Palaeoenvironment and palaeoclimate of the Middle Miocene lake in the Steinheim basin, SW Germany: A reconstruction from C, O, and Sr isotopes of fossil remains

A differentiated reconstruction of palaeolimnologic, -environmental, and -climatic conditions is presented for the Middle Miocene long-term freshwater lake (14.3 to 13.5 Ma) of the Steinheim basin, on the basis of a combined C, 0, and Sr isotope study of sympatric skeletal fossils of aquatic and terrestrial organisms from the lake sediments. The oxygen isotope composition for lake water of the Steinheim basin (delta O-18(H2O) = +2.0 +/- 0.4 parts per thousand VSMOW, n = 6) was reconstructed from measurements of delta O-18(PO4) of aquatic turtle bones. The drinking water calculated from the enamel of large mammals (proboscideans, rhinocerotids, equids, cervids, suids) has delta O-18(H2O) values (delta(OH2O)-O-18 = -5.9 +/- 1.7 parts per thousand VSMOW, n = 31) typical for Middle Miocene meteoric water of the area. This delta O-18(H2O) value corresponds to a mean annual air temperature (MAT) of 18.8 +/- 3.8 degrees C, calculated using a modem-day delta(OH2O)-O-18-MAT relation. Hence, large mammals did not use the lake water as principal drinking water. In contrast, small mammals, especially the then abundant pika Prolagus oeningensis drank from O-18-enriched water sources (delta O-18(H2O) = +2.7 +/- 2.3 parts per thousand VSMOW, n = 7), such as the lake water. Differences in Sr and 0 isotopic compositions between large and small mammal teeth indicate different home ranges and drinking behaviour and support migration of some large mammals between the Swabian Alb plateau and the nearby Molasse basin, while small mammals ingested their food and water locally. Changes in the lake level, water chemistry, and temperature were inferred using isotopic compositions of ostracod and gastropod shells from a composite lake sediment profile. Calcitic ostracod valves (Ilyocypris binocularis; delta O-18 = +1.7 +/- 1.2 parts per thousand VPDB, delta C-18 = -0.5 +/- 0.9 parts per thousand, VPDB, n = 68) and aragonitic, gastropod shells (Gyraulus spp.; delta O-18 = +2.0 +/- 13 parts per thousand VPDB, delta C-13 = -1.1 +/- 1.3 parts per thousand VPDB, n = 89) have delta O-18 and delta C-13 values similar to or even higher than those of marine, carbonates. delta C-13 values:of the biogenic carbonates parallel lake level fluctuations while delta O-18 values scatter around +2 +/- 2 parts per thousand and reflect the short term variability of meteoric water inflow vs. longer term evaporation. Sr-87/Sr-86 ratios of aragonitic Gyraulus spp. gastropod shells parallel the lake level fluctuations, reflecting variable inputs of groundwater and surface waters. Using a water delta O-18(H2O) value of +2.0 parts per thousand VSMOW, water temperatures calculated from skeletal tissue delta O-18 values of ostracods are 16.7 +/- 5.0 degrees C, gastropods 20.6 +/- 5.6 degrees C, otoliths 21.8 +/- 1.4 degrees C, and fish teeth 17.0 +/- 2.7 degrees C. The calculated MAT (similar to 19 degrees C), lake water temperatures (similar to 17 to 22 degrees C) and the O-18-enriched water compositions are indicative of warm-temperate climatic conditions, possibly with a high humidity during this period. Vegetation in the area surrounding the basin was largely of the C-3-type, as indicated by carbon isotopic compositions of tooth enamel from large mammals (delta C-13 = -11.1 +/- 1.1 parts per thousand VPDB, n = 40). (c) 2006 Elsevier B.V. All rights reserved.

Elevated levels of MIC-1/GDF15 in the cerebrospinal fluid of patients are associated with glioblastoma and worse outcome.

