Two patients with history of STEC-HUS, posttransplant recurrence and complement gene mutations.

Hemolytic uremic syndrome (HUS) is a disease of microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. About 90% of cases are secondary to infections by Escherichia coli strains producing Shiga-like toxins (STEC-HUS), while 10% are associated with mutations in genes encoding proteins of complement system (aHUS). We describe two patients with a clinical history of STEC-HUS, who developed end-stage renal disease (ESRD) soon after disease onset. They received a kidney transplant but lost the graft for HUS recurrence, a complication more commonly observed in aHUS. Before planning a second renal transplantation, the two patients underwent genetic screening for aHUS-associated mutations that revealed the presence of a heterozygous CFI mutation in patient #1 and a heterozygous MCP mutation in patient #2, and also in her mother who donated the kidney. This finding argues that the two cases originally diagnosed as STEC-HUS had indeed aHUS triggered by STEC infection on a genetic background of impaired complement regulation. Complement gene sequencing should be performed before kidney transplantation in patients who developed ESRD following STEC-HUS since they may be undiagnosed cases of aHUS, at risk of posttransplant recurrence. Furthermore, genetic analysis of donors is mandatory before living-related transplantation to exclude carriers of HUS-predisposing mutations.

Crisponi syndrome: a new mutation in CRLF1 gene associated with moderate outcome.

SUMMARY: Crisponi syndrome (CS) is a rare, autosomal recessive disorder, characterized by hyperthermia, extensive muscular contractions in the face after even minimal stimuli or crying, hypertonia, opisthotonus, camptodactyly, and typical facial features. Recently, it has been demonstrated that CRLF1 (cytokine receptor-like factor 1) gene mutation is associated with CS. Here we report a case of CS with a new mutation in the CRLF1 gene associated with moderate clinical phenotype.

The cohort of the multiple sclerosis center of Cagliari.

We report our experience in long-term treatment of relapsing remitting multiple sclerosis patients with natalizumab (N). In November 2009 we evaluated 141 patients (126 AIFA criterion A, 15 AIFA criterion B). We paid particular attention to the treatment period and the patients follow-up; during the whole therapeutic program, they undergone to clinical and radiological evaluation for every 3 months. The compliance was optimal and we found no significant side effects. 26 patients completed the 24 monthly doses. After 24 months 51% of patients were free from disease activity. We found a reduction in relapses and EDSS, moreover the clinical improvement was also confirmed by radiological examinations. Our results show that the best therapeutic results are achieved by early initiation of treatment.

Sex-related differences in olfactory function and evaluation of possible confounding factors among patients with Parkinson's disease.

The role of specific sex-related patterns in olfactory dysfunctions of Parkinson's disease (PD) patients is unclear. The aim of this study was to assess the presence of specific sex-related patterns in olfactory dysfunctions excluding the possibility of confounding effects in patients with Parkinson's disease. One hundred and sixty-eight participants (99 PD patients and 69 controls) were enrolled and evaluated using Sniffin' Sticks Extended test (SSET). There was no significant sex difference in the control group for the SSET parameters. By contrast, in the PD group male patients scored significantly lower on odor discrimination (OD), identification (OI), and Threshold-Discrimination-Identification (TDI) score than females. On multivariable linear regression analysis, the only significant predictors of TDI score were sex and apathy. Among PD patients, men showed a significantly greater impairment compared to women in OI, OD and TDI score, but not in odor threshold (OT). These findings highlighted the possible role of sex differences in the development of associated PD non-motor symptoms.

Treatment with gut bifidobacteria improves hippocampal plasticity and cognitive behavior in adult healthy rats.

