Effect of zinc imbalance and salicylic acid co-supply on Arabidopsis response to fungal pathogens with different lifestyles.

In higher plants, Zn nutritional imbalance can affect growth, physiology and response to stress, with effect variable depending on host-pathogen interaction. Mechanisms through which Zn operates are not yet well known. The hormone salicylic acid (SA) can affect plant ion uptake, transport and defence responses. Thus, in this study the impact of Zn imbalance and SA co-supply on severity of infection with the necrotrophic fungal pathogen B. cinerea or the biotroph G. cichoracearum was assessed in A. thaliana Col-0. Spectrophotometric assays for pigments and malondialdehyde (MDA) content as a marker of lipid peroxidation, plant defensin 1.2 gene expression by semi-quantitative PCR, callose visualization by fluorescence microscopy and diseases evaluation by macro- and microscopic observations were carried out. Zinc plant concentration varied with the supplied dose. In comparison with the control, Zn-deficit or Zn-excess led to reduced chlorophyll content and PDF 1.2 transcripts induction. In Zn-deficient plants, where MDA increased, also the susceptibility to B. cinerea increased, whereas MDA decreased in G. cichoracearum. Zinc excess increased susceptibility to both pathogens. Co-administration of SA positively affected MDA level, callose deposition, PDF 1.2 transcripts and plant response to the two pathogens. The increased susceptibility to B. cinerea in both Zn-deficient and Zn-excess plants could be related to lack of induction of PDF 1.2 transcripts; oxidative stress could explain higher susceptibility to the necrotroph and lower susceptibility to the biotroph in Zn-deficient plants. This research shows that an appropriate evaluation of Zn supply according to the prevalent stress factor is desirable for plants.

Reference Protocol to Assess Analytical Performance of Higher Order Structural Analysis Measurements: Results from an Interlaboratory Comparison.

Measurements of protein higher order structure (HOS) provide important information on stability, potency, efficacy, immunogenicity, and biosimilarity of biopharmaceuticals, with a significant number of techniques and methods available to perform these measurements. The comparison of the analytical performance of HOS methods and the standardization of the results is, however, not a trivial task, due to the lack of reference protocols and reference measurement procedures. Here, we developed a protocol to structurally alter and compare samples of somatropin, a recombinant biotherapeutic, and describe the results obtained by using a number of techniques, methods and in different laboratories. This, with the final aim to provide tools and generate a pool of data to compare and benchmark analytical platforms and define method sensitivity to structural changes. Changes in somatropin HOS, induced by the presence of zinc at increasing concentrations, were observed, both globally and at more localized resolution, across many of the methods utilized in this study and with different sensitivities, suggesting the suitability of the protocol to improve understanding of inter- and cross-platform measurement comparability and assess analytical performance as appropriate.

Niveles de Interferón Tipo I en pacientes con Lupus Eritematoso Sistémico / Type I interferon levels in patients with systemic lupus erythematosus

Introducción: El interferón (IFN) tipo I es una citoquina que juega un rol fundamental en la patogenia del Lupus Eritematoso Sistémico (LES). Diferentes niveles de esta citoquina podrían explicar la heterogeneidad de esta patología y ser útil para evaluar la actividad de la misma. Objetivos: Determinar los niveles de IFN tipo I sérico en pacientes con LES y evaluar su utilidad como biomarcador de actividad. Material y Métodos: 16 pacientes con LES (ACR 1997) y 16 controles. Métodos: Actividad de la enfermedad (SLEDAI-2K), daño orgánico (SLICC), IFN tipo I (HEK-Blue-IFNα/β), anticuerpos anti-DNAdc (Inmunofluorescencia Indirecta), anticuerpos anti-ENA (ELISA), C3-C4 (Inmunoturbidimetría). Estadística: InfoStat/Instat/MedCalc. Valores de p<0,05 fueron considerados estadísticamente significativos. Resultados: Se observó un aumento de la concentración de IFN en el grupo LES con respecto al control (p<0,05). Los pacientes con valores de IFN superiores al punto de corte, se asociaron con la presencia de anticuerpos anti-DNAdc (OR:13,33; p<0,05). Pacientes con hipocomplementemia y aquellos con puntaje de SLEDAI-2K mayor a 8 presentaron mayores niveles de IFN comparados con pacientes con complemento normal y menor puntaje de índice, respectivamente (p<0,05). Conclusiones: Estos resultados sugieren la importancia que podría tener la determinación de IFN tipo I para el monitoreo de la actividad del LES.

