Duration of fecal shedding of Shiga toxin-producing Escherichia coli O104:H4 in patients infected during the 2011 outbreak in Germany: a multicenter study.

BACKGROUND: In May-July 2011, Germany experienced a large food-borne outbreak of Shiga toxin 2-producing Escherichia coli (STEC O104:H4) with 3842 cases, including 855 cases with hemolytic uremic syndrome (HUS) and 53 deaths. METHODS: A multicenter study was initiated in 5 university hospitals to determine pathogen shedding duration. Diagnostics comprised culture on selective media, toxin enzyme-linked immunosorbent assay, and polymerase chain reaction. Results were correlated with clinical and epidemiologic findings. Testing for pathogen excretion was continued after discharge of the patient. RESULTS: A total of 321 patients (104 male, 217 female) were included (median age, 40 years [range, 1-89 days]). Median delay from onset of symptoms to hospitalization was 4 days (range, 0-17 days). Two hundred nine patients presented with HUS. The estimate for the median duration of shedding was 17-18 days. Some patients remained STEC O104:H4 positive until the end of the observation time (maximum observed shedding duration: 157 days). There was no significant influence of sex on shedding duration. Patients presenting with HUS had a significantly shortened shedding duration (median, 13-14 days) compared to non-HUS patients (median, 33-34 days). Antimicrobial treatment was also significantly associated with reduced shedding duration. Children (age≤15 years) had longer shedding durations than adults (median, 35-41 vs 14-15 days). CONCLUSIONS: STEC O104:H4 is usually eliminated from the human gut after 1 month, but may sometimes be excreted for several months. Proper follow-up of infected patients is important to avoid further pathogen spread.

Beneficial effects of integrin αvß3-blocking RGD peptides in early but not late phase of experimental glomerulonephritis.

BACKGROUND: Integrin αvß3 plays an important role in the regulation of cell proliferation and neoangiogenesis. We found mesangial de novo expression of integrin αvß3 in mesangioproliferative glomerulonephritis (MesGN). The aim of the study was to clarify if blockade of αvß3 integrin with the specific αvß3-blocking cyclic peptide RGDdFV (cRGD) has beneficial effects on the course of this disease. METHODS: Habu snake venom (Habu) GN was induced in male C57BL/6 mice 1 week after uninephrectomy (6 mg Habu toxin/kg body weight intravenously). After 24 h, nephritic animals received αvß3-inhibitory cRGD or cRAD control peptides for 3 or 7 days, respectively. The kidneys were investigated using morphometry, immunohistochemistry and TaqMan polymerase chain reaction. RESULTS: At Day 3, serum creatinine and albuminuria were lower after cRGD compared to cRAD treatment. At Day 3, glomerulosclerosis index, percentage of glomerular injury, mesangial cell (MC) number and volume density of mesangial matrix were significantly lower (P < 0.05) in cRGD-treated mice than in cRAD-treated controls. At Day 7, only a mild effect of cRGD on mesangial matrix expansion and fibronectin messenger RNA was still detectable (P < 0.05). Complementary in vitro studies in MCs revealed that inhibition of αvß3 by cRGD-blocked adhesion, reduced proliferation and increased apoptosis of MCs. CONCLUSION: Habu GN inhibition of integrin αvß3 by cRGD partly ameliorates early injury but has no or only mild effects on late glomerular lesions.

Induction and coexpression of latent transforming growth factor beta-binding protein-1 and fibrillin-1 in experimental glomerulonephritis.