At the present time, gut microbiota inspires great interest in the field of neuroscience as a function of its role in normal physiology and involvement in brain function. This aspect suggests a specific gut-brain pathway, mainly modulated by gut microbiota activity. Among the multiple actions controlled by microbiota at the brain level, neuronal plasticity and cognitive function represent two of the most interesting aspects of this cross-talk communication. We address the possible action of two-months implementation of gut Bifidobacteria using a mixture of three different strains (B-MIX) on hippocampal plasticity and related cognitive behavior in adult healthy Sprague Dawley rats. B-MIX treatment increases the hippocampal BDNF with a parallel gain in dendritic spines' density of hippocampal CA1 pyramidal neurons. Electrophysiological experiments revealed a significant increment of HFS-induced LTP formation on the CA1 hippocampal region in B-MIX treated rats. All these effects are accompanied by a better cognitive performance observed in B-MIX treated animals with no impairments in locomotion activity. Therefore, in adult rats, the treatment with different strains of bifidobacteria is able to markedly enhance neuronal plasticity and the CNS function influencing cognitive behavior, an effect that may suggest a potential therapeutic treatment in brain diseases associated with cognitive functions.

Cure of short- and long-term experimental Chagas' disease using D0870.

Chagas' disease, a protozoan infection by the kinetoplastid Trypanosoma cruzi, constitutes a major public health problem in Latin America. With the use of mouse models of both short- and long-term forms of the disease, the efficacy of D0870, a bis-triazole derivative, was tested. D0870 was able to prevent death and induced parasitological cure in 70 to 90 percent of animals, in both the short- and long-term disease. In contrast, currently used drugs such as nifurtimox or ketoconazole prolonged survival but did not induce significant curing effects. D0870 may be useful in the treatment of human long-term Chagas' disease, a condition that is currently incurable.

Thrombotic microangiopathy without renal involvement: two novel mutations in complement-regulator genes.

UNLABELLED: ESSENTIALS: The differential diagnosis among thrombotic microangiopathies (TMAs) is challenging. We studied a case of TMA with neurologic symptoms, no renal impairment and normal ADAMTS-13 levels. Two novel mutations in complement factor I and thrombomodulin genes were identified. Complement-regulator genes can be involved in TMAs with normal ADAMTS-13 regardless of renal damage. BACKGROUND: Thrombotic microangiopathies (TMAs) often represent a challenge for clinicians, because clinical, laboratory, and even genetic features are not always sufficient to distinguish among different TMAs. OBJECTIVES: The aim of this study was to investigate the pathogenetic mechanisms underlying an acute case of TMA with features of both thrombotic thrombocytopenic purpura (TTP) and atypical hemolytic uremic syndrome (aHUS). PATIENTS/METHODS: We report the case of a 49-year-old woman who developed an acute TMA with neurologic involvement and no renal impairment. ADAMTS-13, von Willebrand factor, and complement-system biochemical characterization was performed on acute phase samples. Exome sequencing and direct Sanger sequencing of previously aHUS-associated genes were performed. The functional consequences of the thrombomodulin (THBD) mutation were investigated by in vitro expression studies. RESULTS: Despite a clinical diagnosis of TTP, the patient had normal ADAMTS-13 levels and increased VWF antigen levels with ultra-large von Willebrand factor multimers. C3, C4, and complement factors H and I (CFI) were normal. Molecular analysis confirmed two novel heterozygous mutations in CFI (c.805G>A, p.G269S) and THBD (c.1103C>T, p.P368L), and in vitro expression studies showed a reduction in the generation of activated thrombin-activatable fibrinolysis inhibitor (TAFIa) caused by mutated THBD. This proinflammatory condition, associated with the p.G269S mutation in CFI, probably leads to a complement-mediated endothelial activation, with a relevant prothrombotic potential in case of transient environmental triggers. CONCLUSIONS: This study identified the first case of acute TMA without renal involvement but with neurological damage carrying two novel mutations in complement-regulator genes, highlighting the possible role of the complement system as a common pathogenetic mechanism in TMAs.

PEN-2 gene mutation in a familial Alzheimer's disease case.

