Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy with manifestations of hemolytic anemia, thrombocytopenia, and renal impairment. Genetic studies have shown that mutations in ...complement regulatory proteins predispose to non–Shiga toxin–associated HUS (non-Stx–HUS). We undertook genetic analysis on membrane cofactor protein (MCP), complement factor H (CFH), and factor I (IF) in 156 patients with non-Stx–HUS. Fourteen, 11, and 5 new mutational events were found in MCP, CFH, and IF, respectively. Mutation frequencies were 12.8%, 30.1%, and 4.5% for MCP, CFH, and IF, respectively. MCP mutations resulted in either reduced protein expression or impaired C3b binding capability. MCP-mutated patients had a better prognosis than CFH-mutated and nonmutated patients. In MCP-mutated patients, plasma treatment did not impact the outcome significantly: remission was achieved in around 90% of both plasma-treated and plasma-untreated acute episodes. Kidney transplantation outcome was favorable in patients with MCP mutations, whereas the outcome was poor in patients with CFH and IF mutations due to disease recurrence. This study documents that the presentation, the response to therapy, and the outcome of the disease are influenced by the genotype. Hopefully this will translate into improved management and therapy of patients and will provide the way to design tailored treatments.
Mutations in factor H (HF1) have been reported in a consistent number of diarrhoea-negative, non-Shiga toxin-associated cases of haemolytic uraemic syndrome (DHUS). However, most patients with D-HUS ...have no HF1 mutations, despite decreased serum concentrations of C3. Our aim, therefore, was to assess whether genetic abnormalities in other complement regulatory proteins are involved.
We screened genes that encode the complement regulatory proteins—ie, factor H related 5, complement receptor 1, and membrane cofactor protein (MCP)—by PCRsingle-strand conformation polymorphism (PCR-SSCP) and by direct sequencing, in 25 consecutive patients with D-HUS, an abnormal complement profile, and no HF1 mutation, from our International Registry of Recurrent and Familial HUS/TTP (HUS/thrombotic thrombocytopenic purpura).
We identified a heterozygous mutation in MCP, a surface-bound complement regulator, in two patients with a familial history of HUS. The mutation causes a change in three aminoacids at position 233–35 and insertion of a premature stop-codon, which results in loss of the transmembrane domain of the protein and severely reduced cell-surface expression of MCP.
Results of previous studies on HF1 indicate an association between HF1 deficiency and D-HUS. Our findings of an MCP mutation in two related patients suggest that impaired regulation of complement activation might be a factor in the pathogenesis of genetic forms of HUS. MCP could be a second putative candidate gene for D-HUS. The protein is highly expressed in the kidney and plays a major part in regulation of glomerular C3 activation. We propose, therefore, that reduced expression of MCP in response to complement-activating stimuli could prevent restriction of complement deposition on glomerular endothelial cells, leading to microvascular cell damage and tissue injury.
ABSTRACT—Less nitric oxide (NO)-dependent vasodilation and excess formation of reactive oxygen species could explain poor placenta perfusion in preeclampsia, but the pathways involved are unknown. We ...tested the hypothesis that reduced NO activity and increased oxidative stress in preeclamptic placenta is related to a low bioavailability of l-arginine. Placental endothelial NO synthase (ecNOS) expression (by immunoperoxidase) and activity (by diaphorase and HL-citrulline formation) were comparable in normotensive pregnancy and in preeclampsia, whereas nitrotyrosine staining, a marker of peroxynitrite, was stronger in preeclamptic villi, confirming previously reported data. Oxidative tissue damage was documented in preeclamptic villi by strong 4-hydroxynonenal-lysine staining (by immunoperoxidase), which closely colocalized with nitrotyrosine. Concentration of the NO precursor l-arginine (by HPLC) in umbilical blood and in villous tissue was lower in preeclampsia than in normotensive pregnancy. This was not caused by a defective l-arginine transport, because gene expression of the CAT-1, 4F2hc, and LAT-1 cationic amino acid transporters (by real-time reverse-transcription polymerase chain reaction RT-PCR) was normal. Instead, gene expression (by real-time RT-PCR) and protein tissue content (by immunoperoxidase and Western blot) of arginase II—the enzyme that degrades arginine to ornithine—were higher in preeclamptic villi than in normotensive pregnancy. These results provide a biochemical explanation for defective NO activity and increased oxidative stress in preeclamptic placenta. In normal placenta, adequate concentration of l-arginine orients ecNOS toward NO. In preeclampsia, a lower than normal l-arginine concentration caused by arginase II overexpression redirects ecNOS toward peroxynitrite.
