A Late Pleistocene coastal ecosystem in French Guiana was hyperdiverse relative to today.

Warmer temperatures and higher sea level than today characterized the Last Interglacial interval [Pleistocene, 128 to 116 thousand years ago (ka)]. This period is a remarkable deep-time analog for temperature and sea-level conditions as projected for 2100 AD, yet there has been no evidence of fossil assemblages in the equatorial Atlantic. Here, we report foraminifer, metazoan (mollusks, bony fish, bryozoans, decapods, and sharks among others), and plant communities of coastal tropical marine and mangrove affinities, dating precisely from a ca. 130 to 115 ka time interval near the Equator, at Kourou, in French Guiana. These communities include ca. 230 recent species, some being endangered today and/or first recorded as fossils. The hyperdiverse Kourou mollusk assemblage suggests stronger affinities between Guianese and Caribbean coastal waters by the Last Interglacial than today, questioning the structuring role of the Amazon Plume on tropical Western Atlantic communities at the time. Grassland-dominated pollen, phytoliths, and charcoals from younger deposits in the same sections attest to a marine retreat and dryer conditions during the onset of the last glacial (ca. 110 to 50 ka), with a savanna-dominated landscape and episodes of fire. Charcoals from the last millennia suggest human presence in a mosaic of modern-like continental habitats. Our results provide key information about the ecology and biogeography of pristine Pleistocene tropical coastal ecosystems, especially relevant regarding the-widely anthropogenic-ongoing global warming.

Mortality attributable to different Klebsiella susceptibility patterns and to the coverage of empirical antibiotic therapy: a cohort study on patients admitted to the ICU with infection.

PURPOSE: To evaluate the prognostic importance of different Klebsiella spp. sensitivity patterns: multi-susceptible Klebsiella (MS-K), extended-spectrum cephalosporin-resistant, but carbapenem-susceptible Klebsiella (ESCR-CS-K), and carbapenem-resistant Klebsiella (CR-K). METHODS: We developed a prognostic model to predict hospital mortality in patients with infection on admission to the intensive care units (ICUs), and assessed its calibration in the subgroups of interest: patients with infections due to MS-K, ESCR-CS-K, CR-K. We assessed the calibration of the model also in ESCR-CS-K treated empirically with carbapenems and with piperacillin-tazobactam. RESULTS: A total of 13,292 adults with an ongoing infection were admitted to 137 Italian ICUs in 2012-2013. Of 801 Klebsiella spp. infected patients, 451 had MS-K, 116 ESCR-CS-K, and 234 CR-K. The prognostic model calibrated well for the MS-K and ESCR-CS-K subgroups. In the CR-K subgroup there were more deaths than predicted (standardized mortality ratio 1.20; 95% CI 1.08-1.31), indicating a negative prognostic role of the infection, mainly in the medium and high risk-of-death patients. When infection was caused by ESCR-CS-K, treatment with piperacillin-tazobactam increased adjusted mortality among the most severe patients (similarly to CR-K), while treatment with carbapenems did not (similarly to MS-K). CONCLUSIONS: In low risk-of-death patients admitted to the ICU with a Klebsiella spp. infection, the appropriateness of empirical antibiotic therapy seemed uninfluential to eventual mortality, while it appeared to be crucial in high-risk ones. The use of piperacillin-tazobactam may be inappropriate in severe patients with ESCR-CS-K infection. CR-K is associated to a significant 20% increase of adjusted mortality, only for patients at higher risk of death.

Epidural analgesia for cytoreductive surgery with peritonectomy and heated intraperitoneal chemotherapy.

