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.

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.

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.

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.

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.

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.

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%.

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.

Hybrid polar histone deacetylase inhibitor induces apoptosis and CD95/CD95 ligand expression in human neuroblastoma.

Inhibitors of histone deacetylase (HDAC) have been shown to have both apoptotic and differentiating effects on various tumor cells. M-carboxycinnamic acid bishydroxamide (CBHA) is a recently developed hybrid polar compound structurally related to hexamethylene bisacetamide. CBHA is a potent inhibitor of HDAC activity. CBHA induces cellular growth arrest and differentiation in model tumor systems. We undertook an investigation of the effects of CBHA on human neuroblastoma cell lines in vitro. When added to cultures of a panel of neuroblastoma cell lines, CBHA induced the accumulation of acetylated histones H3 and H4, consistent with the inhibition of HDAC. Concentrations of CBHA between 0.5 microM and 4 microM led to apoptosis in nine of nine neuroblastoma cell lines. Apoptosis was assessed by DNA fragmentation analysis and the appearance of a sub-G1 (<2N ploidy) population by flow cytometric analysis. The addition of a caspase inhibitor (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) completely abrogated CBHA-induced apoptosis in three of three cell lines. The addition of cycloheximide greatly reduced CBHA-induced apoptosis, suggesting that apoptotic induction was dependent on de novo protein synthesis. In addition, CBHA induced the expression of both CD95 (APO-1/Fas) and CD95 ligand within 12 h. The effect of CBHA on human neuroblastoma cells suggests that this agent and structurally related synthetic hybrid polar compounds have therapeutic potential for the treatment of this malignancy.

The histone deacetylase inhibitor, CBHA, inhibits growth of human neuroblastoma xenografts in vivo, alone and synergistically with all-trans retinoic acid.

Histone deacetylase inhibitors (HDACIs) inhibit the growth of a variety of transformed cells in culture. We demonstrated previously that the hybrid-polar HDACI m-carboxycinnamic acid bis-hydroxamide (CBHA) induces apoptosis of human neuroblastoma in vitro and is effective in lower doses when combined with retinoids. The current study investigates the effect of CBHA on the growth of human neuroblastoma in vivo, both alone and in combination with all-trans retinoic acid (atRA), using a severe combined immunodeficiency-mouse xenograft model. CBHA (50, 100, and 200 mg/kg/day) inhibited growth of SMS-KCN-69n tumor xenografts in a dose-dependent fashion, with 200 mg/kg CBHA resulting in a complete suppression of tumor growth. The efficacy of 50 and 100 mg/kg CBHA was enhanced by the addition of 2.5 mg/kg atRA. This dose of atRA was ineffective when administered alone. Treatment was accompanied by mild weight loss in all groups except the lowest dose of CBHA. Our results suggest HDACIs alone or combined with retinoids may have therapeutic utility for neuroblastoma.

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.

Very-high-dose short-term chemotherapy for poor-risk peripheral primitive neuroectodermal tumors, including Ewing's sarcoma, in children and young adults.

PURPOSE: To improve the prognosis of patients with poor-risk peripheral primitive neuroectodermal tumors (pPNETs; including peripheral neuroepithelioma and Ewing's sarcoma), while testing the feasibility of intensive use in adolescents and young adults of high-dose cyclophosphamide, doxorubicin, and vincristine (HD-CAV). PATIENTS AND METHODS: This report concerns previously untreated patients with newly diagnosed pPNET deemed poor-risk because of a tumor volume more than 100 cm3 or metastases to bone or bone marrow. The P6 protocol consists of seven courses of chemotherapy. Courses 1, 2, 3, and 6 include 6-hour infusions of cyclophosphamide on days 1 and 2 for a total of 4,200 mg/m2 per course (140 mg/kg per course for patients < 10 years old), plus 72-hour infusions of doxorubicin 75 mg/m2 and vincristine 2.0 mg/m2 beginning on day 1 (HD-CAV). Courses 4, 5, and 7 consist of 1-hour infusions of ifosfamide 1.8 g/m2/d and etoposide (VP-16) 100 mg/m2/d, for 5 days. Granulocyte colony-stimulating factor (G-CSF) and mesna are used. Courses start after neutrophil counts reach 500/microL and platelet counts reach 100,000/uL. Surgical resection follows course 3 and radiotherapy follows completion of all chemotherapy. RESULTS: Among the first 36 consecutive assessable patients (median age, 17 years), HD-CAV achieved excellent histopathologic or clinical responses in 34 patients and partial responses (PRs) in two patients. For 24 patients with locoregional disease, the 2-year event-free survival rate was 77%; adverse events were two locoregional relapses, one distant relapse, and one secondary leukemia. All six patients with metastatic disease limited to lungs achieved a complete response (CR) and did not relapse; one is in remission 36+ months from diagnosis, but the other patients are not assessable in terms of long-term efficacy of the P6 protocol because of short follow-up time (n = 3), additional systemic therapy (bone marrow transplantation), or septic death (autopsy showed no residual pPNET). All six patients with widespread metastases had major responses, including eradication of extensive bone marrow involvement, but distant relapses ensued. Myelosuppression was severe, but most patients received the first three courses of HD-CAV within 6 to 7 weeks. Major nonhematologic toxicities were mucositis and peripheral neuropathy. CONCLUSION: Excellent antitumor efficacy and manageable toxicity support the dose-intensive use of HD-CAV for pPNET in children, as well as in young adults. Consolidation of remissions of pPNET metastatic to bone and bone marrow remains a therapeutic challenge.

Pediatric desmoid tumor: retrospective analysis of 63 cases.