For patients with brain tumors identification of diagnostic and prognostic markers in easy accessible biological material, such as plasma or cerebrospinal fluid (CSF), would greatly facilitate patient management. MIC-1/GDF15 (growth differentiation factor 15) is a secreted protein of the TGF-beta superfamily and emerged as a candidate marker exhibiting increasing mRNA expression during malignant progression of glioma. Determination of MIC-1/GDF15 protein levels by ELISA in the CSF of a cohort of 94 patients with intracranial tumors including gliomas, meningioma and metastasis revealed significantly increased concentrations in glioblastoma patients (median, 229 pg/ml) when compared with control cohort of patients treated for non-neoplastic diseases (median below limit of detection of 156 pg/ml, p < 0.0001, Mann-Whitney test). However, plasma MIC-1/GDF15 levels were not elevated in the matching plasma samples from these patients. Most interestingly, patients with glioblastoma and increased CSF MIC-1/GDF15 had a shorter survival (p = 0.007, log-rank test). In conclusion, MIC-1/GDF15 protein measured in the CSF may have diagnostic and prognostic value in patients with intracranial tumors.

Cytokine levels in bronchoalveolar lavage and serum in 3 patients with 2009 influenza A(H1N1)v severe pneumonia.

Introduction: Pandemic Influenza A (H1N1)v pneumonia has led to a notable increase of admissions to intensive care units. A cytokine-mediated inflammatory response has been well documented in pneumonia and acute respiratory distress syndrome. However, few studies have focused on the role of these inflammatory mediators in infections caused by the Influenza A (H1N1)v. In this study, we assess the inflammatory response mediated by cytokines at the local and systemic levels in three cases of severe pneumonia caused by Influenza A (H1N1) virus. Methodology: Serum and bronchoalveolar lavage samples were obtained from three mechanically ventilated patients diagnosed with Influenza A (H1N1) virus pneumonia by bronchoscopic bronchoalveolar lavage. Levels of interleukin 6 (IL-6), interleukin 8 (IL-8), tumour necrosis factor alpha (TNFα) and interleukin 1 beta (IL-1ß) were meassured in these samples by enzyme-linked immunosorbent assay (ELISA). Results: High levels of C Reactive Protein, Procalcitonin below 1 ng/ml and absence of leukocytosis were common findings in all patients. TNF α and IL-1ß were not detected in the serum. IL-6 levels in serum were (94, pg/ml, 77 pg/ml and 84 pg/ml) respectively in the three patients, while IL-8 levels were (30,2 pg/ml, 128 pg/ml and 40,5 pg/ml). In the BAL samples, only one of the analysed cytokines, IL-1ß was present at detectable levels in two patients (21 pg/ml and 11 pg/ml respectively). Conclusions: Our results support previous findings which suggest that high levels of IL-6 and IL-8 in serum somehow participate in the inflammatory response in severe cases of pandemic influenza pneumonia.

Decreased PM10 exposure attenuates age-related lung function decline: genetic variants in p53, p21, and CCND1 modify this effect.

BACKGROUND: Decreasing exposure to airborne particulates was previously associated with reduced age-related decline in lung function. However, whether the benefit from improved air quality depends on genetic background is not known. Recent evidence points to the involvement of the genes p53 and p21 and of the cell cycle control gene cyclin D1 (CCND1) in the response of bronchial cells to air pollution. OBJECTIVE: We determined in 4,326 participants of the Swiss Cohort Study on Air Pollution and Lung and Heart Diseases in Adults (SAPALDIA) whether four single-nucleotide polymorphisms in three genes [CCND1 (rs9344 [P242P], rs667515), p53 (rs1042522 [R72P]), and p21 (rs1801270 [S31R])] modified the previously observed attenuation of the decline in the forced expiratory flow between 25% and 75% of the forced vital capacity (FEF(25-75)) associated with improved air quality. METHODS: Subjects of the prospective population-based SAPALDIA cohort were assessed in 1991 and 2002 by spirometry, questionnaires, and biological sample collection for genotyping. We assigned spatially resolved concentrations of particulate matter with aerodynamic diameter < or = 10 microm (PM(10)) to each participant's residential history 12 months before the baseline and follow-up assessments. RESULTS: The effect of diminishing PM(10) exposure on FEF(25-75) decline appeared to be modified by p53 R72P, CCND1 P242P, and CCND1 rs667515. For example, a 10-microg/m(3) decline in average PM(10) exposure over an 11-year period attenuated the average annual decline in FEF(25-75) by 21.33 mL/year (95% confidence interval, 10.57-32.08) among participants homozygous for the CCND1 (P242P) GG genotype, by 13.72 mL/year (5.38-22.06) among GA genotypes, and by 6.00 mL/year (-4.54 to 16.54) among AA genotypes. CONCLUSIONS: Our results suggest that cell cycle control genes may modify the degree to which improved air quality may benefit respiratory function in adults.