Genetic evidence indicates a central role of cerebral accumulation of beta-amyloid (Abeta) in the pathogenesis of Alzheimer's disease (AD). Beside presenilin 1 and 2, three other recently discovered proteins (Aph 1, PEN 2 and nicastrin) are associated with gamma-secretase activity, the enzymatic complex generating Abeta. Alterations in genes encoding these proteins were candidates for a role in AD. The PEN 2 gene was examined for unknown mutations and polymorphisms in sporadic and familial Alzheimer patients. Samples from age-matched controls (n=253), sporadic AD (SAD, n=256) and familial AD (FAD, n=140) were screened with DHPLC methodology followed by sequencing. Scanning the gene identified for the first time a missense mutation (D90N) in a patient with FAD. Three intronic polymorphisms were also identified, one of which had a higher presence of the mutated allele in AD subjects carrying the allele epsilon4 of apolipoprotein E than controls. The pathogenic role of the PEN-2 D90N mutation in AD is not clear, but the findings might lead to new studies on its functional and genetic role.

The effect of fetuin and other sialoglycoproteins on the in vitro penetration of Trypanosoma cruzi trypomastigotes into fibroblastic cells.

Heat-inactivated calf-, human-, and especially fetal calf serum stimulate infection of Vero cells by cell culture-derived trypomastigotes of Trypanosoma cruzi: the stimulatory effect is more marked with extracellular activated parasites or trypsinized trypomastigotes than with recently released parasites. The augmented invasion is not the consequence of a stimulation of attachment of trypomastigotes to host cells. Various sialoglycoproteins like fetuin, transferrin, fibrinogen, alpha-1-antitrypsin, mucin and goat-IgG are also effective in enhancing in vitro infectivity. Colominic acid also stimulates invasion, but other non-sialic polyanionic compounds are either ineffective (chondroitin sulfate, poly-aspartic acid) or inhibitory (heparin, phytic acid, myo-inositol hexasulfate). Fetuin, the best stimulatory compound tested, gives half-maximal activation with approximately 0.03 mg ml-1, and total activation with 0.5-1 mg ml-1. The enhancement of infectivity is time-dependent (2-3 h for maximal activation) at 37 degrees C and does not occur at 0 degrees C. Desialidated-fetuin or -fetal calf serum do not stimulate infectivity at all. Treatment with fetuin of parasites alone (or Vero cells alone), followed by removal of free fetuin and by interaction with untreated Vero cells (or parasites) indicates that the stimulation effect of fetuin occurs mainly on the trypomastigotes. No specific binding of [125I]fetuin to the parasites could be demonstrated, and incubation with exogenous neuraminidase of trypomastigotes previously activated by fetuin, reverses nearly completely the stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of inhibitors of macromolecular biosynthesis on the in vitro infectivity and morphology of Trypanosoma cruzi trypomastigotes.

The effect of inhibitors of RNA, protein, and glycoprotein biosynthesis has been studied on the development of in vitro infectivity and the transformation to spheromastigotes occurring when recently isolated trypomastigotes of Trypanosoma cruzi are incubated extracellularly. Puromycin (1 microgram/ml) blocks the development of parasite adhesion and penetration of Vero cells, as well as the transformation. Actinomycin D (8 ng/ml) and tunicamycin (30 ng/ml) inhibit completely the development of infectivity, without blocking adhesion and transformation. The last two parameters are inhibited by higher actinomycin D concentrations, but are unaffected by tunicamycin. The results obtained suggest that a parasite glycoprotein is involved in the penetration step of T. cruzi trypomastigotes into fibroblastic cells, and that adhesion, penetration, and transformation to spheromastigotes are three different processes, each one of them requiring de novo synthesis of distinct proteins.

The effect of surface membrane modifications of fibroblastic cells on the entry process of Trypanosoma cruzi trypomastigotes.

