α (TNFα), interleukin (IL) 1β and IL6 gene expression. Plasma samples were collected at 0 and 180 min for detection of mature cytokines by enzyme immunoassay with plates pre-coated with monoclonal antibodies to TNFα, IL1β and IL6. A significant increase in TNFα mRNA was detected at 15 min of hemodialysis in 12 of 22 patients: 5 of 9 treated with cuprophan; 3 of 3 with cellulose triacetate; 3 of 5 with polysulfone, and only 1 of 5 treated with polymethylmethacrylate membranes. A parallel increase in IL1β or IL6 mRNA was detected, and significant relationships were found between TNFα and IL1β (p < 0.001), and IL1β and IL6 gene expression (p < 0.05). Increased levels of mature TNFα and IL1β molecules in plasma were detected in the majority of patients showing an increased cytokine gene expression. However, the absolute amount of cytokine mRNA transcription at 15 min did not predict the levels of mature molecules reached in plasma at 180 min. Cytokine mRNA transcription is quite common at the beginning of a dialysis run. Possibly due to intracellular degradation of critical sequences of cytokine mRNA, gene expression does not necessarily imply translation into mature protein. It is suggested that mechanisms related to cell-to-cell interaction, which may possibly involve procytokine biology, are needed to drive phenomena of cytokine activation to clinical effectiveness.

Genetic Factors Associated with Rheumatoid Arthritis and Systemic Vasculitis: Evaluation of a Panel of Polymorphisms

Immune and inflammatory response activation is a common feature of connective tissue diseases and systemic vasculitis. The aim of our study was to evaluate the possible involvement of TNFα c.-308A > G, IL-10 c.-1082A > G, uteroglobin c.38A > G, TGFβ 1 c.869C > T and NFκB2 c.-1837T > C gene polymorphisms in susceptibility to connective tissue diseases. Our study cohort included 68 unrelated patients affected by rheumatoid arthritis (RA) (37 patients) and ANCA-positive [micropolyangiitis (mPA) 17 patients] or ANCA-negative systemic vasculitis [including 8 patients with Henoch-Schönlein purpura (HSP) and 6 patients with mixed cryoglobulinaemia (MC)] as well as 98 control subjects. Allele frequency analysis of uteroglobin c.38G > A polymorphism showed a significant increase in the c.38A allele in patients (p= 0.002). Genotype frequency analysis of uteroglobin and NF-κB2 gene polymorphisms in patients showed an increase in c.38GA and c.38AA genotypes in the uteroglobin gene (p=0.02) coupled with an increase in homozygous c.-1837CC in the NF-κB2 gene (p=0.02). Our data suggest that genetic variation in UG and NF-κB2 pathways could have effects in connective tissue disease susceptibility.

Failure to Relate Mononuclear Phagocyte System Function to HLA-A, B, C, DR, DQ Antigens in Membranous Nephropathy

Nineteen patients with idiopathic membranous nephropathy were typed for HLA pattern and analyzed for the Fc receptor function of splenic macrophages by detecting in vivo the clearance of IgG-sensitized 51Cr-labelled autologous erythrocytes. Seven out of 19 patients were found to have a macrophage dysfunction. This defect was not related to any HLA-A, B, C, DR, DQ antigen tested nor to the levels of IgG-containing immune complexes, as detected by a Clq solid phase test, nor to the magnitude of proteinuria. Since HLA-B8 and HLA-DR3 antigens were significantly more frequent in patients than in the control group, the factors that may impair the macrophage system in individuals predisposed to this nephropathy are discussed.

Absence of NF-κB Activation by a New Polystyrene-Type Adsorbent Designed for Hemoperfusion

Aim: The aim of the study was to evaluate biocompatibility of anew polystyrene-type adsorbent (BetaSorbTM) designed for hemoperfusion, using second-level biomolecular analyses. The device has recently been developed to enhance β2-microglobulin removal during hemodialysis. Molecular structure and chemical modifications of the surface beads of this cartridge should prevent exposure of dense hydrophobic surface sites to proteins, and avoid the major drawbacks of previous polystyrene-type adsorbent materials. Methods: Whole blood of healthy donors was incubated in sterile minicolumns packed with BetaSorb Cuprophan, Hemophan, polysulfone and cellulose acetate. In parallel experiments, whole blood was recirculated for 180 min in a sham dialysis circuit equipped with the study sorbent or Hemophan or polysulfone. Biocompatibility was assessed by means of new biomolecular approaches focused on nuclear factor ĸB (NF-ĸB) activation (assessed by electrophoretic mobility shift assay), TNF-α and IL-1β gene expression (evaluated by real-time PCR), TNF-α and IL-1β production (measured by Western blot assay and ELISA), nitric oxide (NO) generation (detected by electron paramagnetic resonance), free oxygen radical production (by chemiluminescence in a biological assay) and the generation of the complement breakdown product C3d. Results:In coincubation experiments, 5-min contact with any dialysis device, but BetaSorb, was enough to induce activation of NF-ĸB. The amount of TNF-α precursor form was found to increase after 5 min of exposure to each tested polymer, but no traces of mature forms of TNF-α or IL-1β were detected in in vitro experimental conditions using healthy blood. NO and free oxygen radical generation were significantly lower in blood samples exposed to BetaSorb than in control dialysis devices. C3d levels were found to be increased with Hemophan, unaffected by polysulfone, and remarkably decreased with the BetaSorb device. In the sham hemodialysis experiments, NF-ĸB activation and C3d and NO profiles were similar to direct incubation experiments. Compared to basal levels, quantitation of TNF-α and IL-1β mRNA revealed a 15- and 9-fold increase, respectively, in samples exposed to Hemophan for 180 min. Conclusions: The new BetaSorb device not only appears to be highly biocompatible, but shares properties that make it probably able to interfere with the activation of the inflammatory state.