Genetic investigation of the functions of c-Myc and N-Myc in Hematopoietic stem cells

Résumé : c-Myc, le premier facteur de transcription de la famille Myc a été découvert il y a maintenant trente ans. Il reste à l'heure actuelle parmi les plus puissants proto-oncogènes connus. c-Myc est dérégulé dans plus de 50% des cancers, où il promeut la prolifération, la croissance cellulaire, et la néoangiogenèse. Myc peut aussi influencer de nombreuses autres fonctions de par sa capacité à activer ou à réprimer la transcription de nombreux gènes, et à agir globalement sur le génome à travers des modifications épigénétiques de la chromatine. La famille d'oncogènes Myc comprend, chez les mammifères, trois protéines structurellement proches: c-Myc, N-Myc et L-Myc. Ces protéines ont les mêmes proprietés biochimiques, exercent les mêmes fonctions mais sont le plus souvent exprimées de façon mutuellement exclusive. Myc a été récemment identifié comme un facteur clef dans la maintenance des cellules souches embryonnaires et adultes ainsi que dans la réacquisition des proprietés des cellules souches. Nous avons précédemment démontré que l'élimination de c-Myc provoque une accumulation de cellules souches hématopoïétiques (CSH) suite à un défaut de différenciation lié à la niche. Les CSH sont responsables de la production de tous les éléments cellulaires du sang pour toute la vie de l'individu et sont définies par leur capacité à s'auto-renouveler tout en produisant des précurseurs hématopoïétiques. Afin de mieux comprendre la fonction de Myc dans les CSH, nous avons choisi de combiner l'utilisation de modèles de souris génétiquement modifiées à une caractérisation systématique des schémas d'expression de c-Myc, N-Myc et L-Myc dans tout le système hématopoïétique. Nous avons ainsi découvert que les CSH les plus immatures expriment des quantités équivalentes de transcrits de c-myc et N-myc. Si les CSH déficientes en N-myc seulement ont une capacité d'auto-renouvellement à long-terme réduite, l'invalidation combinée des gènes c-myc et N-myc conduit à une pan-cytopénie suivie d'une mort rapide de l'animal, pour cause d'apoptose de tous les types cellulaires hématopoïétiques. En particulier, les CSH en cours d'auto-renouvelemment, mais pas les CSH quiescentes, accumulent du Granzyme B (GrB), une molécule fortement cytotoxique qui provoque une mort cellulaire rapide. Ces données ont ainsi mis au jour un nouveau mécanisme dont dépend la survie des CSH, à savoir la répression du GrB, une enzyme typiquement utilisée par le système immunitaire inné pour éliminer les tumeurs et les cellules infectées par des virus. Dans le but d'évaluer l'étendue de la redondance entre c-Myc et N-Myc dans les CSH, nous avons d'une part examiné des souris dans lesquelles les séquences codantes de c-myc sont remplacées par celles de N-myc (NCR) et d'autre part nous avons géneré une série allèlique de myc en éliminant de façon combinatoire un ou plusieurs allèles de c-myc et/ou de N-myc. Alors que l'analyse des souris NCR suggère que c-Myc et N-Myc sont qualitativement redondants, la série allélique indique que les efficiences avec lesquelles ces deux protéines influencent des procédés essentiels à la maintenance des CSH sont différentes. En conclusion, nos données génétiques montrent que l'activité générale de MYC, fournie par c-Myc et N-Myc, contrôle plusieurs aspects cruciaux de la fonction des CSH, notamment l'auto-renouvellement, la survie et la différenciation. Abstract : c-Myc, the first Myc transcription factor was discovered 30 years ago and is to date one of the most potent proto-oncogenes described. It is found to be misregulated in over 50% of all cancers, where it drives proliferation, cell growth and neo-angiogenesis. Myc can also influence a variety of other functions, owing to its ability to activate and repress transcription of many target genes and to globally regulate the genome via epigenetic modifications of the chromatin. The Myc family of oncogenes consists of three closely related proteins in mammals: c-Myc, N-Myc and L-Myc. These proteins share the same biochemical properties, exert mostly the same functions, but are most often expressed in mutually exclusive patterns. Myc is now emerging as a key factor in maintenance of embryonic and adult stem cells as well as in reacquisition of stem cell properties, including induced reprogramming. We previously showed that c-Myc deficiency can cause the accumulation of hematopoietic stem cells (HSCs) due to a niche dependent differentiation defect. HSCs are responsible for life-long replenishment of all blood cell types, and are defined by their ability to self-renew while concomitantly giving rise to more commited progenitors. To gain further insight into the function of Myc in HSCs, in this study we combine the use of genetically-modified mouse models with the systematic characterization of c-myc, N-myc and L-myc transcription patterns throughout the hematopoietic system. Interestingly, the most immature HSCs express not only c-myc, but also about equal amounts of N-myc transcripts. Although conditional deletion of N-myc alone in the bone marrow does not affect steady-state hematopoiesis, N-myc null HSCs show impaired long-term self-renewal capacity. Strikingly, combined deficiency of c-Myc and N-Myc results in pan-cytopenia and rapid lethality, due to the apoptosis of most hematopoietic cell types. In particular, self-renewing HSCs, but not quiescent HSCs or progenitor cell types rapidly up-regulate and accumulate the potent cytotoxic molecule GranzymeB (GrB), causing their rapid cell death. These data uncover a novel pathway on which HSC survival depends on, namely repression of GrB, a molecule typically used by the innate immune system to eliminate tumor and virus infected cells. To evaluate the extent of redundancy between c-Myc and N-Myc in HSCs, we examined mice in which c-myc coding sequences are replaced by that of N-myc (NCR) and also generated an allelic series of myc, by combinatorially deleting one or several c-myc and/or N-myc alleles. While the analysis of NCR mice suggests that c-Myc and N-Myc are qualitatively functionally redundant, our allelic series indicates that the efficiencies with which these two proteins affect crucial HSC maintenance processes are likely to be distinct. Collectively, our genetic data show that general "MYC" activity delivered by c-Myc and N-Myc controls crucial aspects of HSC function, including self-renewal, survival and niche dependent differentiation.