Treatment prior to infection with Trypanosoma cruzi trypomastigotes of Vero, MA-103, and chick muscle cells with concanavalin A, phytohemagglutinin, wheat germ agglutinin and ricin I results in a diminished parasite interiorization in these cells; succinylated concanavalin A is also inhibitory. The effect of these lectins is abolished by the corresponding sugar haptens. Trypsin and periodate treatment of the cells also inhibits infection, as well as calcium ionophore A23187 and drugs that disrupt microtubules and microfilaments directly, like colchicine, vinblastine and cytochalasin B. These results show that alteration(s) of a surface glycoprotein(s) and/or of the plasma membrane architecture of fibroblastic host cells inhibit infection, suggesting that the surface membrane of these cells does not play a passive role in the process of infection by T. cruzi.

The effect of proteolytic enzymes and protease inhibitors on the interaction Trypanosoma cruzi-fibroblasts.

It has been shown previously that the capability to adhere to and infect fibroblastic cells by Trypanosoma cruzi is expressed only partially in trypomastigotes recently liberated from infected fibroblasts, but these parasites can increase several-fold their adhesion and infectivity by a time-dependent extracellular incubation. It is now shown that polyacrylamide gel electrophoresis patterns of 125I-labelled surface proteins of the parasites change during the activation process and that protease inhibitors of diverse specificity can block both these changes and the development of adhesion and infectivity. Treatment of fresh trypomastigotes with different proteases increases immediately adhesion and infection. The effect of trypsin has been studied in detail and it was found that this protease stimulates adhesion 4- to 6-fold, even in trypomastigotes obtained and assayed in the absence of serum. Trypomastigotes incubated for various periods and then exposed to trypsin increase their adhesion to values similar to those attained by prolonged incubation of trypomastigotes alone, but infection is stimulated in fresh trypomastigotes only. Trypomastigotes whose development of activation has been inhibited either by protease inhibitors, puromycin, and tunicamycin, and are thereafter trypsinized, show respectively, that: adhesion and infection are restored immediately to the same high values obtained when untreated controls are trypsinized, adhesion is restored, but not infection, and infection is not restored. These results suggest that the adhesion step of T. cruzi trypomastigotes to fibroblastic cells depends on a membrane protein(s) that is (are) already present in an inactive or hidden form in parasites recently liberated from infected fibroblasts. Upon extracellular maturation of these trypomastigotes this proteins(s) is activated or unmasked, probably through an endogenous proteolytic process, whose expression requires protein synthesis. The penetration step requires biosynthesis of a tunicamycin-sensitive glycoprotein(s) of the parasite and its full expression necessitates serum.

The interaction of a Trypanosoma cruzi surface protein with Vero cells and its relationship with parasite adhesion.

Previous studies have shown that adhesion to fibroblastic cells of cell culture-derived trypomastigotes of Trypanosoma cruzi probably occurs through a ligand-receptor interaction. The results now obtained indicate that solubilization with a mild detergent ('Chaps', 0.8%) of 125I-surface proteins of trypomastigotes, followed by detergent removal and interaction of the solubilized proteins with a monolayer of intact Vero cells, brings about binding to the cells of a parasite surface protein, which exhibits a molecular weight of 83,000 and isoelectric point of 8.1-8.6 upon two-dimensional polyacrylamide gel electrophoresis. This polypeptide was detected in extracts of highly adherent, extracellularly incubated parasites, but not in extracts of poorly adhesive, recently released trypomastigotes. The detergent-free extracts of incubated trypomastigotes inhibit attachment of live parasites to Vero cells, while extracts of fresh trypomastigotes are nearly ineffective. Binding of the parasite polypeptide to the cells is stimulated by parasite trypsinization or activation in the presence of tunicamycin, and it is inhibited by the presence of mannan or by Vero cell trypsinization, thus showing a similar behaviour to that observed for parasite attachment to Vero cells under these conditions. These results suggest that the surface membranes of activated, highly adherent T. cruzi trypomastigotes contain an 83 kDa polypeptide which acts as a lectin-like protein that can interact with the surface of Vero fibroblasts, probably through mannose residues of a glycoprotein receptor of the host cell.

Changes in morphology and infectivity of cell culture-derived trypomastigotes of Trypanosoma cruzi.