BACKGROUND: Latent transforming growth factor-beta-binding protein 1 (LTBP-1) and fibrillin-1 were shown to colocalize and interact in the extracellular matrix of the skin and vasculature. This interaction may regulate transforming growth factor-beta (TGF-beta) activity. TGF-beta is an important progression factor for glomerular diseases. We hypothesized that LTBP-1 and fibrillin-1 are coexpressed in the glomerulus and upregulated during glomerulonephritis. METHODS: Acute anti-Thy1.1 glomerulonephritis was induced with a single intravenous injection (1 mg/kg body weight) of a monoclonal anti-Thy1.1 antibody in rats. Real-time RT-PCR and immunohistochemical analyses for LTBP-1 and fibrillin-1 were performed. RESULTS: Induction of glomerular LTBP-1 mRNA was detected on day 2 of disease, while mRNA for fibrillin-1 was already upregulated 1 day after induction of disease. Both LTBP-1 and fibrillin-1 showed a mesangial distribution. An expansion of the LTBP-1 and fibrillin-1-positive mesangial area was seen on day 6 of disease, when transient matrix accumulation was most prominent. On day 12 of disease, glomerular LTBP-1 and fibrillin-1 immunoreactivities had returned to control levels. In serial sections, some colocalization of LTBP-1 and fibrillin-1 was detected in control as well as in nephritic glomeruli. CONCLUSION: Mesangial expression of LTBP-1 and fibrillin-1 is induced early in experimental nephritis and LTBP-1 and fibrillin-1 are partially colocalized in the nephritic glomerulus. An interaction of these molecules could stabilize latent TGF-beta complexes and thus attenuate the activation of TGF-beta during this self-limited glomerular disease.

Dynamic expression patterns of transforming growth factor-beta(2) and transforming growth factor-beta receptors in experimental glomerulonephritis.

Numerous studies have demonstrated the involvement of the transforming growth factor (TGF) isoform beta(1) in the pathogenesis of renal fibroproliferative diseases. Although in vitro studies suggest that TGF-beta(2) is equally potent to TGF-beta(1) in terms of its antimitogenic and fibrogenic effects, much less is known about the regulation of TGF-beta(2) in renal diseases associated with glomerular cell hyperplasia and matrix expansion. Here we investigated the glomerular expression patterns of TGF-beta(2) and of the TGF-beta receptors I, II, and III during the course of rat anti-Thy1.1 nephritis (days 2, 6, 12, and 56), a model characterized by transient mesangial hypercellularity and extracellular matrix accumulation. TGF-beta(2) exhibited dynamic changes in expression. Immunohistochemical double-staining of renal sections revealed that most TGF-beta(2)-positive cells in control glomeruli were podocytes with few TGF-beta(2)-positive mesangial cells. This staining pattern could also be observed in human kidney. On day 6 of anti-Thy1.1 nephritis both TGF-beta(2) positive podocytes and mesangial cells were more abundant. By western blot analysis of isolated glomeruli from nephritic rats, protein expression of TGF-beta(2) was upregulated tenfold over control glomeruli, peaking on day 6 of the disease. In cultured rat mesangial cells we found that the TGF-beta(2) and TGF-beta(1) isoforms were equally potent in terms of nuclear accumulation of phosphorylated Smad 2/3, inhibition of DNA synthesis, and induction of beta(1)-integrin and type I collagen protein synthesis. Protein expression of the TGF-beta receptor I was not detected by immunohistochemistry in control glomeruli but was markedly induced in the mesangium on day 6 of nephritis. Mesangial staining for TGF-beta receptors II and III was detected in normal kidneys. Expression of TGF-beta receptor II was strongly enhanced on days 6 and 12 of disease, while TGF-beta receptor III was upregulated only on day 6. In summary, we report marked yet transient upregulation of TGF-beta(2) protein and of TGF-beta receptors I, II, and III in glomerular cells during anti-Thy1.1 nephritis. These results are in keeping with the notion that TGF-beta(2) and its receptors participate in the pathogenesis and/or resolution of this transient form of glomerulonephritis.

Requirement of cyclin D1 in mesangial cell mitogenesis.