Introduction: Type I interferon (IFN) is a cytokine that plays a fundamental role in the pathogenesis of Systemic Lupus Erythematosus (SLE). Different levels of this cytokine could explain the heterogeneity of this pathology and be useful to evaluate its activity. Objectives: To determine the serum type I IFN levels in patients with SLE and evaluate its usefulness as a biomarker of activity. Material and Method: 16 patients with SLE (ACR 1997) and 16 controls. Methods: Disease activity (SLEDAI-2K), organ damage (SLICC), type I IFN (HEK-Blue-IFNα/β), anti-dsDNA antibodies (Indirect Immunofluorescence), anti-ENA antibodies (ELISA), C3-C4 (Immunoturbidimetry). Statistics: InfoStat/Instat/MedCalc. P values <0.05 were statistically significant. Results: An increase in IFN concentration was observed in the SLE group respect to the control (p <0.05). Patients with IFN values above the cut-off point were associated with the presence of anti-dsDNA antibodies (OR: 13.33; p<0.05). Hypocomplementemic patients and those with a SLEDAI-2K score greater than 8 had higher IFN levels compared to patients with normal complement and a lower index score, respectively (p<0.05). Conclusions: These results suggest the importance that the determination of IFN type I could have for the monitoring of SLE activity.

Polineuropatía desmielinizante inflamatoria crónica como forma de presentación de lupus eritematoso sistémico / Chronic inflammatory demyelinating polyneuropathy as a form of presentation of systemic lupus erythematosus

Se describe el caso de una mujer de 35 años que presenta polineuropatía desmielinizante inflamatoria crónica como compromiso neurológico en su diagnóstico inicial de lupus eritematoso sistémico (LES). Si bien el compromiso neurológico es de una prevalencia variable en lupus, la asociación que se describe no es frecuente y tiene importantes connotaciones en el tratamiento.

We described a 35 years old female, who developed Chronic inflammatory demyelinating polyneuropathy as neurologic commitment during the early diagnosis in Systemic Lupus Erithematosus (SLE). While the neuropsychiatric commitment has a variable prevalence in SLE, the association that we describe is infrequent and it has important concerns during its treatment.

Polineuropatía desmielinizante inflamatoria crónica como forma de presentación de lupus eritematoso sistémico / Chronic inflammatory demyelinating polyneuropathy such as presentation of systemic lupus erythematosus

Se describe el caso de una mujer de 35 años que presenta polineuropatía desmielinizante inflamatoria crónica como compromiso neurológico en su diagnóstico inicial de lupus eritematoso sistémico (LES). Si bien el compromiso neurológico es de una prevalencia variable en lupus, la asociación que se describe no es frecuente y tiene importantes connotaciones en el tratamiento.

We described a 35 years old female, who developed Chronic inflammatory demyelinating polyneuropathy as neurologic commitment during the early diagnosis in Systemic Lupus Erithematosus (SLE). While the neuropsychiatric commitment has a variable prevalence in SLE, the association that we describe is infrequent and it has important concerns during its treatment.

Immune function assay (immunknow) drop over first 6 months after renal transplant: a predictor of opportunistic viral infections?

BACKGROUND: The Immuknow assay (IKA; Cylex) is a T-cell immune function assay that evaluates immunoreactivity in immunocompromised patients. The aim of this study was to analyze IKA values in a cohort of kidney transplantation (KT) recipients to investigate correlations between single-time point low IKA values and their trend over time with cytomegalovirus (CMV) or BK virus (BKV) reactivation. METHODS: A total of 118 adult patients receiving deceased-donor KT were enrolled (55.6±11.9 years old; 79 [66.9%] male). IKA CMV and BKV viremia determinations and were performed at months 1, 3, and 6 after surgery. RESULTS: Overall, 272 IKA determinations were performed: IKA values significantly decreased from month 1 (422±184 ng/mL) to month 3 (330±159 ng/mL; P<.001) and from month 3 to month 6 (300±128 ng/mL; P=.030). IKA values did not correlate with renal function or viral reactivation at any time. However, patients with either CMV or BKV viremia had a trend to higher IKA values at month 1 and lower IKA values at month 6, even if the difference did not reach a statistical significance (P=.115). CONCLUSIONS: Our study suggests that presence of low immunologic reactivity (IKA<225 ng/mL) is not associated with an increased risk of CMV and BKV reactivation over the 1st 6 months after KT. However, a trend to a more pronounced drop in IKA values over time was observed in patients with viral reactivation. These preliminary results suggests that drop in IKA values within the 1st post-KT months, unlike single-time point immune function assay, may predict the risk of opportunistic viral infections.