Trypomastigotes of Trypanosoma cruzi, obtained from the first burst of infected Vero cells, have only a limited invasive capability for fibroblastic cells. Intracellular amastigotes and epimastigotes do not infect these cells at all. Preincubation of the isolated trypomastigotes with Eagle's minimal essential medium/10% fetal calf serum increases 5- to 15-fold their in vitro infectivity. This increased invasive capability is accompanied, in the case of the EP strain of T. cruzi, by a morphological transformation into an amastigote-like or spheromastigote form, which is similar, but not identical to replicating intracellular amastigotes. Trypomastigotes from another isolate (BEC) also increase their infectivity several fold upon preincubation, but before any morphological differentiation occurs, suggesting that these two events are independent. The phenomenon of increased infective capability of the parasite is expressed similarly in different host cells. Parasite adhesion is stimulated 4- to 12-fold upon preincubation of the trypomastigotes. The type of serum used (fetal calf, calf, human) affects the development of infectivity, as well as the process of cell infection in itself, but not the morphological differentiation. These processes are also temperature-dependent. The highly infective parasitic forms do not synthetize DNA, but are active in RNA and protein synthesis. The results obtained indicate the existence in T. cruzi trypomastigotes of an active system for infecting fibroblastic cells, which is only partially expressed in trypomastigotes recently released from host fibroblasts but which can undergo a further extracellular maturation, thus allowing studies on the mechanism of infection in cell-free media.

Possible role of cAMP in the differentiation of Trypanosoma cruzi.

To assess the possible action of cAMP on the cell differentiation of Trypanosoma cruzi, we determined both cAMP levels and cAMP-binding activities of epimastigotes and trypomastigotes of this parasite. Trypomastigotes showed a 4-fold higher cAMP content and a 2.5-fold increase in the specific activity of a cAMP-binding protein with identical properties to that of epimastigotes. The high levels of cAMP present in trypomastigotes strongly suggest a role of this cyclic nucleotide on the differentiation of T. cruzi.

Sterol composition and biosynthesis in Trypanosoma cruzi amastigotes.

A detailed analysis of the endogenous sterols present in the clinically relevant intracellular (amastigote) stages of Trypanosoma cruzi, is presented. The parasites were grown in cultured Vero cells in the absence or presence of different sterol biosynthesis inhibitors, including the C14alpha demethylase inhibitor ketoconazole and two inhibitors of delta24(25)-sterol methyl transferase, 20 piperidin-2-yl-5alpha-pregnan-3beta-20-R-diol (22,26-azasterol) and 24-(R,S),25-epiminolanosterol. Amastigotes were isolated and purified from their host cells and neutral lipids were extracted, separated and analyzed by chromatographic and mass spectrometric methods. Control (untreated) amastigotes contained as main endogenous sterols 24-methyl-cholesta-7-en-3beta-ol (ergosta-7-en-3beta-ol) and its 24-ethyl analog, plus smaller amounts of their precursor, ergosta-7,24(28)dien-3beta-ol; these cells also contained cholesterol (up to 80% by weight of total sterols), probably derived from host cells. Amastigotes that proliferated in the presence of 10 nM ketoconazole (minimal inhibitory concentration, MIC) for 24 h had a sharply reduced content of endogenous 4-desmethyl sterols with a concomitant accumulation of 24-methyl-dihydrolanosterol and 24-methylene-dihydrolanosterol. On the other hand, amastigotes incubated during the same period of time with the two inhibitors of 24(25)-SMT at their respective MICs (100-300 nM) accumulated large amounts of C27 sterols whose structure suggested, in the case of 22,26-azasterol, that delta14 sterol reductase was also inhibited. Ketoconazole produced a dose-dependent reduction in the incorporation of [2-(14)C]-acetate into the parasite's endogenous C4-desmethyl sterols with an IC50 of 50 nM, indistinguishable from the value reported previously for the extracellular epimastigote form. Taken together, the results showed that amastigotes have a simpler sterol biosynthetic pathway than that previously described for epimastigotes, lacking both delta5 and delta22 reductases. They also suggest that the 100-fold higher potency of antifungal azoles as antiproliferative agents against amastigotes, when compared with epimastigotes, is most probably due to a smaller pool of endogenous sterols in the intracellular parasites.

