Inflammatory and oxidative stress biomarkers in alkaptonuria: data from the DevelopAKUre project.

OBJECTIVE: The aim of this work was to assess baseline serum levels of established biomarkers related to inflammation and oxidative stress in samples from alkaptonuric subjects enrolled in SONIA1 (n = 40) and SONIA2 (n = 138) clinical trials (DevelopAKUre project). METHODS: Baseline serum levels of Serum Amyloid A (SAA), IL-6, IL-1ß, TNFα, CRP, cathepsin D (CATD), IL-1ra, and MMP-3 were determined through commercial ELISA assays. Chitotriosidase activity was assessed through a fluorimetric method. Advanced Oxidation Protein Products (AOPP) were determined by spectrophotometry. Thiols, S-thiolated proteins and Protein Thiolation Index (PTI) were determined by spectrophotometry and HPLC. Patients' quality of life was assessed through validated questionnaires. RESULTS: We found that SAA serum levels were significantly increased compared to reference threshold in 57.5% and 86% of SONIA1 and SONIA2 samples, respectively. Similarly, chitotriosidase activity was above the reference threshold in half of SONIA2 samples, whereas CRP levels were increased only in a minority of samples. CATD, IL-1ß, IL-6, TNFα, MMP-3, AOPP, thiols, S-thiolated protein and PTI showed no statistically significant differences from control population. We provided evidence that alkaptonuric patients presenting with significantly higher SAA, chitotriosidase activity and PTI reported more often a decreased quality of life. This suggests that worsening of symptoms in alkaptonuria (AKU) is paralleled by increased inflammation and oxidative stress, which might play a role in disease progression. CONCLUSIONS: Monitoring of SAA may be suggested in AKU to evaluate inflammation. Though further evidence is needed, SAA, chitotriosidase activity and PTI might be proposed as disease activity markers in AKU.

Local climate explains degree of seed dormancy in Hypericum elodes L. (Hypericaceae).

Seed dormancy and germination characteristics may vary within species in response to several factors. Knowledge of such variation is crucial to understand plant evolution and adaptation to environmental changes. We examined the correlation of climate and population genetic differentiation (ISSR) with primary seed dormancy and germination behaviour in populations of the Atlantic-European soft-water pool specialist Hypericum elodes. Primary dormancy was measured by analysing seed germination response of fresh seeds and after various periods of cold stratification. Laboratory germination experiments revealed that the single most important factor for promoting germination was cold stratification prior to placing at the germination temperature. However, in agreement with their weaker primary dormancy, the seeds germinated well when fresh, and the benefit of cold stratification was more relaxed for the southern populations. Seeds of all populations demonstrated a near absolute requirement for a light and alternating temperature regime in order to germinate. The promoting effect of alternating temperatures was particularly effective at warm temperatures (mean 20 °C) but not at cool temperatures. Whilst seed germination requirements were similar among populations, the degree of primary dormancy varied considerably and was not associated with population genetic differentiation. Primary dormancy degree was instead associated with local climate: higher temperature in summer and rainfall in winter predicted weak and rapid loss of dormancy. These results suggest that seed maturation environment may play a substantial role in explaining the degree of dormancy in H. elodes, highlighting that physiological dormancy can be modulated by local climate.

Seed dormancy and germination in three Crocus ser. Verni species (Iridaceae): implications for evolution of dormancy within the genus.

The aim of this work was to examine whether seed ecophysiological traits in three closely related Crocus species were associated with ecological niche differentiation and species divergence. Seeds of the temperate tetraploid cytotype of Crocus neapolitanus, the sub-Mediterranean C. etruscus and the Mediterranean C. ilvensis were placed either on agar in the laboratory under different periods of simulated seasonal conditions or in nylon mesh bags buried outdoors to examine embryo growth, radicle and shoot emergence. In agreement with the phenology observed outdoors, in the laboratory embryos required a cool temperature (ca. 10 °C) to grow to full size (embryo length:seed length, E:S ratio ca. 0.75) but only after seeds received a warm stratification; radicle emergence then followed immediately (November). Shoot emergence is a temporally separated phase (March) that was promoted by cold stratification in C. neapolitanus while in the other two species this time lag was attributed to a slow continuous developmental process. These species have similar embryo growth and radicle phenology but differ in their degree of epicotyl dormancy, which is related to the length of local winter. Conclusions from laboratory experiments that only consider root emergence could be misleading; evaluating the phenology of both root and shoot emergence should be considered in order to demonstrate ecologically meaningful differences in germination behaviour and to develop effective propagation protocols. Although these taxa resulted from recent speciation processes, the outcomes suggest an early onset of adaptation to local ecological factors and that phylogeny may represent a significant constraint in the evolution and expression of seed traits in Crocus.