Schmallenberg virus in Italy: a retrospective survey in Culicoides stored during the bluetongue Italian surveillance program.

Following the first case of Schmallenberg (SBV) in northern Italy in February 2012, virus detection was conducted on midges collected during the national entomological surveillance program for bluetongue (BT). Six cattle farms, within a radius of 50 km from the SBV case, were selected for a 12 month study, aiming to determine when the virus entered the area, if it was capable of overwintering, and the possible role played by each species of the Obsoletus complex in disseminating the infection. A total of 33,724 Culicoides were collected at the six sites between June 2011 and June 2012. Species belonging to the Obsoletus Complex were the most abundant (94.44%) and, within the complex, Culicoides obsoletus was the most prevalent species in the studying area (65.4%). Nearly 7000 Culicoides midges were screened, either in pools or individually, for SBV by real-time RT-PCR. Viral genome was detected in six pools of the Obsoletus complex, collected at three sites between September and November 2011, and in a single parous female of C. obsoletus, collected in May 2012. As a result of the BT surveillance program in Italy it was possible to demonstrate, retrospectively, that SBV has circulated in at least three Italian provinces since early September 2011, nearly 5 months prior and as far as 40 km away from the first detected case. Similarly, the survey confirmed the presence of SBV in the vector population 3 months after the outbreak, following a cold winter during which the blacklight traps failed to catch active adult midges.

Severe acute nephrotoxicity in a kidney transplant patient despite low tacrolimus levels: a possible interaction between donor and recipient genetic polymorphisms.

WHAT IS KNOWN AND OBJECTIVE: Tacrolimus has a narrow therapeutic index and shows large interindividual variations in pharmacokinetics, which may be partly explained by genetic variability in metabolic enzymes of the cytochrome P450 (mainly CYP3A4 and CYP3A5) and transport P-glycoprotein (encoded by the ABCB1 gene). Genetic variability in the expression of biotransformation enzymes and drug transporters may also predispose individuals to tacrolimus-induced nephrotoxicity. CASE SUMMARY: We report a case of severe biopsy-proven Tacrolimus (TAC) nephrotoxicity that occurred 1 month after renal transplantation despite persistently low TAC levels. The donor genotype was CYP3A5*3/*3 (loss-of-function genotype), whereas that of the recipient was CYP3A5*1/*3. The donor and recipient genotypes did not differ with respect to either CYP3A4 rs35599367C>T (both were CC homozygotes) or ABCB1 gene polymorphisms (both TT homozygotes for the 1236C>T polymorphism and CT heterozygotes for the 3435C>T polymorphism). WHAT IS NEW AND CONCLUSION: This case study suggests that donor/recipient genetic mismatch in metabolic enzymes may have an important role in modulating tacrolimus nephrotoxicity. It provides a possible explanation for the intriguing observation that for a subset of patients, cumulative TAC doses appear to correlate better with nephrotoxicity than trough levels.

Anti-IFI16 antibodies and their relation to disease characteristics in systemic lupus erythematosus.