PURPOSE: To evaluate epidural analgesia role after cytoreductive surgery with peritonectomy combined with heated intraperitoneal chemotherapy. METHODS: 101 patients were retrospectively studied (between 2008 and 2012) to evaluate epidural analgesia effectiveness, tolerability and safety in this surgical context through the assessment of pain, detection of adverse events (nausea, vomiting, itching), temporary motor block, respiratory failure and coagulation profile in the post-operative period. RESULTS: The median duration of epidural analgesia was 5 [range 1-10] days. As regards pain relief, the median verbal numerical scale scores at rest and on movement were below 2 and 5 until the fifth post-operative day, respectively. 13% of patients suffered nausea, 4% vomit, and 1% itching. No bradycardia or respiratory failure event was reported. 9.9% of patients had hypotension episodes. Coagulation reached normality only 3-4 days after surgery. 5 risky accidental dislodgments of epidural catheter occurred (prothrombine time INR > 1.5) without neurological complications. CONCLUSIONS: Epidural analgesia ensures adequate pain relief and is well tolerated by patients after cytoreductive surgery with peritonectomy combined with heated intraperitoneal chemotherapy. Hypotension is common in this context and careful monitoring of coagulation parameters, especially in the first 3 days after surgery, is advisable to reduce the risk of neuraxial complications.

Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC) in a patient with peritoneal mesothelioma and HIV infection.

BACKGROUND: High rates of septic complications have been associated with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy, which has been suggested as the treatment of choice for isolated peritoneal malignancies. Patients infected by the human immunodeficiency virus (HIV) are still considered at a high operative risk. METHOD: A 58-year-old man with HIV infection and diffuse peritoneal mesothelioma underwent optimal cytoreductive surgery and hyperthermic intraperitoneal chemotherapy. RESULTS: The patient experienced a complete clinical response to therapy with no adverse effect on disease course or markers for HIV (CD4 count, beta2-microglobulin, neopterin, p24 antigen, and viral load). CONCLUSION: This report suggests that this innovative approach can be successfully performed also in this clinical setting. In selected patients who respond to all criteria, surgery is possible and is a safe and effective therapeutic option.

Muscle strength quantification in small animals: a new transcutaneous technique in rabbits.

The objective of this study was to develop a new, simple, and noninvasive technique to measure the force produced by dorsi-flexion of the foot in small animals. In addition, this study aimed to quantitatively describe changes in muscle and soft tissue structures using histomorphometry. The recovery of the dorsi-flexing muscles in the tibialis anterior compartment in New Zealand White rabbits was evaluated after musculoskeletal trauma by measuring isometric contractions after submaximal transcutaneous electrical stimulation of the peroneal nerve. The trauma included muscle and bone trauma that was treated with limb shortening followed by distraction osteogenesis. Muscle contractions were initiated at an amplitude of 5.1 mA for a duration of 2.56 ms at intervals of 50 ms. Based on consecutive measurements of the force on days 5, 10, 15, 20, 25, and 30 postsurgery, a positive trend in recovery of the stimulated force produced by dorsi-flexion of the foot was observed. The muscle strength at 30 days postsurgery was compared to that measured presurgery (baseline): 55% of the animals had dorsi-flexion strength that was 60% below that of the presurgery baseline muscle strength; 36% of the animals had dorsi-flexion strength that was greater than 60% of the baseline measure, indicating that there was a significant decrease in force produced by dorsi-flexion of the foot after trauma on all testing days (p < 0.01) and that a severe muscular injury was set with limited recovery. This technique provides a new option for examining muscle regeneration and rehabilitation in small animals.

Pseudomyxoma peritonei: clinical pathological and biological prognostic factors in patients treated with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC).