Trypanosoma cruzi: polypeptide markers of epimastigotes and trypomastigotes.

We compared the major polypeptides of epimastigotes and trypomastigotes of T. cruzi, by submitting total parasite lysates to electrophoresis in polyacrylamide gels (SDS-PAGE), protein staining with Coomassie brilliant blue, laser densitometry, or immunoblotting with sera derived from infected individuals (Chagas' disease). Epimastigotes and trypomastigotes displayed extensive homology, the differences being quantitative, except for a trypomastigote-specific band of Mr 75,000 which reacted with chagasic sera. Immunoblotting with chagasic sera confirmed the electrophoretic homology of epimastigotes and trypomastigotes. Upon antigenic dilution, a cluster of antigenic bands in the range of Mr 150,000 to 75,000 prevailed in the trypomastigotes, whereas the epimastigotes displayed more abundance of antigenic bands in the range of Mr 72,000 to 36,000.

Inhibition of phosphatidylcholine biosynthesis and cell proliferation in Trypanosoma cruzi by ajoene, an antiplatelet compound isolated from garlic.

Ajoene [(E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide], a potent antiplatelet compound derived from garlic, inhibits the proliferation of both epimastigotes and amastigotes of Trypanosoma cruzi, the causative agent of Chagas' disease. The growth of the epimastigote form was immediately arrested by 80 microM ajoene, while 100 microM induced cell lysis in 24 hr. In the amastigote form proliferating inside VERO cells, 40 microM ajoene was sufficient to eradicate the parasite from the host cells in 96 hr. Growth inhibition of the epimastigotes was accompanied by a gross alteration of the phospholipid composition of the treated cells in which phosphatidylcholine (PC), the major phospholipid class present in control cells, dropped to the least abundant phospholipid in cells treated with 60 microM ajoene for 96 hr, while its immediate precursor, phosphatidylethanolamine (PE), became the predominant species; this was correlated with a marked drop in the incorporation of [14C-U]acetate in PC and a corresponding increase in PE. Concomitant with the change in the phospholipid headgroup composition of the cells, the fatty acids esterified to this lipid fraction underwent a dramatic alteration due to the increase in the content of saturated fatty acids and a marked reduction in the content of linoleic (18:2) acid, which is the predominant fatty acid in control cells. We also found that ajoene inhibited the de novo synthesis of neutral lipids and, in particular, of sterols in the epimastigotes, but the resultant changes in the sterol composition were not sufficient to explain the antiproliferative effects of the drug. Electron-microscopy showed a concentration-dependent alteration of intracellular membranous structures, particularly the mitochondrion and endoplasmatic reticulum. The results suggest that one important factor associated with the antiproliferative effects of ajoene against T. cruzi is its specific alteration of the phospholipid composition of these cells.

Synthesis and antitumor evaluation of 6-thioxo-, 6-oxo- and 2,4-dioxopyrimidine derivatives.

A series of 6-thioxopyrimidines (5, 6), their 6-oxo- analogs (11-14), and pyrimidine-2,4-diones (20-26), were synthesized and evaluated for their antitumoral activity against 60 tumoral cell lines. The activity of propenethioamide (3, 4) and propeneamide (7-10 and 15-19) intermediates is also reported. Among the tested compounds the thioxopyrimidine 5c, bearing an N'-benzyl group, showed the best cytostatic activity. Furthermore, high selectivity and cytotoxic activity on the HOP-92 cell line of non-small cell lung cancer was exhibited by 3-amino-2-[(methylamino)thioxamethyl]-3-pyrrolidino-2-propenenitrile (3a).