OBJECTIVE: Several studies have shown the presence of anti-IFI16 antibodies in systemic lupus erythematosus (SLE), Sjögren Syndrome (SjS), systemic sclerosis (SSc) and other autoimmune diseases. However, the significance of anti-IFI16 antibodies in SLE has not been fully characterized. The aim of this study was to investigate associations between anti-IFI16 antibodies and clinical and serologic parameters of SLE. METHODS: An enzyme-linked immunosorbent assay (ELISA) kit was used to measure anti-IFI16 antibodies in the sera of 168 SLE patients, 46 patients with any type of primary glomerulonephritis (GN) and 182 healthy controls (HCs). Associations between anti-IFI16 antibodies and clinical and serologic parameters of SLE were statistically evaluated using both univariate and multivariate analysis. RESULTS: Significantly higher anti-IFI16 titres were observed in SLE patients compared to both non-SLE GN and HCs (median levels: 270.1 U/ml vs 132.1 U/ml, p = 0.001, and 52.9 U/ml, p < 0.0001, respectively). With cut-off levels corresponding to the 95th percentile of the control population (113 U/ml), 63% of the SLE patients tested positive for anti-IFI16 autoantibodies, compared to just 24% of patients with primary non-SLE GN and 5% of HCs. The presence of anti-IFI16 antibodies inversely correlated with proteinuria (univariate analysis) and C3 hypocomplementaemia (univariate and multivariate analyses). CONCLUSIONS: The inverse correlations observed between anti-IFI16 positivity, proteinuria and C3 hypocomplementaemia suggest that anti-IFI16 antibodies do not contribute to renal inflammation in SLE; indeed they may even prevent complement consumption. Anti-IFI16 antibodies hold the potential to serve as a new biomarker of disease activity in SLE.

Middle and long-term outcomes of dual kidney transplant: a multicenter experience.

INTRODUCTION: Dual kidney transplantation (DKTx) to reduce the disparity between demand and supply of organs was evaluated in two Italian centers (Bari and Novara). MATERIALS AND METHODS: Between October 2000 and October 2011, we performed 97 DKT (26 ipsilateral/71 bilateral) following routine biopsy of all kidneys obtained from expanded criteria donors by Remuzzi-Karpinsky scores. The reference group was 379 single grafts from donors older than 60 years single kidney transplantation ([SKT] × > 60). RESULTS: Good postoperative renal function was observed in 56 DKTx (57.7%); whereas acute tubular necrosis requiring dialysis was observed in 41 (42.3%) patients. After a mean follow-up of 60 months, DKTx graft survivals were 96%, 93%, and 90% and patient survivals, 96%, 91%, and 91% at 1, 3, and 5 years, respectively. Complications in expanded criteria donor kidney transplantations included a high rate of cytomegalovirus (CMV) disease especially dual kidney cases. DKTx represented the only independent risk factor for CMV disease upon multivariate analysis (odds ratio [OR] 2.33, 95% confidence interval [CI] 1.28-4.2; P = .006). We did not observe any significant difference in graft or patient survival between DKTx and SKTx > 60 years. CONCLUSIONS: We observed good outcomes up to 5 years after transplantation in terms of graft and patient survival despite the use of inferior grafts. Comparing DKTx and SKT > 60, we noted that the mean Karpinski score for SKTx was significantly better than DKTx, although patient and graft survivals were similar. This trend confirms that the use of a biopsy to allocate expanded criteria donor kidneys may be too protective; therefore, the criteria to select DKTx require further refinement.

Structure-function relationships of iodinated contrast media and risk of nephrotoxicity.

Characteristics of CM have greatly changed over time. First-generation ionic CM have many-fold (5-7) greater osmolalities than plasma. Subsequently non ionic CM generations were looked for to reduce osmolality, and encompass nonionic monomers and nonionic dimers reaching osmolality as low as that of plasma (iso-osmolar CM) but paying however dear, as viscosity is considerably increased. Intrarenal microcirculation has its "Achilles" heel in the outer medulla, where the smallness of capillary lumen and the slackness of the capillary mesh render regular blood flow at high risk, mainly because it is the same area in which the only renal work needing oxygen is made and? Iodinated CM may exert their nephrotoxic effects in three different ways: by interfering with vascular hemodynamics, by interfering with intratubular fluid volume and composition, and by producing direct cytotoxic effects to glomerular and tubular cells due to iodine by-itself. Furthermore, effects of oxygen free radical can damage glomerular cells by increasing the permeability and tubular cells impairing specific function and leading to apoptosis. Although clinical nephrotoxicity has considerably improved over time, there is no evidence for an a priori superiority of a specific CM. In general, low-osmolar (2-3 times blood) and iso-osmolar (the same as blood) CM are recommended, keeping in mind that within last generation CM dimeric iso-somolar compounds reach viscosity values higher than monomeric low-osmolar compounds and hyperviscosity is a neglected mechanisms of nephrotoxicity. We suggest that CM should be classified not only by osmolality, but also by viscosity.