Abstract. Hyperplasia of mesangial cells (MC) is a frequent finding in glomerulonephritis. The control and function of cyclin D1, a regulator of cell cycle progression, in MC proliferation in vivo and in vitro were investigated. In a rat model of mesangioproliferative glomerulonephritis, increases in the number of cyclin D1-positive MC nuclei were prominent on day 5 of the disease, preceding the peak of MC hyperplasia. In growth-arrested rat MC in culture, mitogenic stimulation with serum or platelet-derived growth factor (PDGF) led to rapid increases in cyclin D1 protein expression. Transforming growth factor-beta1 inhibited PDGF induction of cyclin D1 protein at 12 h. In an examination of the subcellular distribution of cyclin D1, it was observed that stimulation of MC with PDGF for 6 h caused translocation of cyclin D1 from the cytoplasm into the nucleus. Coincubation with PDGF and transforming growth factor-beta1 completely inhibited this effect, without altering the cellular cyclin D1 protein abundance at that time point. To test whether reduction of cyclin D1 protein levels was sufficient to inhibit mitogenesis, MC were transfected with antisense oligonucleotides (ODN) complementary to rat cyclin D1 mRNA. Antisense ODN against cyclin D1 reduced the serum- or PDGF-induced protein expression of cyclin D1 to 27 or 10% of control levels, respectively. These inhibitory effects were correlated with diminished cyclin-dependent kinase 4 activity. Antisense ODN against cyclin D1 also decreased the PDGF-induced increase in p21(Waf-1) protein levels. The MC proliferation caused by serum or PDGF was markedly inhibited by antisense ODN against cyclin D1, as measured by [(3)H]thymidine uptake and cell counts. It is concluded that increased cyclin D1 protein expression of MC is required for MC proliferation. Targeting cyclin D1 expression may represent an effective means to inhibit MC proliferation in vitro and in vivo.

Low-dose sirolimus in combination with mycophenolate mofetil improves kidney graft function late after renal transplantation and suggests pharmacokinetic interaction of both immunosuppressive drugs.

AIMS: Chronic allograft nephropathy and/or calcineurin inhibitor toxicity are common problems after organ transplantation. The aim of this study was to examine the safety and efficacy of switching from a calcineurin inhibitor-based to a calcineurin inhibitor-free immunosuppressive regimen consisting of sirolimus and mycophenolate mofetil (MMF) late after renal transplantation. METHODS: Kidney biopsies were performed in renal-transplanted patients with increasing serum creatinine levels at least 6 months after transplantation (mean time +/- SD after renal transplantation: 76.4 +/- 50.4 months). Patients with no signs of acute rejection were switched to MMF (500-2,000 mg/day) in combination with a low dose of sirolimus (1 mg/day). Renal function, serum chemistry, blood trough levels of sirolimus and MMF, and blood pressure were monitored. RESULTS: 13 patients were investigated. During our observation period (mean observation time +/- SD: 11.2 +/- 5.9 months), an improvement in renal function was observed in 10/13 patients. In 3/13 patients, renal function deteriorated further and hemodialysis was initiated in 2 patients within the next 6 months. However, a serum creatinine concentration above 3.5 mg/dl was measured in 2 of those 3 patients prior to the switch of the immunosuppressive protocol. Administration of a low dosis of sirolimus (1 mg/day) led to relevant sirolimus (4.16 +/- 1.85 ng/ml) and MMF blood trough levels (month 1: 6.8 +/- 3.46; month 3: 4.67 +/- 1.78 mg/l). The following adverse events were observed: borderline acute rejection (1/11 patients), anemia responding to higher dosage of erythropoietin (3/11), hyperlipidemia (1/11), and urinary tract infections (4/11). CONCLUSIONS: Low-dose sirolimus therapy in combination with concentration-adjusted MMF therapy leads to improvement of organ function late after renal transplantation. The follow-up of those patients should include assessments of blood cell counts, serum lipids and urinalysis to recognize the possible side effects.

Tacrolimus and cerivastatin pharmacokinetics and adverse effects after single and multiple dosing with cerivastatin in renal transplant recipients.