Pretreatment neuropsychological deficits in children with brain tumors.

Treatment of childhood brain cancer has been associated with long-term cognitive morbidity in children. In the present study, the cognitive status of children with brain tumors was examined prior to any treatment to single out the role of tumor and tumor-related factors in cognitive deficits. Eighty-three children with newly diagnosed brain tumors (mean age, 8.6 years; range, 7 months to 16.6 years; median, 9.4 years) completed an extensive battery of age-related tests to assess cognitive function before any therapeutic intervention. Magnetic resonance imaging (MRI) was used to determine tumor site and volume and tumor-related factors. Performance under test was compared with symptom duration, neurological status, epilepsy, and MRI. Cognitive difficulties are detected at diagnosis in as many as 50% of patients for some cognitive domains; 6% of patients present with true-diagnosed mental retardation. The location of the tumor is the principal determinant of cognitive deficits, with major impairment in children with cortical tumors. Symptom duration and the presence of epilepsy are significantly associated with neuropsychological disabilities, while neuroradiological tumor-related variables do not correlate clearly with neurocognitive performance. The knowledge of the pre-existing cognitive deficits is critical to evaluate the results of treatment, providing a baseline for assessing the true impact of therapy in determining cognitive decline. In addition, the study suggests that some clinical variables require careful monitoring, because they could be specifically implicated in the neuropsychological outcome; the efforts to reduce the impact of these factors could ameliorate long-term prognosis.

Oxidative stress in IgA nephropathy.

IgA nephropathy (IgAN) is characterized by mesangial deposits of IgA1, likely due to accumulation of IgA immune complexes. The activation of intracellular signaling mostly results in oxidative stress, as detected in mesangial cells cultured with aberrantly glycosylated IgA or IgA aggregates and in renal biopsies of patients with IgAN. Signs of altered oxidation/antioxidation balance have been detected in sera and/or in erythrocytes of patients with IgAN, including increased levels of lipoperoxide or malondialdehyde and reduced activity of superoxide dismutase, catalase and glutathione peroxidase. Moreover, increased levels of a marker of oxidative stress, advanced oxidation protein products (AOPPs), have been reported to be significantly associated with proteinuria and disease progression in patients with IgAN. AOPPs are often carried by albumin and can in turn enhance the oxidative stress in the circulation. Recent research suggests that the nephrotoxicity of aberrantly glycosylated IgA1 in IgAN is enhanced in the presence of systemic signs of oxidative stress, and it is tempting to hypothesize that the level of the oxidative milieu conditions the different expression and progression of IgAN.

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 m(2)/day in association with severe microscopic haematuria) and inactive patients (proteinuria < 0.5 g/1.73 m(2)/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.

Investigating genetic diversity and habitat dynamics in Plantago brutia (Plantaginaceae), implications for the management of narrow endemics in Mediterranean mountain pastures.