The interactions of age, sex, body mass index, genetics, and steroid weight-based doses on tacrolimus dosing requirement after adult kidney transplantation.

PURPOSE: The aim of this study was to evaluate the effect of different clinical covariates on tacrolimus dose requirements in adult kidney transplant patients with a specific focus on drug interactions. PATIENTS: Tacrolimus dosing requirement, normalized by drug levels and expressed as the concentration/dose (C/D) ratio as a surrogate index of tacrolimus bioavailability, was employed to identify four categories of tacrolimus dosing requirement, namely, very high, high, small, and very-small, in very fast, fast, slow, and very slow metabolizers, respectively. Steroid weight-based doses were analyzed instead of fixed doses, and genetic analysis of cytochrome P450 (CYP) 3A5*1/*3 and multi-drug resistance 1 (MDR1) C3435T and C1236T polymorphisms were performed RESULTS: Multivariate analysis on 450 adult transplant patients identified six risk factors for being slow metabolizers and therefore requiring small tacrolimus doses: male sex (OR 1.615, p = 0.020); age >60 years (OR 2.456, p = 0.0005); body mass index ≥ 25 (OR 1.546, p = 0.046), hepatitis C virus positivity (OR 2.800, p = 0.0004); low steroid dose <0.06 mg/kg (OR 3.101, p < 0.0001). Patients with a small tacrolimus requirement were at increased risk for multiple infections (OR 1.533, p = 0.0008) and higher systolic blood pressure (OR 1.385, p = 0.022) and showed a significant association with the CYP3A5*3/*3 genotype adjusted by MDR1 polymorphisms C3435T and C1236T (OR 8.104, p = 0.0001). CONCLUSIONS: Our results demonstrate the importance of the interaction among genetic and clinical factors in conditioning tacrolimus disposition, with corticosteroid weight-based dose being the only modifiable risk factor for tacrolimus requirement. As the tacrolimus dosing requirement increases with increasing tacrolimus clearance through concomitant steroid use, undesirable changes in tacrolimus levels may occur when steroid doses are tapered, predominantly in slow metabolizers. This often neglected drug interaction has to be monitored to optimize tacrolimus exposure in kidney transplant patients.

First Report of Clonostachys rhizophaga as a Pathogen of Dendrocalamus giganteus in Mozambique.

Dendrocalamus giganteus Wall. ex Munro, a wild plant belonging to the family Poaceae, is widespread in Mozambique where it is used as a construction material. At the end of 2007, disease symptoms have been observed on D. giganteus plants growing in the neighborhood of Maputo. Diseased plants showed longitudinal dark streaks on the stem surface to which corresponded internal vascular browning and chlorosis in wide leaves that gradually developed into necrosis. At the final stage of the disease plants died. To isolate the pathogen, stem segments collected during September 2008 were surface sterilized with 1% HgCl2 for 30 s, rinsed with sterile deionized water for 30 s, and incubated on potato dextrose agar (PDA) medium at 22°C in the dark for 2 weeks. Monosporic cultures of the isolated fungus formed dimorphic Verticillium-like (primary) or penicillate (secondary) conidiophores and ovoidal to elongate, minutely curved, hyaline conidia, 5 to 9.5 × 2.5 to 4.5 µm, with laterally displaced hilum. These characteristics are typical of Clonostachys rhizophaga Schroers (3). Identification was confirmed by the Centraalbureau voor Schimmelcultures (Utrecht, the Netherlands) on the basis of the ß-tubulin (tub2) gene sequence (3). For our isolate CBS 125416, the tub2 sequence was 100% similar to that of the C. rizhophaga strain CBS 124511 (GenBank Accession No. FJ 593883) (1). To verify the pathogenicity of the fungus, a 5-mm-diameter mycelial plug obtained from 2-week-old colonies grown on PDA was affixed to a portion of the stem of D. giganteus from which the superficial tissues had been removed and the inoculation site was covered with wet cotton and wrapped with Parafilm. Control plants were treated by the same method but using PDA plugs without mycelium. Twenty plants were used, ten of which were controls. They were grown in a controlled climatic chamber at 22°C with a photoperiod of 16 h at 40 µE·m-2·s-1. Two months after inoculation, all plants showed a dark area surrounded by an idropic halo on the stem surface and internal browning, whereas control plants remained healthy. C. rhizophaga was recovered from all infected plants. C. rhizophaga is apparently rare. The fungus (as Verticillium rhizophagum Tehon & Jacobs, nom. invalid.) has been previously reported from the United States, Chile, and Ecuador (4) and as a culture contaminant in Switzerland (3). More recently C. rhizophaga has been found to be associated with Pinus canariensis in Argentina (2) and it has been reported as a causal agent of chickpea wilt in Syria (1). To our knowledge, this is the first report of C. rhizophaga for subsaharian Africa. It may be under reported and more common than has been thought because of the difficulty in identifying Clonostachys species, but with the ability to identify species using tub2 (3), there can be no doubt of its identity. References: (1) M. M. Abang et al. Plant Dis. 93:666, 2009. (2) L. Eduardo Piontelli and G. Giusiano. Bol. Micol. 18:89, 2003. (3) H. J. Schroers. Stud. Mycol. 46:85, 2001. (4) L. R. Tehon and H. L. Jacobs. Bull. Davey Tree Expert Company, Kent, OH. 6:3, 1936.