PURPOSE: This study was conducted to evaluate clinical prognostic factors predictive of the probability of recurrence of desmoid tumor (DT). PATIENTS AND METHODS: Sixty-three patients with histologically confirmed diagnosis of DT were retrospectively studied. Median age at diagnosis was 13 years. Patient distribution by site was as follows: 61% extremities, 18% head and neck, 13% trunk (including 5% intraabdominal), and 8% multicentric lesions. All patients had partial or complete resections; 20 patients also received radiotherapy and/or chemotherapy. RESULTS: At a median follow-up time of 6 years since first treatment, the overall actuarial probability of having one or more recurrences was 75%. Age, sex, site, size, or number of previous recurrences had no significant impact on the likelihood of recurrence. The only factor associated with an increased proportion of recurrence-free patients was a negative margin of resection (70% v 15% with positive margins; P = .006). Of the four patients with more than 3 years follow-up since chemotherapy, two recurred, and of the 11 patients with the same follow-up after radiotherapy, four recurred, including two of five patients who received a dose of 50 Gy or more. No deaths directly related to tumor invasion were observed. CONCLUSION: A surgical approach aiming at clear margins is presently the best treatment option. When this cannot be accomplished without severe disfigurement or function impairment, partial resection is an acceptable alternative, but one associated with a high risk of regrowth. Whether adjuvant strategies should be used in this situation remains to be addressed.

Use of vancomycin chiral stationary phase for the enantiomeric resolution of basic and acidic compounds by nano-liquid chromatography.

In this paper we studied the potentiality of nano-liquid chromatography (nano-LC) for the enantiomeric resolution of both basic and acidic compounds of pharmaceutical interest using a vancomycin modified silica stationary phase. Experiments were carried out in a fused silica capillary of 75 microm I.D. packed with chiral modified silica particles of 5 microm diameter, the detection, was done on-line at 195 nm. Enantiomeric resolution of alprenolol, atenolol, metoprolol, oxprenolol, pindolol, propranolol (basic compounds) and some acidic analytes, namely 2-[(5'-benzoyl-2'-hydroxy)phenyl]propionic acid (DF1738Y), 2-[(4'-benzoyloxy-2'-hydroxy)phenyl]propionic acid (DF1770Y), ketoprofen, indoprofen and suprofen was studied by nano-LC utilizing mobile phases containing methanol-acetonitrile-ammonium formate or acetate. The effect of mobile phase composition (buffer type and concentration, organic modifier type and concentration) on chiral resolution (Rs), retention factor (k) and retention time (tR) was also investigated. Good enantiomeric resolution was achieved for basic compounds utilizing the mobile phase containing 90% (MeCN-MeOH)/5% water/5% of 100 mM ammonium acetate pH 4.5. Acidic compounds such as DF1738Y and DF1770Y were better resolved at lower pH 3.5 while ketoprofen, indoprofen and suprofen exhibited the highest resolution at pH 4.5; in this case the mobile phase contained MeOH or MeCN (90%), 5% buffer and 5% of water. The nano-LC method was validated using R-(+)-propranolol as an internal standard finding good repeatability, detection limit, correlation coefficient and recovery and applied to the assay of a pharmaceutical formulation containing a racemic mixture of metoprolol.

Dynamic electric field assisted multi-dimensional liquid chromatography of biological samples.

Complex biological samples require very high resolution separation strategies. The platform introduced here capitalises on the hyphenation of liquid chromatographic (LC) and electric potential gradient electrochromatographic multi-dimensional separation genres. First-dimension selectivity is provided by simultaneous size exclusion (SEC) and strong cation exchange (SCX) chromatography modes, while the second dimension comprises reversed phase (RP) characteristics in a dynamic (time-variant) electric field. The time-variant potential gradient with reversal of polarity is applied across the second dimension monolithic capillary throughout the duration of the solvent strength gradient elution. Hence, the platform offers comprehensive on-line sample clean-up (matrix depletion, analyte enrichment), fractionation (first dimention LC), and separation (second dimension LC) with the prospect of altering selectivity via polarity reversal dynamic electric field tuning.

Analysis of diltiazem and its related substances by HPLC and HPLC/MS.

Diltiazem (DTZ) is an optically active calcium channel blocker having a benzodiazepine structure. The drug used in therapy is (+)-cis-diltiazem with configuration (2S,3S). To describe the analytical profile of DTZ different stationary phases (RP-18, RP-8, monolithic support) were tested. The best separation of DTZ from A, B, E and F was obtained using as stationary phase a RP-8 or a monolithic RP-18. The characterization of impurities was carried out using two analytical systems, HPLC and HPLC/MS.

Direct determination by capillary electrophoresis of cardiovascular drugs, previously included in liposomes.

The lipophilicity of some cardiovascular drugs was determined by capillary electrophoresis (CE). Mexiletine, amlodipine and indapamide, the drugs considered, were in contact with liposomial vescicles for 2, 4 or 6 h. After the contact time the drugs, penetrated into liposomial vesicles, were determined by CE using phosphate buffer (pH 6.3 or 7.4) or borate buffer (pH 9). The lipophilicity of three drugs was determined considering the drug percentage penetrated into liposomial vesicles. The found lipohilicity order was amlodipine > mexiletine > indapamide.

Micellar electrokinetic chromatography for determination of drug partition in phospholipids.

The lipophilicity of pipemidic, nalidixic and oxolinic acids was determined by forming phospholipidic micelles directly in an electrophoretic capillary. Phosphatidylcholine derivatives, namely L-alpha-dilauroyl phosphatidylcholine (DLPC) or L-alpha-dimiristoyl phosphatidylcholine (DMPC), were added in the run buffer (50 mM phosphate buffer at pH 7.4). To obtain a mixed micelle, phospholipidic derivatives and sodium cholate were together added in the run buffer. Considering the increasing of migration time when phosphatidylcholine derivative is added in the run buffer, Ks can be determined and then quinolones lipophilicity.