Teresa Rampino

Monitoring of human cytomegalovirus-specific CD4 and CD8 T-cell immunity in patients receiving solid organ transplantation.

Absolute and human cytomegalovirus (HCMV)‐specific CD4+ and CD8+ T‐cell counts were monitored in 38 solid organ (20 heart, 9 lung and 9 kidney) transplant recipients during the first year after transplantation by a novel assay based on T‐cell stimulation with HCMV‐infected autologous dendritic cells. According to the pattern of T‐cell restoration occurring either within the first month after transplantation or later, patients were classified as either early (n = 21) or late responders (n = 17). HCMV‐specific CD4+ and CD8+ T‐cell counts were consistently lower in late compared to early responders from baseline through 6 months after transplantation. In addition, in late responders, while HCMV infection preceded immune restoration, HCMV‐specific CD4+ restoration was significantly delayed with respect to CD8+ T‐cell restoration. The number of HCMV‐specific CD4+ and CD8+ T‐cells detected prior to transplantation significantly correlated with time to T‐cell immunity restoration, in that higher HCMV‐specific T‐cell counts predicted earlier immune restoration. Clinically, the great majority of early responders (18/21, 85.7%) underwent self‐resolving HCMV infections (p = 0.004), whereas the great majority of late responders (13/17, 76.5%) were affected by HCMV infections requiring antiviral treatment (p = <0.0001). Simultaneous monitoring of HCMV infection and HCMV‐specific T‐cell immunity predicts T‐cell‐mediated control of HCMV infection.

Toll‐like receptor 4 expression is increased in circulating mononuclear cells of patients with immunoglobulin A nephropathy

We investigated Toll‐like receptors (TLR‐3, ‐4 and ‐7) expression in circulating mononuclear cells of patients with immunoglobulin A nephropathy (IgAN), a disease with debated relationships with mucosal immunity. TLR‐4 expression (detected by fluorescence activated cell sorter) and mRNA transcriptional levels (Taqman) were significantly higher in patients with IgAN than in healthy controls (P = 0·00200 and P = 0·0200). TLR‐3 and TLR‐7 were not modified significantly. In IgAN patients proteinuria was correlated significantly with TLR‐4 expression (P = 0·0312). In a group of nephrotic syndromes, TLR‐3, ‐4 and ‐7 expression was similar to healthy controls. A significant difference in TLR‐4 expression and mRNA levels was found between very active IgAN patients (proteinuria > 1 g/1·73 m2/day in association with severe microscopic haematuria) and inactive patients (proteinuria < 0·5 g/1·73 m2/day, with absent or minimal haematuria). No correlation with levels of aberrantly glycosylated IgA1, age, renal biopsy features or therapy was found. This study shows for the first time an up‐regulation of TLR‐4 in circulating mononuclear cells of patients with IgAN, particularly in association with proteinuria and heavy microscopic haematuria.

Hepatocyte growth factor/scatter factor released during peritonitis is active on mesothelial cells.

Peritonitis causes mesothelial detachment that may result in persistent peritoneal denudation and fibrosis. We investigated whether hepatocyte growth factor (HGF), a scatter factor that induces detachment from substrate and fibroblastic transformation of several cell types, is produced during peritonitis and is active on mesothelial cells. We studied 18 patients on peritoneal dialysis, 9 uncomplicated, 9 with peritonitis. HGF was measured in serum, peritoneal fluid, and supernatant of peripheral blood mononuclear cells and peritoneal mononuclear cells. Primary culture of human peritoneal mesothelial cells and the human mesothelial cell line MeT-5A were conditioned with recombinant HGF, serum, and peritoneal fluid. HGF levels were significantly higher in serum and peritoneal fluid of peritonitic than uncomplicated patients. Mononuclear cells of peritonitic patients produced more HGF than cells of uncomplicated patients. Recombinant HGF, serum, and peritoneal fluid of peritonitic patients caused mesothelial cell growth, detachment, transformation from epithelial to fibroblast-like shape, overexpression of vimentin, and synthesis of type I and III collagen. In conclusion, HGF released during peritonitis causes a change in mesothelial cell phenotype and function. HGF may affect the healing process facilitating repair through mesothelial cell growth, but may contribute to peritoneal fibrosis inducing cell detachment with mesothelial denudation and collagen synthesis.