Substrate specificity of human kallikrein 2 (hK2) as determined by phage display technology.

Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease expressed predominantly in the prostate epithelium. Recently, hK2 has proven to be a useful marker that can be used in combination with prostate specific antigen for screening and diagnosis of prostate cancer. The cleavage by hK2 of certain substrates in the proteolytic cascade suggest that the kallikrein may be involved in prostate cancer development; however, there has been very little other progress toward its biochemical characterization or elucidation of its true physiological role. In the present work, we adapt phage substrate technology to study the substrate specificity of hK2. A phage-displayed random pentapeptide library with exhaustive diversity was generated and then screened with purified hK2. Phages displaying peptides susceptible to hK2 cleavage were amplified in eight rounds of selection and genes encoding substrates were transferred from the phage to a fluorescent system using cyan fluorescent protein (derived from green fluorescent protein) that enables rapid determination of specificity constants. This study shows that hK2 has a strict preference for Arg in the P1 position, which is further enhanced by a Ser in P'1 position. The scissile bonds identified by phage display substrate selection correspond to those of the natural biological substrates of hK2, which include protein C inhibitor, semenogelins, and fibronectin. Moreover, three new putative hK2 protein substrates, shown elsewhere to be involved in the biology of the cancer, have been identified thus reinforcing the importance of hK2 in prostate cancer development.

Mutation prevalence of cerebral cavernous malformation genes in Spanish patients.

OBJECTIVE To study the molecular genetic and clinical features of cerebral cavernous malformations (CCM) in a cohort of Spanish patients. METHODS We analyzed the CCM1, CCM2, and CCM3 genes by MLPA and direct sequencing of exons and intronic boundaries in 94 familial forms and 41 sporadic cases of CCM patients of Spanish extraction. When available, RNA studies were performed seeking for alternative or cryptic splicing. RESULTS A total of 26 pathogenic mutations, 22 of which predict truncated proteins, were identified in 29 familial forms and in three sporadic cases. The repertoire includes six novel non-sense and frameshift mutations in CCM1 and CCM3. We also found four missense mutations, one of them located at the third NPXY motif of CCM1 and another one that leads to cryptic splicing of CCM1 exon 6. We found four genomic deletions with the loss of the whole CCM2 gene in one patient and a partial loss of CCM1and CCM2 genes in three other patients. Four families had mutations in CCM3. The results include a high frequency of intronic variants, although most of them localize out of consensus splicing sequences. The main symptoms associated to clinical debut consisted of cerebral haemorrhage, migraines and epileptic seizures. The rare co-occurrence of CCM with Noonan and Chiari syndromes and delayed menarche is reported. CONCLUSIONS Analysis of CCM genes by sequencing and MLPA has detected mutations in almost 35% of a Spanish cohort (36% of familial cases and 10% of sporadic patients). The results include 13 new mutations of CCM genes and the main clinical symptoms that deserves consideration in molecular diagnosis and genetic counselling of cerebral cavernous malformations.