Many factors have contributed to the richness of narrow endemics in the Mediterranean, including long-lasting human impact on pristine landscapes. The abandonment of traditional land-use practices is causing forest recovery throughout the Mediterranean mountains, by increasing reduction and fragmentation of open habitats. We investigated the population genetic structure and habitat dynamics of Plantago brutia Ten., a narrow endemic in mountain pastures of S Italy. Some plants were cultivated in the botanical garden to explore the species' breeding system. Genetic diversity was evaluated based on inter-simple sequence repeat (ISSR) polymorphisms in 150 individuals from most of known stands. Recent dynamics in the species habitat were checked over a 14-year period. Flower phenology, stigma receptivity and experimental pollinations revealed protogyny and self-incompatibility. With the exception of very small and isolated populations, high genetic diversity was found at the species and population level. amova revealed weak differentiation among populations, and the Mantel test suggested absence of isolation-by-distance. Multivariate analysis of population and genetic data distinguished the populations based on genetic richness, size and isolation. Landscape analyses confirmed recent reduction and isolation of potentially suitable habitats. Low selfing, recent isolation and probable seed exchange may have preserved P. brutia populations from higher loss of genetic diversity. Nonetheless, data related to very small populations suggest that this species may suffer further fragmentation and isolation. To preserve most of the species' genetic richness, future management efforts should consider the large and isolated populations recognised in our analyses.

Chromosome diversity and evolution in Liliaceae.

BACKGROUND AND AIMS: There is an extensive literature on the diversity of karyotypes found in genera within Liliaceae, but there has been no attempt to analyse these data within a robust phylogenetic framework. In part this has been due to a lack of consensus on which genera comprise Liliaceae and the relationships between them. Recently, however, this changed with the proposal for a relatively broad circumscription of Liliaceae comprising 15 genera and an improved understanding of the evolutionary relationships between them. Thus there is now the opportunity to examine patterns and trends in chromosome evolution across the family as a whole. METHODS: Based on an extensive literature survey, karyo-morphometric features for 217 species belonging to all genera in Liliaceae sensu the APG (Angiosperm Phylogeny Group) were obtained. Included in the data set were basic chromosome number, ploidy, chromosome total haploid length (THL) and 13 different measures of karyotype asymmetry. In addition, genome size estimates for all species studied were inferred from THLs using a power regression model constructed from the data set. Trends in karyotype evolution were analysed by superimposing the karyological data onto a phylogenetic framework for Liliaceae. KEY RESULTS AND CONCLUSIONS: Combining the large amount of data enabled mean karyotypes to be produced, highlighting marked differences in karyotype structure between the 15 genera. Further differences were noted when various parameters for analysing karyotype asymmetry were assessed. By examining the effects of increasing genome size on karyotype asymmetry, it was shown that in many but not all (e.g. Fritillaria and all of Tulipeae) species, the additional DNA was added preferentially to the long arms of the shorter chromosomes rather than being distributed across the whole karyotype. This unequal pattern of DNA addition is novel, contrasting with the equal and proportional patterns of DNA increase previously reported. Overall, the large-scale analyses of karyotype features within a well-supported phylogenetic framework enabled the most likely patterns of chromosome evolution in Liliaceae to be reconstructed, highlighting diverse modes of karyotype evolution, even within this comparatively small monocot family.

[Non-immunological therapy in nephropathies leading to chronic renal failure and in post-transplant chronic renal dysfunction]. / La terapia non immunologica delle nefropatie con insufficienza renale cronica e della disfunzione cronica da trapianto.

With its aging population, the Western world is experiencing a significant increase in the prevalence of chronic kidney disease, which has actually been defined as ''pandemic''. The high mortality and comorbidity, especially in terms of cardiovascular disease, have led to a significant increase in hospitalizations and rising public health expenditure, setting a trend that will become unsustainable in the next decades, even in the most developed countries. These epidemiological data have underlined the need for prevention campaigns and urged researchers and clinicians to develop new and more specific treatments able to slow down the progression of chronic kidney disease towards dialysis. To obtain such results, a deeper understanding of the pathogenetic mechanisms of nephropathy progression is mandatory. Once sclerosis is established and the initial pathogenetic noxa, whether or not involving the glomeruli, has been extinguished, the sclerotic progression of different nephropathies follows a standard pathway, irrespective of the initial cause. The achievement of an effective therapy for this condition, in native kidneys as well as transplanted organs, is the third-millennium challenge for nephrologists. In this review we will first describe the main risk factors and pathogenetic mechanisms involved in nephropathy progression and then discuss the results obtained with drugs that basic research has identified as potentially useful in experimental animal models as well as rigorous clinical trials.