Amine-reactive isobaric tagging reagents: requirements for absolute quantification of proteins and peptides.

Amine-reactive isobaric tagging reagents such as iTRAQ (isobaric tags for relative and absolute quantitation) have recently become increasing popular for relative protein quantification, cell expression profiling, and biomarker discovery. This is due mainly to the possibility of simultaneously identifying and quantifying multiple samples. The principles of iTRAQ may also be applied to absolute protein quantification with the use of synthetic peptides as standards. The prerequisites that must be fulfilled to perform absolute quantification of proteins by iTRAQ have been investigated and are described here. Three samples of somatropin were quantified using iTRAQ and synthetic peptides as standards, corresponding to a portion of the protein sequence. The results were compared with those obtained by quantification of the same protein solutions using double exact matching isotope dilution mass spectrometry (IDMS). To obtain reliable results, the appropriate standard peptides needed to be selected carefully and enzymatic digestion needed to be optimized to ensure complete release of the peptides from the protein. The kinetics and efficiency of the iTRAQ derivatization reaction of the standard peptides and digested proteins with isobaric tagging reagents were studied using a mixture of seven synthetic peptides and their corresponding labeled peptides. The implications of incomplete derivatization are also presented.

[Early diagnosis and prevention of renal disease: collaboration between nephrologists and general practitioners]. / Diagnosi precoce e prevenzione delle nefropatie: collaborazione integrata con i medici di medicina generale.

Our outpatient clinic activity has taught us that a working relationship between general practitioners (GPs) and nephrologists may improve the definition of the diagnostic-therapeutic course for the benefit of the patient. We have therefore contacted the 7 teams comprising 104 GPs and pediatricians working in the area of the Agnelli Hospital in Pinerolo (132,000 inhabitants in 1,404 square kilometers) to assess the possibility of improving and strengthening the collaboration between GPs and nephrologists. The starting point was a direct telephone link aimed at dealing with patients' kidney problems in real time, evaluating history and clinical data, establishing the best timing of treatment, and defining the diagnostic and therapeutic options. The initiative was welcomed at all team meetings and it stimulated further requests for collaboration. One of the main requests was for simple clinical guidelines to deal with the most frequent clinical nephrological issues. This is the program we are carrying out: 1) We have established consulting hours during which GPs can call nephrologists at the hospital to discuss the best diagnostic-therapeutic approach for individual kidney patients. 2) We have identified diseases of common interest (isolated urinary abnormalities; hypertension; nephrotoxicity; abnormal renal function; chronic renal failure; urinary infections; kidney stones). 3) We have planned to draw up clinical guidelines. 4) We will discuss each draft with the team of GPs. On the basis of the gathered suggestions, we will prepare a final version of the guidelines to be sent to the GPs and pediatricians of our area.

Risk management of renal biopsy: 1387 cases over 30 years in a single centre.