HCF-1 self-association via an interdigitated Fn3 structure facilitates transcriptional regulatory complex formation.

Host-cell factor 1 (HCF-1) is an unusual transcriptional regulator that undergoes a process of proteolytic maturation to generate N- (HCF-1(N)) and C- (HCF-1(C)) terminal subunits noncovalently associated via self-association sequence elements. Here, we present the crystal structure of the self-association sequence 1 (SAS1) including the adjacent C-terminal HCF-1 nuclear localization signal (NLS). SAS1 elements from each of the HCF-1(N) and HCF-1(C) subunits form an interdigitated fibronectin type 3 (Fn3) tandem repeat structure. We show that the C-terminal NLS recruited by the interdigitated SAS1 structure is required for effective formation of a transcriptional regulatory complex: the herpes simplex virus VP16-induced complex. Thus, HCF-1(N)-HCF-1(C) association via an integrated Fn3 structure permits an NLS to facilitate formation of a transcriptional regulatory complex.

Basic calcium phosphate crystals induce monocyte/macrophage IL-1(beta) secretion through the NLRP3 inflammasome in vitro.

Basic calcium phosphate (BCP) crystals are associated with severe osteoarthritis and acute periarticular inflammation. Three main forms of BCP crystals have been identified from pathological tissues: octacalcium phosphate, carbonate-substituted apatite, and hydroxyapatite. We investigated the proinflammatory effects of these BCP crystals in vitro with special regard to the involvement of the NLRP3-inflammasome in THP-1 cells, primary human monocytes and macrophages, and mouse bone marrow-derived macrophages (BMDM). THP-1 cells stimulated with BCP crystals produced IL-1β in a dose-dependent manner. Similarly, primary human cells and BMDM from wild-type mice also produced high concentrations of IL-1β after crystal stimulation. THP-1 cells transfected with short hairpin RNA against the components of the NLRP3 inflammasome and mouse BMDM from mice deficient for NLRP3, apoptosis-associated speck-like protein, or caspase-1 did not produce IL-1β after BCP crystal stimulation. BCP crystals induced macrophage apoptosis/necrosis as demonstrated by MTT and flow cytometric analysis. Collectively, these results demonstrate that BCP crystals induce IL-1β secretion through activating the NLRP3 inflammasome. Furthermore, we speculate that IL-1 blockade could be a novel strategy to inhibit BCP-induced inflammation in human disease.

IRF6 is a mediator of Notch pro-differentiation and tumour suppressive function in keratinocytes.

While the pro-differentiation and tumour suppressive functions of Notch signalling in keratinocytes are well established, the underlying mechanisms remain poorly understood. We report here that interferon regulatory factor 6 (IRF6), an IRF family member with an essential role in epidermal development, is induced in differentiation through a Notch-dependent mechanism and is a primary Notch target in keratinocytes and keratinocyte-derived SCC cells. Increased IRF6 expression contributes to the impact of Notch activation on growth/differentiation-related genes, while it is not required for induction of 'canonical' Notch targets like p21(WAF1/Cip1), Hes1 and Hey1. Down-modulation of IRF6 counteracts differentiation of primary human keratinocytes in vitro and in vivo, promoting ras-induced tumour formation. The clinical relevance of these findings is illustrated by the strikingly opposite pattern of expression of Notch1 and IRF6 versus epidermal growth factor receptor in a cohort of clinical SCCs, as a function of their grade of differentiation. Thus, IRF6 is a primary Notch target in keratinocytes, which contributes to the role of this pathway in differentiation and tumour suppression.