Early activation of the hypothalamic-pituitary-adrenal-axis in very-low-birth-weight infants with small thymus at birth.

BACKGROUND: An acute thymic involution in human fetuses and newborns has been described in very-low-birth-weight (VLBW) infants with histological chorioamnionitis. However, the mechanisms of thymic involution remain to be clarified. Here, we tested the hypothesis that an activation of the hypothalamic-pituitary-adrenal (HPA) axis occurs in VLBW infants with acute thymic involution at birth. METHODS: A total of 180 randomly selected VLBW newborns (28.8 +/- 3.15 wk gestation; 1093 +/- 305 g) entered the study. Thymic size was measured on standard chest radiographs at birth, and expressed as the ratio between the transverse diameter of the cardiothymic image at the level of the carina (CT) and that of the thorax (T). CT/T < 0.28 was considered to indicate a small thymic size. Plasma cortisol and adrenocorticotropic hormone (ACTH) concentrations were determined on days 1 (d-1) and 7 (d-7), and at 1 month (mo-1). Results. A total of 66 (36.7%) newborns had CT/T < 0.28. Infants with small thymus had significantly increased cortisol on d-1 ( approximately 5.2-folds) [median: 18.95 (95% CI: 11.20-39.4) microg/dl vs. 3.66 (1.94-6.82) microg/dl, p < 0.0001)] and d-7( approximately 1.7-folds) [12.0 (4.39-22.97) microg/dl vs. 7.8 (3.63-12.8) microg/dl, p = 0.0384)], as compared with those with normal thymic size, together with higher adrenocorticotropic hormone (ACTH) concentrations on d-1 ( approximately 1.9-folds) [28 (15.6-61.07) pg/ml vs. 14.9 (9.0-23.42) pg/ml, p = 0.0005)], while no significant differences for cortisol at mo-1 or ACTH concentrations on d-7 and mo-1 were evidenced (p > 0.50). From a multivariate logistic regression analysis, a small thymus at birth was a significant independent predictor of plasma cortisol concentrations in the top-quartile (OR = 14.4; 95% CI: 6.079-34.11), and plasma ACTH concentrations in the top-quartile (OR = 4.40 (95% CI: 1.99-9.74) on d-1 (results adjusted for variables significant at univariate analysis). CONCLUSIONS: Our data indicated the presence of a previously unrecognized, early activation of the HPA axis in VLBW newborns with a small thymus at birth.

Angiotensin antagonists and fish oil for treating IgA nephropathy.

In IgA nephropathy (IgAN), ACE inhibitors (ACE-I) and angiotensin receptor blockers (ARB) are beneficial against hypertension, and their anti-proteinuric effect has been clearly demonstrated. However, sub-analyses of IgAN patients enrolled in large studies failed to prove a benefit against progression to renal failure. The European Community Biomed Concerted Action - a placebo-controlled randomized controlled trial begun in 1995 - in children and adults (9-35 years old) with proteinuria > 1 < 3.5 g/day/1.73 m(2) and normal or moderately reduced renal function proved the significant benefit of ACE-I on progression of kidney disease. The combination of ACE-I and ARB in proteinuric normotensive IgAN patients showed greater antiproteinuric effect and the COOPERATE trial also reported a superior effect of combination therapy in protecting against renal function deterioration. Treating IgAN with fish oil has a good rationale for renal inflammation as well as for prevention of cardiovascular morbidity. However, the published reports gave conflicting conclusions and also very recent data did not show significant benefits. In conclusion, ACE-I and ARB have a definite role in treating IgAN, particularly the hypertensive and proteinuric forms. These patients should be treated to target BP to <130/70 mm Hg and proteinuria <0.5 g/day.

[Kidney transplantation in children]. / Il trapianto di rene nel bambino.