The Ron proto-oncogene product is a phenotypic marker of renal oncocytoma

The proto-oncogene product Ron is the receptor for macrophage stimulating protein, a scatter factor that stimulates cell proliferation, prevents apoptosis, and induces an invasive cell phenotype. We investigated the expression of Ron, Ki-67 (proliferation index), p53, and bcl-2 (proapoptotic and antiapoptotic proteins, respectively) in 50 renal tumors (19 clear cell carcinomas, 18 oncocytomas, 7 papillary cell carcinomas, 5 chromophobe cell carcinomas, and 1 carcinoma with sarcomatoid areas). In addition, we studied Ron in normal kidney and in the renal carcinoma cell line Caki-1. Immunostaining and Western blot showed Ron in normal kidney and in all oncocytomas but never in renal cell carcinomas or in Caki-1. In addition, Western blot showed that Ron was expressed in phosphorylated, i.e., active, form. Bcl-2 was strongly expressed in oncocytomas, whereas Ki-67 and p53 were much less expressed in oncocytomas than in carcinomas. These results indicate in Ron a marker that differentiates oncocytoma from the other renal epithelial tumors. We therefore think that Ron may prove to be a new tool for a sound and precise diagnosis of oncocytoma, a benign tumor that cannot always be distinguished from carcinomas at histologic examination. The overexpression of bcl-2, but not p53 in oncocytoma, suggests that the MSP/Ron system sustains the growth of oncocytoma by opposing apoptosis.

Mesenchymal Stem Cells Infusion Prevents Acute Cellular Rejection in Rat Kidney Transplantation

Mesenchymal stem cells (MSC) are multipotent cells that differentiate into various mature cell lineages. MSC show immunomodulatory effects by inhibiting T-cell proliferation. We evaluated the effect of the infusion of MSC in rats experimental kidney transplantation. Sprague-Dawley transgenic rats (SD) able to express the green fluorescent protein (EGFP) were used as MSC donors. Syngeneic (Lewis to Lewis, n = 10) and allogeneic (Fischer to Lewis, n = 10) kidney transplantations were performed after bilateral nephrectomy. Five transplanted rats who received syngeneic grafts, were treated with 3 x 10(6) MSC (Gr B), while the other 5 did not received MSC (Gr A). Five rats with allogenic grafts received 3 x 10(6) MSC (Gr C) and another 5 did not receive MSC (Gr D). The MSC were infused directly into the renal artery of the graft. No immunosuppressive therapy was provided. The animals were killed after 7 days. Biochemical analysis for renal function, histological (Banff criteria) and immunohistological analysis (ED1+ and CD8+) were performed on treated animals. MSC improved kidney function in Gr B and D vs Gr A and C. The tubular damage appeared to be less severe among Gr B and Gr D with respect to Gr A and C (P < .01). Vasculitis was more accentuated in Gr A and C (P < .01). MSCs reduced the inflammatory infiltrate; in Gr B and D, the number of ED1+ cells was lower than in Gr A and C (P < .005), which was also observed for CD8+ cells (P < .05). Our study demonstrated that the infusion of MSC attenuated histological damage from acute rejection by reducing the cellular infiltration.

Risk Factors for Chronic Renal Dysfunction in Cardiac Allograft Recipients

Renal dysfunction is one of the most common and threatening complications in heart transplant recipients. Even if ciclosporin seems to play a central role in inducing renal damage, other factors may concur or predispose to renal injury. In order to identify factors responsible for renal dysfunction, we retrospectively studied a cohort of 114 cardiac transplant recipients during a follow-up period of at least 3 years. The patients had a normal renal function before and 0.5 months after heart transplantation. Doubling of baseline serum creatinine or attainment of serum creatinine steadily above 176.8 µmol/l (2.0 mg/dl) was used as criterion to define the end-point renal dysfunction. A series of clinical and laboratory variables were obtained from the patients’ charts at different time intervals, and their prognostic value for the occurrence of renal dysfunction was calculated by Cox proportional hazards models. 23 out of 114 patients reached the end point after a median time period of 21 months. High serum triglyceride, alanine aminotransferase, alkaline phosphatase, ciclosporin, urea, glucose, and hemoglobin levels were shown to be associated with the development of renal dysfunction. Four variables, i.e., triglyceride, ciclosporin, urea, and alkaline phosphatase, had an independent prognostic value. Our results confirm a role for ciclosporin in inducing renal dysfunction and identify hyperlipidemia and an increased plasma urea level as risk factors for renal dysfunction in heart transplant recipients.

Which Is the Most Suitable and Effective Route of Administration for Mesenchymal Stem Cell-Based Immunomodulation Therapy in Experimental Kidney Transplantation: Endovenous or Arterial?