Origin of mineralizing fluids of the sediment-hosted Navachab gold mine, Namibia: Constraints from stable (O, H, C, S) isotopes

The Navachab gold mine in the Damara belt of central Namibia is characterized by a polymetallic Au-Bi-As-Cu-Ag ore assemblage, including pyrrhotite, chalcopyrite, sphalerite, arsenopyrite, bismuth, gold, bismuthinite, and bismuth tellurides. Gold is hosted by quartz sulfide veins and semimassive sulfide lenses that are developed in a near-vertical sequence of shelf-type metasedimentary rocks, including marble, calcsilicate rock, and biotite schist. The sequence has been intruded by abundant syntectonic lamprophyre, aplite, and pegmatite dikes, documenting widespread igneous activity coeval with mineralization. The majority of quartz from the veins has delta(18)O values of 14 to 15 per mil (V-SMOW). The total variations in delta(18)O values of the biotite schist and calcsilicate rock are relatively small (12-14 parts per thousand), whereas the marble records steep gradients in delta(18)O values (17-21 parts per thousand), the lowest values being recorded at the vein margins. Despite this, there is no correlation between delta(18)O and delta(13)C values and the carbonate content of the rocks, indicating that fluid-rock interaction alone cannot explain the isotopic gradients. In addition, the marble records increased delta(13)C values at the contact to the veins, possibly related to a change in the physicochemical conditions during fluid-rock interaction. Gold is interpreted to have precipitated in equilibrium with metamorphic find (delta(18)O 12-14 parts per thousand; delta D = -40 to -60 parts per thousand) at peak metamorphic conditions of ca. 550 degrees C and 2 kbars, consistent with isotopic fractionations between coexisting calcite, garnet, and clinopyroxene in the alteration halos. The most likely source of the mineralizing fluid was a midcrustal fluid in equilibrium with the Damaran metapelites that underwent prograde metamorphism at amphibolite- to granulite-facies grades. Although there is no isotopic evidence for the contribution of magmatic fluids, they may have been important in contributing to the overall hydraulic regime and high apparent geothermal gradients (ca. 80 degrees C/km(-1)) in the mine area.

A validated assay for measuring doxorubicin in biological fluids and tissues in an isolated lung perfusion model: matrix effect and heparin interference strongly influence doxorubicin measurements.

Doxorubicin is an antineoplasic agent active against sarcoma pulmonary metastasis, but its clinical use is hampered by its myelotoxicity and its cumulative cardiotoxicity, when administered systemically. This limitation may be circumvented using the isolated lung perfusion (ILP) approach, wherein a therapeutic agent is infused locoregionally after vascular isolation of the lung. The influence of the mode of infusion (anterograde (AG): through the pulmonary artery (PA); retrograde (RG): through the pulmonary vein (PV)) on doxorubicin pharmacokinetics and lung distribution was unknown. Therefore, a simple, rapid and sensitive high-performance liquid chromatography method has been developed to quantify doxorubicin in four different biological matrices (infusion effluent, serum, tissues with low or high levels of doxorubicin). The related compound daunorubicin was used as internal standard (I.S.). Following a single-step protein precipitation of 500 microl samples with 250 microl acetone and 50 microl zinc sulfate 70% aqueous solution, the obtained supernatant was evaporated to dryness at 60 degrees C for exactly 45 min under a stream of nitrogen and the solid residue was solubilized in 200 microl of purified water. A 100 microl-volume was subjected to HPLC analysis onto a Nucleosil 100-5 microm C18 AB column equipped with a guard column (Nucleosil 100-5 microm C(6)H(5) (phenyl) end-capped) using a gradient elution of acetonitrile and 1-heptanesulfonic acid 0.2% pH 4: 15/85 at 0 min-->50/50 at 20 min-->100/0 at 22 min-->15/85 at 24 min-->15/85 at 26 min, delivered at 1 ml/min. The analytes were detected by fluorescence detection with excitation and emission wavelength set at 480 and 550 nm, respectively. The calibration curves were linear over the range of 2-1000 ng/ml for effluent and plasma matrices, and 0.1 microg/g-750 microg/g for tissues matrices. The method is precise with inter-day and intra-day relative standard deviation within 0.5 and 6.7% and accurate with inter-day and intra-day deviations between -5.4 and +7.7%. The in vitro stability in all matrices and in processed samples has been studied at -80 degrees C for 1 month, and at 4 degrees C for 48 h, respectively. During initial studies, heparin used as anticoagulant was found to profoundly influence the measurements of doxorubicin in effluents collected from animals under ILP. Moreover, the strong matrix effect observed with tissues samples indicate that it is mandatory to prepare doxorubicin calibration standard samples in biological matrices which would reflect at best the composition of samples to be analyzed. This method was successfully applied in animal studies for the analysis of effluent, serum and tissue samples collected from pigs and rats undergoing ILP.