Indications, procedures, complications, pharmacokinetics and outcomes of renal transplantation are different in children and in adults. Subjects <18 yrs old, are often included in a unique list as in Italy, benefiting from donors <15 yrs old, and the waiting time is reduced to <12 months in 71% of cases. The risk of thrombosis limits the use of donors <2 yrs and trans-plantation in infants <1 yr. The age at kidney transplantation is <5 yrs in 20-30% of children. In Italy living-related trans-plantation (LRT) is performed in 7% of cases, while in the USA it is more common (57%) and is often pre-emptive before entering dialysis (24%). Current therapy tends to reduce steroid treatment doses and, optimizing induction therapy with IL-2R inhibitors, using tacrolimus or mycophenolate or sirolimus. Transplanted patient survival is better in children than in adults (94-98% at 5 yrs). Infections, cardiovascular diseases and neoplasia induce 34, 15 and 12% of deaths, respectively, at 10 yrs; morbidity for infections and lymphoproliferative disease is increasing. Acute rejections declined from 70% in 1987 to 31% in 2002 in cadaveric transplantation (CT) and renal survival at 3 yrs increased from 50% in 1985 to 82% for CT and up to 92% in LRT. In adolescents (11-17 yrs old) renal survival is lower than in infants and in adults <65 yrs old. Renal losses are due to chronic transplant nephropathy (32%), vascular thrombosis (13%) and the recurrence of the original nephropathy (focal glomerulosclerosis up to 50%, membrano-proliferative glomerulonephritis up to 30%, and primary hyperoxaluria up to 90% if combined kidney-liver transplantation is not performed). Growth improves after transplantation particularly in children <5 yrs, while it is not completely satisfactory in adolescents. Overall, results indicate that kidney transplantation in children has very much improved and will offer in the near future even more favorable outcomes.

Are allopurinol and metabolites found in HPRT deficient erythrocytes responsible for increased NAD synthesis?

Aim of this study was to ascertain whether allopurinol, usually administered to hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficient patients, or metabolites abnormally increased in HPRT deficient erythrocytes (NAD, PPribP) could be directly responsible for the reported increased activities of nicotinic acid phosphoribosyltransferase (NAPRT) and NADsynthetase (NADs) in these patients. No direct effect of the mentioned metabolites was demonstrated.

[Biocompatibility of the acetate in the dialysis fluid]. / Biocompatibilità dell'acetato contenuto nel bagno di dialisi.

PURPOSE: Dialytic vasculopathy is a major morbidity and mortality risk factor in patients undergoing chronic dialysis treatment. Among the pathogenetic factors some are related to the uremic condition, others are due to biocompatible reactions to dialytic materials. Endothelial cells (EC) are the target of the mediators released during bioincompatible reactions, and the related effects could be considered the initial event eliciting the vasculopathy pathogenesis. Among the others, we focused our attention on the role played in this process by the inducible isoform of nitric oxide (NO) synthase (iNOS). In previous studies we demonstrated that bioincompatible membranes, as well as acetate-containing dialysis buffers stimulate iNOS gene expression and activity in endothelial cells in culture. In this study, we planned to evaluate the potential role of a new dialysis buffer in which acetate has been substituted with HCl as a stabilizer. METHODS: ECs were incubated for 12 h at 37 degrees C with different dialysis buffers: acetate (Acet), standard bicarbonate (Bic), acetate-free buffer (AF) and HCl-bicarbonate (BicHCl). We evaluated in reverse transcriptase polymerase chain reaction (RT-PCR) the gene transcription for iNOS, the NOS activity (as the production of H3 citrulline from H3 arginine by ionic exchange chromatography), EC proliferative (H3 thymidine incorporation) and pro-apoptotic rate (TUNEL analysis) and the nuclear translocation of the transcriptional factor NF-kappaB (EMSA). RESULTS: Acetate, even in the low concentration present in Bic was able to induce a significant iNOS gene transcription (results expressed as relative units and referred to basal values: Acet 1.9 +/- 0.01 fold increase, p<0.01; Bic 1.45 +/- 0.03 p<0.05; BicHCl 1.24 +/- 0.01; AF 1.17 +/- 0.02) and translation. Acetate at concentrations both of 3 mmoL and 38 mmoL (present in the bicarbonate buffer) significantly increased the enzymatic NOS activity vs unconditioned ECs: Acet 3.46 +/- 0.3, p<0.0005; Bic 1.69 +/- 0.2, p<0.005; BicHCl 1.24 +/- 0.15; AF 1.17 +/- 0.05. The EC proliferative index was significantly depressed by acetate containing dialysis buffers (unconditioned ECs 100%, Acet 38 +/- 15%, p<0.01; Bic 65 +/- 6%, p<0.05; AF 87 +/- 8%; BicHCl 75 +/- 6%). The percentage of apoptotic ECs was significantly increased by buffers contain-ing Acet vs BicHCl and AF. Finally, acetate at the concentrations present in Acet and Bic activated and promoted the nuclear translocation of the transcriptional factor NF-kappaB in ECs (p<0.01 vs unconditioned cells). CONCLUSIONS: The acetate-free dialysis buffers have better biocompatibility and potentially down-modulate the flogistic and sclerotic processes responsible for dialytic vasculopathy.