BACKGROUND: Surgical cytoreduction combined with hyperthermic intraperitoneal chemotherapy (HIPEC) has been recently advocated as the standard of care for pseudomyxoma peritonei (PMP). We reviewed our 10-year monoinstitutional case series to identify selection factors predicting postoperative outcome. METHODS: One hundred and four patients with PMP were operated on with the aim of performing adequate cytoreduction (residual tumor nodules < or =2.5 mm) and closed-abdomen HIPEC with mytomicin-C and cisplatin. Previously, 26 patients had systemic chemotherapy. PMP was histologically classified into disseminated peritoneal adenomucinosis (DPAM), peritoneal mucinous carcinomatosis (PMCA), and intermediate/discordant group (ID). Immunohistochemical stains were performed for cytokeratin (CK)-7, CK-20, CDX-2, MUC-2, MUC-5AC, CD-44s. The significance of 22 potential clinical, pathological, and biological prognostic variables was assessed by multivariate analysis. RESULTS: Adequate cytoreduction was performed in 89 patients, suboptimal cytoreduction in six, palliative surgery in nine. Operative mortality was 1%. Seventy-eight patients were diagnosed with DPAM, 26 with PMCA, and none with ID. Median follow-up was 37 months (range, 1-110) for the overall series. Five-year overall survival (OS) and progression-free survival (PFS) were 78.3% and 31.1%, respectively. At multivariate analysis, adequate cytoreduction, no previous systemic chemotherapy, and DPAM correlated to better OS and PFS, elevated serum CA19.9 correlated only to better PFS. In most cases, CK20, CDX-2, and MUC-2 were diffusely positive, while CK-7, MUC-5AC, and CD44s were variably expressed. CK20 expression correlated to prognosis at univariate analysis. CONCLUSIONS: Favorable outcome after comprehensive treatment can be expected in patients with DPAM, not treated with preoperative systemic chemotherapy and amenable to adequate cytoreduction. MUC-2, CK-20, and CD44s expression may be related to PMP unique biologic behavior.

Binding of recombinant mistletoe lectin (aviscumine) to resected human adenocarcinoma of the lung.

BACKGROUND: Lectins, carbohydrate proteins, bind to glycoconjugates of all mammalian cells, including cancer cells. Aberrant glycosylation, detected by lectin histochemistry, can predict outcome in some tumour entities. One such lectin is aviscumine (recombinant mistletoe lectin). Aviscumine has cytotoxic effects and can therefore be used as anti-tumour therapy. MATERIALS AND METHODS: Lectin histochemistry with aviscumine was performed on primary tumour sections from resected adenocarcinoma of the lung. Staining results were then correlated with the clinical course of the patients. RESULTS: Most of the adenocarcinomas (92.5%) bound aviscumine. Kaplan-Meier analysis revealed no correlation between aviscumine binding and progression-free survival or overall survival. CONCLUSION: These results suggest that for the selected group of patients with adenocarcinoma of the lung aviscumine binding activity can not serve as a prognostic factor. More strikingly, however, aviscumine binds to malignant cells in 92.5% of the patients. This is an indicator for the use of aviscumine as a possible target for tumour therapy.

Adaptation and performance of an immuno-PCR assay for the quantification of Aviscumine in patient plasma samples.

An immuno-polymerase chain reaction (IPCR) assay is used to evaluate the kinetic behaviour of the novel anti-cancer drug Aviscumine in plasma samples taken from 41 patients during a 3-year clinical trial. The ultrasensitive IPCR assay employed the amplification of a detection-antibody linked marker-DNA and an internal competitor DNA for standardization, thus enabling the detection of the antigen in concentrations far below the detection limit of conventional enzyme-linked immuno-sorbent assay (ELISA). The quantification of Aviscumine was carried out using external calibration curves obtained from individual patient plasma samples, collected previous to the administration of Aviscumine, which were spiked with known amounts of the reference substance Aviscumine. Additional controls were measured containing standardized human serum spiked with Aviscumine to assure the continuous general reproducibility of the assay as well as to estimate differences between individual patients. Average recovery was found to be 95+/-19% and the average deviation in precision of the assay was determined to be 9+/-5%. Data for the quantification of Aviscumine were obtained from all patient samples investigated with the exception of a single patient. The collected data provided the basis for the valid routine quantification of patient samples for the calculation of the pharmacokinetic behaviour of Aviscumine in patient plasma.

Tumor-associated CD75s gangliosides and CD75s-bearing glycoproteins with Neu5Acalpha2-6Galbeta1-4GlcNAc-residues are receptors for the anticancer drug rViscumin.