Immunomodulating cell therapy represents a new perspective for the control of cellular immune responses that determine the occurrence of acute rejection (ACR) in allo-transplantation. Mesenchymal stem cells (MSC) demonstrate immunoregulatory effects by inactivating T-cell components that regulate tissue damage in transplantation models. The presumed mechanism of action is recruitment of cells by a cytokine network. The purpose of this study was to test which route of administration (intra-arterial vs intravenous) was the most effective route to achieve immunomodulating effects in experimental rat kidney transplantation. Transgenic Sprague-Dawley rats (SD) expressing the enhanced green fluorescent protein (EGFP) at the somatic level were used as MSC donors: Allogeneic Fischer to Lewis grafts (n = 4 per group) were performed in rats after bilateral nephrectomy. In Gr B, 3 x 10(6) MSCs were infused into the renal graft artery, whereas in Gr C, they were infused into the tail vein. The untreated Gr A were a control group. No immunosuppressive therapy was administered. The animals were sacrificed at day 7 postoperatively. Biochemical analysis for renal function, histological (Banff criteria) and immunohistological (anti-EGFP-Immunoglobulin) analysis were performed on the transplanted animals. In Gr B, functional recovery was more rapid (creatinine: Gr B vs Gr C, P < .05). The inflammatory infiltrate in the graft was less in Gr B vs Gr C, with preservation of tubules, arteries, and glomeruli (P < .01). Intra-arterial infusion of MSCs was more effective to control ACR.

Stimulation of Hepatocyte Growth Factor in Human Acute Renal Failure

Hepatocyte growth factor (HGF) is a potent mitogen for tubular cells. Experimental injury to the kidney is associated with HGF release both locally and by distant organs stimulated by circulating ‘injurins’. In this study, the serum HGF concentration was measured in patients with acute renal failure (ARF). Normal subjects and chronic renal failure patients either not on dialysis or on regular dialysis treatment served as controls. Human mesangial cells were incubated with sera from ARF patients and controls. The serum HGF concentration was strikingly increased in ARF patients (478 ± 68 ng/dl) and was normal in chronic renal failure patients not on dialysis (20 ± 3 ng/dl) and in those on regular dialysis treatment (25 ± 3 ng/dl). Serum of ARF patients strongly stimulated HGF release from mesangial cells (1,384 ± 55 ng/ml) in comparison with normal serum (67 ± 10 ng/ml). These results indicate that in ARF HGF participates in tubular repair both as an endocrine factor, released in the circulation, and as a paracrine substance, diffusing to the tubules from the mesangium.

KCNA1 and TRPC6 ion channels and NHE1 exchanger operate the biological outcome of HGF/scatter factor in renal tubular cells

Hepatocyte growth factor (HGF) is a glycoprotein that induces in vitro epithelial tubular cell growth, motility, scattering and branching morphogenesis. The cell machineries that account for HGF biological effects are still unclear. In previous study, we found that HGF upregulated in epithelial tubular cell line (HK2) 3 genes: potassium channel KCNA1, calcium channel (transient receptor potential channel, subfamily C, member 6, TRPC6) and Na+/H+ exchanger-1 (NHE1). In this study, we validated these results with reverse transcription PCR and WB analysis. To investigate whether KCNA1, TRPC6, NHE1 mediate the changes induced by HGF in HK2, we studied the effects of their inhibitors: 4-aminopyridine, charybdotoxin, dendrotoxin K inhibitors of KCNA1, lanthanum, N-(p-amylcinnamoyl) anthranilic acid inhibitors of TRPC6, 5-(N-ethyl-N-isopropyl)amiloride, cariporide inhibitors of NHE1. The inhibitors prevented HGF-induced growth, migration, cytoskeletal reorganization and tubulogenesis in HK2. These results indicate that KCNA1, TRPC6 and NHE1 are cell machineries that are exploited by HGF to effect its biological outcome in renal tubular cells.

Mesenchymal stromal cells improve renal injury in anti-Thy 1 nephritis by modulating inflammatory cytokines and scatter factors

MSC (mesenchymal stromal cells) can differentiate into renal adult cells, and have anti-inflammatory and immune-modulating activity. In the present study, we investigated whether MSC have protective/reparative effects in anti-Thy1 disease, an Ab (antibody)-induced mesangiolysis resulting in mesangioproliferative nephritis. We studied five groups of rats: (i) rats injected with anti-Thy1.1 Ab on day 0 (group A); (ii) rats injected with anti-Thy1.1 Ab on day 0+MSC on day 3 (group B); (iii) rats injected with anti-Thy1.1 Ab on day 0+mesangial cells on day 3 (group C); (iv) rats injected with saline on day 0+MSC on day 3 (group D); and (v) rats injected with saline on day 0 (group E). Rats were killed on days 1, 3, 7 and 14. MSC prevented the increase in serum creatinine, proteinuria, glomerular monocyte influx and glomerular histopathological injury. Furthermore, MSC suppressed the release of IL-6 (interleukin-6) and TGF-β (transforming growth factor-β), modulated glomerular PDGF-β (platelet-derived growth factor-β), and reset the scatter factors and their receptors, potentiating HGF (hepatocyte growth factor)/Met and inactivating MSP (macrophage-stimulating protein)/Ron (receptor origin nantaise). Few MSC were found in the kidney. These results indicate that MSC improve anti-Thy 1 disease not by replacing injured cells, but by preventing cytokine-driven inflammation and modulating PDGF-β and the scatter factors, i.e. systems that regulate movement and proliferation of monocytes and mesangial cells.