[Neonatal chronic kidney failure associated with cyclo-oxygenase-2 inhibitors administered during pregnancy]. / Insufficienza renale cronica neonatale associata ad assunzione di un inibitore della ciclo-ossigenasi-2 durante le gravidanza.

Non-steroidal anti-inflammatory drugs (NSAID) are used since years as tocolytic due to their capacity to inhibit cyclo-oxygenase (COX) expressed in uterus and fetal membranes, fundamental for labour initiation and maintenance. The use of nimesulide, a COX-2 selective NSAID, has been recently proposed due to its capacity to selectively inhibit the enzyme expressed in the myometrium and endometrium. A case of neonatal irreversible end stage renal failure after maternal assumption of nimesulide as tocolytic for 6 week is reported. Cesarean section at the 32nd week due to oligohydramnios gave birth to a baby girl of 2090 g, in good general conditions, without signs of respiratory distress and of visible abnormalities. From birth she displayed oligo-anuria which required dialytic substitutive therapy from the second day of life. At US scan both kidneys had normal diameters for gestational age slightly increased echogenicity and a reduced cortico-medullary differentiation. On the 20th day of life she had a surgical renal biopsy for the persistence of oligo-anuria, showing fetal glomeruli, without lymphocytic interstitial infiltrate, and normal tubuli without evidence of necrosis. She is now 16 months old and under automated peritoneal dialysis on a home dialysis program. The occurrence of chronic renal failure in strict relationship with maternal nimesulide assumption in this case is strongly suggestive for a pharmacological damage, either direct or mediated by renin angiotensin inhibition, and possibly modulated by genetic factors, likely to account for the different outcome of similarly treated patients. A cautious use of this drug as long term tocolytic should be recommended while waiting for ad hoc experimental and clinical evidences of safeness.

Polymorphisms in the promoter region and at codon 54 of the MBL2 gene are not associated with IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN) occurs sporadically in unrelated individuals. Several different polymorphic genes have been investigated in recent years in order to demonstrate their possible association with IgAN. Three recent, different studies with conflicting conclusions have discussed the role of the mannose binding lectin (MBL), a serum lectin involved in natural immunity, in the IgAN pathogenesis by examination of MBL deposits in biopsies. In the present study we investigated several polymorphisms of the MBL gene located in the promoter region and in the first exon. METHODS: MBL polymorphism detection was performed in 22 Italian patients with familial IgA nephropathy and in 138 Italian patients with the sporadic form of the disease. The polymorphisms in the MBL2 promoter region and in the exon 1 were investigated, respectively, by direct sequencing and by amplification refractory mutation system-polymerase chain reaction on genomic DNA collected from peripheral blood. Seventy-four unrelated healthy subjects matched for ethnic origin were used as controls. RESULTS: Allelic and genotypic frequencies of the polymorphisms at position -550, -328, -221 and at codon 54 did not show any differences between patients and controls. Similar frequency distributions of these polymorphisms were also found in the subgroups of IgAN patients subdivided according to the clinical manifestations and the progression of the disease. CONCLUSIONS: This study indicates that the analysed polymorphisms of the MBL gene do not appear to be primarily involved in the susceptibility and severity of IgAN.