BACKGROUND: Although renal biopsy is largely employed, even in old patients with systemic diseases, few clinical studies have addressed its risk management. We aimed to obtain a comprehensive assessment of safety/utility ratio of percutaneous renal biopsy. PATIENTS AND METHODS: Retrospective review of all the 1387 patients who consecutively underwent renal biopsy in a single centre over three decades (1973-2002) was made, with calculation of complications, multivariate logistical analyses to evaluate risk factors of complications, and rate of alteration of clinical hypotheses by pathological diagnosis. RESULTS: There were no deaths and five major complications, (0.36%). One nephrectomy (0.07%), two surgical revisions (0.1%) and two arterial-venous fistulae (0.1%). There were also 337 minor bleeding complications (24.2%) (16.4% gross haematuria and 7.8% clinically relevant haematomas needing at least prolonged bed rest). Multivariate analyses demonstrated that the risk for complications was significantly increased by systemic autoimmune diseases with odds ratio (OR) 2.06, 95% confidence interval (CI)=1.40-3.01, end-stage kidney/acute-tubular necrosis (OR 2.96, 95% CI=1.19-7.30), and prolonged bleeding time test (BTT) (OR 1.87, 95% CI=1.17-2.83). Among the 1288 cases in which a clinical hypothesis before renal biopsy was recorded, renal pathology changed previous diagnoses in 423/1,288 (32.8%) of cases. CONCLUSIONS: Risk assessment demonstrates that renal biopsy is a useful procedure with a low incidence of serious complications. Platelet function is the only modifiable factor significantly related to bleeding complications, suggesting the need for a more standardized alternative to the BTT. Platelet function should be evaluated to select low-risk patients for renal biopsy as 'a day case procedure', in order to build adequate risk management strategies.

Dual effect of methylprednsolone pulses on apoptosis of peripheral leukocytes in patients with renal diseases.

It is well known that change in apoptosis may modulate the natural story of illness, and that many drugs may act through modulation of apoptosis, but the role of steroids in acting through apoptosis in different settings, including renal diseases, has still to be elucidated. We studied the in vivo effects of steroids by oral assumption (10 to 25 mg/deltacortene) or by intravenous pulses (300 to 1000 mg/dose) on apoptosis and cellular subsets of peripheral lymphocytes, by evaluating DNA-fragmentation and lymphocyte subsets in 79 subjects: 22 controls and 57 patients with various renal diseases (25 Lupus-GN, 19 membranous-GN (MGN), 6 rapidly progressive-GN (RPGN), 2 acute interstitial nephritis (AIN), 5 on chronic dialysis. Baseline apoptosis was present in 1/22 (4.5%) of controls, 3/25 (12%) SLE, 2/6 (33.3%) RPGN and 10/19 (52.6%) MGN. A significant decrease in CD3+CD8+ cell count and a significant increase of the CD3+CD4/CD3+CD8+ ratio were found in apoptosis-positive subjects. DNA fragmentation did not change after oral steroids, paralleling a 22 to 32% decrease in total lymphocytes. Following intravenous methylprednisolone pulses, a deeper drop of all lymphocyte subsets was observed, while DNA fragmentation turned from present to absent in 2 MGN, but not in 2 RPGN, and from absent to present in 1 ARF and 1 SLE, independently of the dosage. We demonstrated that the presence of apoptosis in renal diseases is associated with decreased CD3+CD8+ cell count. Furthermore, steroid intravenous pulses, besides inducing a profound decrease in lymphocyte subsets, do exert a dual effect on baseline leukocyte apoptosis, eventually leading to a reversal of baseline patterns, either turning from negative to positive or from positive to negative. Oral steroid therapy did not influence baseline apoptosis.

Identification of an isomer impurity in piperaquine drug substance.

A significant contaminant of the antimalarial drug piperaquine (1,3-bis-[4-(7-chloroquinolyl-4)-piperazinyl-1]propane) has been identified using liquid chromatography-mass spectrometry (LC-MS) and 2D NMR spectroscopy (1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC). The impurity was identified as the positional isomer 1-[(5-chloroquinolin-4)-piperazinyl]-3-[(7-chloroquinolin-4)-piperazinyl]propane. The impurity is formed because of contamination of batches of 4,7-dichloroquinoline (a precursor in the synthesis of piperaquine) with 4,5-dichloroquinoline. The amount of impurity (peak area impurity/peak area piperaquine using LC-UV at 347 nm) in old batches of piperaquine and in Artekin (the combination of dihydroartemisinin-piperaquine) ranged from 1.5 to 5%.