Summary
Endothelial injury is the central factor in the events leading to thrombotic microangiopathy (TMA); however, the mechanisms involved are not fully understood. Here we investigate the role of ...neutrophils (PMNs) and of complement activation in inducing microvascular damage and loss of thromboresistance in TMA associated with ADAMTS-13 deficiency. PMNs isolated during the acute phase of the disease released excessive amounts of reactive-oxygen species (ROS), N-derived oxidants and proteinases and induced damage and thromboresistance loss in human microvascular endothelial cell line (HMEC-1)
ex vivo
. Endothelial cytotoxicity and thromboresistance loss was also induced by TMA serum. Complement-derived products were responsible for the above effects: in fact, TMA serum caused C3 and Membrane Attack Complex (MAC) deposition on HMEC-1 and its cytotoxic effect was abolished by complement inhibition. TMA serum caused surface expression of P-selectin on HMEC-1 which may promote PMN adhesion and resulted in increased PMN cytotoxicity, indicating that complement may have a role in PMN activation. In addition, TMA serum stimulated control PMNs to release ROS and proteinases, and to cause endothelial cell cytotoxicity. All of the above effects were abrogated by complement inactivation. These data document for the first time that complement-initiated PMN activation and endothelial injury may have a crucial role in microvascular thrombosis of TMA associated with ADAMTS-13 deficiency.
During hemodialysis, vascular reactivity is impaired, which can be corrected by lowering dialysate temperature. It has also been shown that nitric oxide (NO) is related to intradialytic hypotension. ...As NO synthesis may be temperature-dependent, this study addressed the influence of dialysate temperature on the NO synthetic capacity of plasma.
NO synthetic capacity was studied during hemodialysis with a dialysate temperature of 37.5 degrees C (dialysis-37.5 degrees C) and programmed extracorporeal blood cooling (cool dialysis; Blood Temperature Monitor; Fresenius C) in 12 stable patients. NO synthetic capacity was assessed ex vivo by 3HL-citrulline formation from 3HL-arginine in cultured endothelial cells after incubation with plasma samples obtained during the respective sessions.
Core temperature decreased (-0.32 +/- 0.10 degrees C) and energy transfer rate was significantly lower (-27.5 +/- 2.8 W; p < 0.05) during cool dialysis compared to dialysis-37.5 degrees C (0.19 +/- 0.06 degrees C and -0.8 +/- 1.2 W respectively; p < 0.05). Systolic blood pressure decreased during dialysis-37.5 degrees C (-19 +/- 4 mm Hg; p < 0.05), but not during cool dialysis (-6 +/- 5 mm Hg). NO synthetic capacity increased during dialysis-37.5 degrees C (55.5 +/- 9.3 to 73.5 +/- 10.2 pmol/10(5) cells; p < 0.05), in contrast to cool dialysis (67.3 +/- 11.1 to 66.2 +/- 10.8 pmol/10(5) cells).
The stimulatory effect of uremic plasma on endothelial NO synthesis was augmented during dialysis-37.5 degrees C but not during cool dialysis.