How Much Chemotherapy Are Patients With Advanced Pancreatic Cancer Receiving at the End of Life?

Background: Advanced pancreatic adenocarcinoma (APC) is a chemoresistant cancer with poor prognosis. We evaluated the use of chemotherapy in the last months of life.Methods: Retrospective analysis of patients with APC treated from 1993 to 2010 at the Oncology Institute of Southern Switzerland. Clinical and laboratory parameters starting from 28 days prior to the last administration of chemotherapy were recorded, including ECOG performance status, presence of ascites, haemoglobin (Hb), white blood cell (WBC) count, platelets, total bilirubin, albumin, LDH, C-reactive protein (C-rp) and Ca 19.9.Results: The characteristics of the 231 patients were: males/females 53%/47%; metastatic/locally advanced disease 80%/20%; median age 66 years (range 32−85). Median overall survival calculated from diagnosis was 6.1 months (95% CI: 5.1−7.2); death was due to disease progression in all cases. At last chemotherapy administration, ECOG performance status was 0−1 in 38% and 2−3 in 62%. Fifty-nine percent of pts received first-line chemotherapy only (gemcitabine in 70%; gemcitabine-based doublets or 5FU in 30%), whilst 32%, 8% and 1% had second- (5FU 37%; oxaliplatinbased doublets 57%; phase I trial 6%), third- and fourth-line therapy (single agent or phase I trial), respectively. The interval between last chemotherapy administration and death was <4 weeks in 24%, _4−12 weeks in 47% and >12 weeks in 29%. Table 1 summarizes the proportion of patients treated according to the interval between last chemotherapy and death refered to chemotherapy line. Median survival from last chemotherapy delivery to death was 7.5 weeks (95% CI 6.7−8.4). In univariate analysis, presence of ascites, elevated WBC, total bilirubin, LDH, C-rp and Ca 19.9, and reduced albumin were found to predict shorter survival (p < 0.05 for each). However, none of them was an independent predictor in the multivariate analysis.Conclusions: A significant proportion of patients with APC received chemotherapy in the last months of life. In our study, none of the clinical and laboratory parameters recorded 28 days priorto the last chemotherapy delivery were found to predict survival.

Vitamin C requirements in parenteral nutrition.

Some biochemical functions of vitamin C make it an essential component of parenteral nutrition (PN) and an important therapeutic supplement in other acute conditions. Ascorbic acid is a strong aqueous antioxidant and is a cofactor for several enzymes. The average body pool of vitamin C is 1.5 g, of which 3%-4% (40-60 mg) is used daily. Steady state is maintained with 60 mg/d in nonsmokers and 140 mg/d in smokers. Shocked surgical, trauma, and septic patients have a drastic reduction of circulating plasma ascorbate concentrations. These low concentrations require 3-g doses/d to restore normal plasma ascorbate concentrations, questioning the recommended PN dose of 100 mg/d. Determination of intravenous requirements is usually based on plasma concentrations, which are altered during the inflammatory response. There is no clear indicator of deficiency: serum or plasma ascorbate concentrations <0.3 mg/dL (20 micromol/L) indicates inadequate vitamin C status. On the basis of available pharmacokinetic data the 100 mg/d dose for patients receiving home PN and 200 mg/d for stable adult patients receiving PN are adequate, but requirements have been shown to be higher in perioperative, trauma, burn, and critically ill patients, paralleling oxidative stress. One recommendation cannot fit all categories of patients. Large vitamin C supplements may be considered in severe critical illness, major trauma, and burns because of increased requirements resulting from oxidative stress and wound healing. Future research should distinguish therapeutic use of high-dose ascorbic acid antioxidant therapy from nutritional PN requirements.