The anticancer drug rViscumin, currently under clinical development, has been shown in previous studies to be a sialic acid specific ribosome inactivating protein (RIP). Comparative binding assays with the CD75s-specific monoclonal antibodies HB6 and J3-89 revealed rViscumin to be a CD75s-specific RIP due to identical binding characteristics toward CD75s gangliosides. The receptor gangliosides are IV6nLc4Cer, VI6nLc6Cer, and the newly characterized ganglioside VIII6nLc8Cer, all three carrying the Neu5Acalpha2-6Galbeta1-4GlcNAc motif. To elucidate the clinical potential of the rViscumin targets, CD75s gangliosides were determined in several randomly collected gastrointestinal tumors. The majority of the tumors showed an enhanced expression of CD75s gangliosides compared with the unaffected tissues. The rViscumin binding specificity was further investigated with reference glycoproteins carrying sialylated and desialylated type II N-glycans. Comparative Western blots of rViscumin and ricin, an rViscumin homologous but galactoside-specific RIP, revealed specific recognition of type II N-glycans with CD75s determinants by rViscumin, whereas ricin failed to react with terminally sialylated oligosaccharides such as CD75s motifs and others. This strict binding specificity of rViscumin and the increased expression of CD75s gangliosides in various tumors suggest this anticancer drug as a promising candidate for an individualised adjuvant therapy of human tumors.

Cytotoxicity of the novel anti-cancer drug rViscumin depends on HER-2 levels in SKOV-3 cells.

rViscumin is a recombinant mistletoe lectin under clinical investigation as new anti-cancer drug. The relationship between oncogene, e.g., HER-2/neu (c-erbB2) receptor activation and tumor cell chemosensitivity, is of considerable importance to better predict the response to chemotherapy. Here, we analyze the cellular and molecular effects of HER-2 expression on rViscumin chemotoxicity in SKOV-3 cells. We show that selective depletion of HER-2 by ribozyme-targeting markedly decreases cellular sensitivity towards rViscumin. These findings are confirmed by treatment with the well-established inhibitory HER-2 antibody trastuzumab (Herceptin). Using clonal ribozyme-transfected cell lines, we establish a 'HER-2 gene dose' dependence of rViscumin cytotoxicity, which is due to differential induction of apoptosis and is not mediated by cell cycle alterations or altered cellular rViscumin binding/internalization. We further demonstrate an rViscumin-mediated, HER-2-dependent down-regulation of bcl-2 and the dose-dependent activation of members of the MAPK family, p42/44, SAPK/JNK, and p38, but not of caspases-3 and -7.

Mistletoe lectin I is a sialic acid-specific lectin with strict preference to gangliosides and glycoproteins with terminal Neu5Ac alpha 2-6Gal beta 1-4GlcNAc residues.

Mistletoe lectin I (ML-I) is a type II ribosome-inactivating protein, which inhibits the protein biosynthesis at the ribosomal level. ML-I is composed of a catalytically active A-chain with rRNA N-glycosidase activity and a B-chain with carbohydrate binding specificities. Using comparative solid-phase binding assays along with electrospray ionization tandem mass spectrometry, ML-I was shown to preferentially bind to terminally alpha2-6-sialylated neolacto series gangliosides from human granulocytes. IV(6)Neu5Ac-nLc4Cer, VI(6)Neu5Ac-nLc6Cer, and VIII(6)Neu5Ac-nLc8Cer were identified as ML-I receptors, whereas the isomeric alpha2-3-sialylated neolacto series gangliosides were not recognized. Only marginal binding of ML-I to terminal galactose residues of neutral glycosphingolipids with a Galbeta1-4Glc or Galbeta1-4GlcNAc sequence was determined, whereas a distal Galalpha1-4Gal, GalNAcbeta1-3Gal, or GalNAcbeta1-4Gal disaccharide did not bind at all. Among the glycoproteins investigated in Western blot and microwell adsorption assays, only those carrying Neu5Acalpha2-6Galbeta1-4GlcNAc residues, exclusively, predominantly, or even as less abundant constituents in an assembly with Neu5Acalpha2-3Galbeta1-4GlcNAc-terminated glycans, displayed high ML-I binding capacity. From our data we conclude that (i) ML-I has to be considered as a sialic acid- and not a galactose-specific lectin and (ii) neolacto series gangliosides and sialoglycoproteins with type II glycans, which share the Neu5Acalpha2-6Galbeta1-4GlcNAc terminus, are true ML-I receptors. This strict preference might help to explain the immunostimulatory potential of ML-I toward certain leukocyte subpopulations and its therapeutic success as a cytotoxic anticancer drug.