The existence of treatment-resistant cancer stem cells contributes to the aggressive phenotype of glioblastoma. However, the molecular alterations that drive stem cell proliferation in these tumors ...remain unknown. In this study, we found that expression of the MET oncogene was associated with neurospheres expressing the gene signature of mesenchymal and proneural subtypes of glioblastoma. Met expression was almost absent from neurospheres expressing the signature of the classical subtype and was mutually exclusive with amplification and expression of the EGF receptor (EGFR) gene. Met-positive and Met-negative neurospheres displayed distinct growth factor requirements, differentiated along divergent pathways, and generated tumors with distinctive features. The Met(high) subpopulation within Met-pos neurospheres displayed clonogenic potential and long-term self-renewal ability in vitro and enhanced growth kinetics in vivo. In Met(high) cells, the Met ligand HGF further sustained proliferation, clonogenicity, expression of self-renewal markers, migration, and invasion in vitro. Together, our findings suggest that Met is a functional marker of glioblastoma stem cells and a candidate target for identification and therapy of a subset of glioblastomas.
The prevalence of ANA and anti-ENA in the general population is not well established, especially their clinical significance in healthy subjects. We herein determined the prevalence and predictive ...value of serum ANA and anti-ENA for connective tissue diseases (CTD), cancer, and mortality. We took advantage of a randomly selected sample of the 1998 general population (Isola I) consisting of 2828 subjects (53% women, age 43±13 years) from a well-defined Northern Italian area. Serum ANA and anti-ENA were tested on the 2690 samples available in 2012 (Isola II, 50% women, age 58±13 years). Administrative databases were searched for CTD, cancer diagnosis, and death cases occurring between enrollment and December 31, 2013. The hazard ratio (HR) was calculated for incident cases. Serum ANA is positive in 18.1% for any titer and 6.1% for titers ≥1:160, 23% in subjects over 50 years and 13.1% and 6.1% for any titer and titers ≥1:160, respectively, in women. The HR for CTD development was significantly high for all ANA titers, with the highest for ANA ≥1:160 (HR 14.19, 95% CI 3.07–65.68). ANA positivity was not associated with cancer (HR 1.03; 95% CI 0.75–1.43), or with mortality (HR adjusted for age and sex 1.40; 95% CI 0.94–2.09). Serum anti-ENA is positive in a minority of subjects with highest figures for anti-nucleosome (1.9%), -histone (1.6%) and -PM/Scl (1.5%). In conclusion, serum ANA prevalence in the general population is highest in senior subjects and in women, while the female predominance is significantly lower compared to overt CTD. Serum ANA is associated with an increased probability of CTD development over time, but does not influence survival or cancer risk.
•Serum ANA is detected in 18.1% of the general population and the prevalence increases with age.•Serum ANA is associated with an increased risk of connective tissue disease over 15 years.•Serum ANA is not related to an increased risk of cancer over 15 years.•Serum ANA positivity is not associated with increased mortality over 15 years.
Abstract The prevalence of ANA and anti-ENA in the general population is not well established, especially their clinical significance in healthy subjects. We herein determined the prevalence and ...predictive value of serum ANA and anti-ENA for connective tissue diseases (CTD), cancer, and mortality. We took advantage of a randomly selected sample of the 1998 general population (Isola I) consisting of 2828 subjects (53% women, age 43 ± 13 years) from a well-defined Northern Italian area. Serum ANA and anti-ENA were tested on the 2690 samples available in 2012 (Isola II, 50% women, age 58 ± 13 years). Administrative databases were searched for CTD, cancer diagnosis, and death cases occurring between enrollment and December 31, 2013. The hazard ratio (HR) was calculated for incident cases. Serum ANA is positive in 18.1% for any titer and 6.1% for titers ≥ 1:160, 23% in subjects over 50 years and 13.1% and 6.1% for any titer and titers ≥ 1:160, respectively, in women. The HR for CTD development was significantly high for all ANA titers, with the highest for ANA ≥ 1:160 (HR 14.19, 95% CI 3.07–65.68). ANA positivity was not associated with cancer (HR 1.03; 95% CI 0.75–1.43), or with mortality (HR adjusted for age and sex 1.40; 95% CI 0.94–2.09). Serum anti-ENA is positive in a minority of subjects with highest figures for anti-nucleosome (1.9%), -histone (1.6%) and -PM/Scl (1.5%). In conclusion, serum ANA prevalence in the general population is highest in senior subjects and in women, while the female predominance is significantly lower compared to overt CTD. Serum ANA is associated with an increased probability of CTD development over time, but does not influence survival or cancer risk.
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