AIMS: In contrast to cyclosporin, only limited information exists on the interaction potential between the immunosuppressive agent tacrolimus and HMG-CoA reductase inhibitors, which are metabolized via the cytochrome P450 system. The aim of this study was to investigate the pharmacokinetics, and adverse effects of cerivastatin combined with tacrolimus in renal transplant patients. METHODS: Ten patients with stable kidney graft functions and LDL-cholesterol serum concentrations > 110 mg dl-1 were included in the study. After an observation period of 3 months, cerivastatin (0.2 mg daily) was administered for an additional 3 months. Tacrolimus steady-state pharmacokinetics and cerivastatin single- and multiple-dose pharmacokinetics were determined. Lipid concentrations, routine laboratory parameters and adverse events were obtained and analysed throughout the study period of 6 months. RESULTS: Blood tacrolimus trough concentrations were not affected by cerivastatin (mean +/- SD 8.6 +/- 2.1 ng ml(-1) before, and 8.7 +/- 2.4 ng ml(-1) at day 90 of cerivastatin dosing, with a 95% confidence interval on the difference = 0.97, 1.08). The mean area under the blood concentration-time curve to 24 h (AUC(0,24 h)) for cerivastatin was 14.5 +/- 2.53 micro g l(-1) h(-1) at day 1 after starting treatment and 19.02 +/- 3.55 micro g l(-1) h(-1) (3 months later), resulting in a 35% higher (AUC(0,24 h)) compared with the first dose. Total cholesterol, LDL-cholesterol and triglyceride concentrations were significantly lowered by cerivastatin whereas no significant effect of cerivastatin on serum creatininkinase concentrations was observed and no adverse effects were documented. CONCLUSIONS: Tacrolimus increased the AUC(0, 24 h) of cerivastatin by a mean of 35% in renal transplant patients. Cerivastatin had no detectable effect on the pharmacokinetics of tacrolimus.

Nitric oxide increases adrenomedullin receptor function in rat mesangial cells.

BACKGROUND: Adrenomedullin (ADM) exerts antiproliferative effects on rat mesangial cells in vitro and, therefore is a possible renoprotective agent. In contrast, nitric oxide (NO) is capable of exerting both cytoprotective and cytotoxic actions. It was the objective of the present study to examine whether NO stimulates the ADM system. METHODS: Rat mesangial cells were incubated with the NO donors GSNO and SNAP, the guanylate cyclase inhibitor ODQ, and the cGMP analog 8-bromo-cGMP. ADM radioligand binding, ADM-induced intracellular cAMP-accumulation (radioimmunoassay) and ADM receptor gene expression (TaqMan real time PCR) were measured. RESULTS: Twenty-four hour treatment of mesangial cells with GSNO and SNAP (100 micromol/L each) increased the maximal binding of ADM to its receptor from 52%+/- 4% to 101%+/- 4% (P < 0.001) and 81%+/- 2% (P < 0.001), respectively. GSNO, SNAP (both 100 micromol/L) and 8-bromo-cGMP (50 micromol/L) increased EC50 from 9.9 x 10-8 to 7.0 x 10-10, 4.8 x 10-10, 1.1 x 10-9, respectively. In contrast, combined pretreatment with GSNO (100 micromol/L) and ODQ (100 micromol/L) reduced EC50 to values similar to the control cells (2.4 x 10-8). In contrast, ADM receptor gene expression was reduced significantly by different concentrations of GSNO, SNAP, and by 50 micromol/L 8-bromo-cGMP, but not by 8-bromo-cAMP. CONCLUSIONS: NO increases ADM signal transduction via a cGMP dependent pathway. This effect is caused, at least in part, by an increase in ADM receptor availability and is counteracted in a feedback manner on the mRNA level. This mechanism might direct the impact of NO on mesangial cell function toward cytoprotection.