Detection of rViscumin in plasma samples by immuno-PCR.

To allow for pharmacokinetic studies in adjunction with the current clinical developments of the potent cytostatic anti-cancer drug rViscumin, a sandwich immuno-PCR (IPCR) assay was developed for the detection of rViscumin in blood plasma. The IPCR was carried out with a commercially available reagent kit, consisting of pre-assembled rViscumin-specific antibody-DNA conjugates as well as a specific competitor DNA fragment to be amplified by PCR. Various combinations of capture- and detection-antibodies were compared for performance in IPCR. Using the optimized assay, as few as 50 zeptomol (approx. 100 fg/ml) rViscumin (MW 57 kDa) was detectable in standardized human serum samples. The IPCR assay was very selective for rViscumin and in spiking experiments in proband plasma samples, signal recovery rates between 70% and 120% were obtained. The linear sensitivity range of the assay covered more than five orders of magnitude. Repeated measurements of rViscumin resulted in a mean standard deviation value of 14.2%.

Preferential binding of the anticancer drug rViscumin (recombinant mistletoe lectin) to terminally alpha2-6-sialylated neolacto-series gangliosides.

Production of biochemically defined recombinant mistletoe lectin was achieved by cloning and separate expression of the single catalytically active A-chain and the B-chain with carbohydrate binding properties in Escherichia coli, yielding an active heterodimeric protein named rViscumin (Eck et al. [1999] Eur. J. Biochem., 265, 788-797). Employing solid phase binding assays, rViscumin was shown to preferentially bind to terminally alpha2-6-sialylated neolacto-series gangliosides IV(6)Neu5Ac-nLc4Cer, VI(6)Neu5Ac-nLc6Cer, and VIII(6)Neu5Ac-nLc8Cer isolated from human granulocytes. Only marginal binding of rViscumin to galactose-terminated neutral GSLs was determined, whereas reinvestigation of ricin specificity demonstrated this lectin as a galactose-binding protein. Human promyelotic HL-60 cells exhibited an IC(50) value (half maximum cytotoxicity) of 1.16 pM and human bladder carcinoma 5637 cells of 12.1 pM rViscumin; CHO-K1 cells were resistant to rViscumin treatment up to a concentration of 5.26 nM tested. Quantification of the predominant receptor ganglioside IV(6)Neu5Ac-nLc4Cer by means of a specific anti-Neu5Acalpha2-6Galbeta1-4GlcNAc-R antibody revealed 3.68 x 10(6) and 1.54 x 10(6) receptor molecules per HL-60 and 5637 cell, respectively; CHO-K1 cells were negative, lacking alpha2-6-sialylated gangliosides. The data imply a direct correlation of rViscumin cytotoxicity and the expression of receptor ganglioside. Moreover, CHO-K1 cells were rendered susceptible toward rViscumin cytotoxicity after exogenous application of human granulocyte gangliosides. Thus, (1) rViscumin has to be considered as a sialic acid-specific rather than a galactose-specific type II ribosome-inactivating protein, and (2) neolacto-series gangliosides with Neu5Acalpha2-6Galbeta1-4GlcNAc-terminus are true functional and physiologically relevant rViscumin receptors.