Pharmacokinetics and pharmacodynamics of ch14.18/CHO in relapsed/refractory high-risk neuroblastoma patients treated by long-term infusion in combination with IL-2

Ch14.18 manufactured in Chinese hamster ovary (CHO) cells is currently being evaluated in clinical trials. Short-term infusion (STI) (8–20 h/day; 4–5 days) of 100 mg/m2 ch14.18/CHO (dinutiximab β) per cycle in combination with cytokines is standard treatment of neuroblastoma (NB) patients. As pain is a limiting factor, we investigated a novel delivery method by continuous long-term infusion (LTI) of 100 mg/m2 over 10 days. 53 NB patients were treated with 5–6 cycles of 6 × 106 IU/m2 subcutaneous interleukin-2 (d 1-5, 8-12), LTI of 100 mg/m2 ch14.18/CHO (d 8-18) and 160 mg/m2 oral 13-cis-retinoic acid (d 22-35). Human anti-chimeric antibody (HACA), antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity were determined. With LTI, we observed a maximum concentration of ch14.18/CHO (Cmax) of 12.56 ± 0.68 µg/ml and a terminal half-life time (t1/2 β) of 32.7 ± 16.2 d. The clearance values for LTI and STI of 0.54 ± 0.13 and 0.41 ± 0.29 L/d m2 and area under the serum concentration-time curve (AUC) values of 189.6 ± 41.4 and 284.8 ± 156.8 µg×d/ml, respectively, were not significantly different. Importantly, we detected ch14.18/CHO trough concentration of ≥ 1 µg/ml at time points preceding subsequent antibody infusions after cycle 1, allowing a persistent activation of antibody effector mechanisms over the entire treatment period of 6 months. HACA responses were observed in 10/53 (19%) patients, similar to STI (21%), indicating LTI had no effect on the immunogenicity of ch14.18/CHO. In conclusion, LTI of ch14.18/CHO induced effector mechanisms over the entire treatment period, and may therefore emerge as the preferred delivery method of anti-GD2 immunotherapy to NB patients.     

Ch14.18 antibody produced in CHO cells in relapsed or refractory Stage 4 neuroblastoma patients

Purpose: This study aimed to assess the safety, pharmacokinetic and activity profiles of the human-mouse chimeric monoclonal anti-disialoganglioside GD2 antibody ch14.18 produced in Chinese hamster ovary (CHO) cells (ch14.18/CHO).

Methods: Sixteen children with recurrent/refractory neuroblastoma (median age 7.6 y) were enrolled in this Phase 1 dose-finding study. Patients received ch14.18/CHO courses of 10, 20 or 30 mg/m²/day as an eight-hour infusion over five consecutive days. Three courses at the same dose level were allowed unless disease progressed. Clearance and biodistribution of radiolabelled ch14.18/CHO in Balb/c and A/J mice were analyzed.

Results: A total of 41 ch14.18/CHO courses were given (10 × 3 courses, 5 × 2 courses, 1 × 1 course). Side effects were similar in expectedness, frequency and magnitude to those reported for ch14.18/SP2/0. The dose level of 20 mg/m²/day was confirmed. Toxicity was reversible and no treatment-related deaths occurred. In children, the peak plasma concentration was 16.51 µg/ml ± 5.9 µg/ml and the half-life was 76.91 h ± 52.5 h. A partial response following ch14.18/CHO was observed in 2/7 patients with residual disease. In mice, the half-lives were 22.7 h ± 1.9h for ch14.18/CHO and 25.0 h ± 1.9 h for ch14.18/SP2/0. The biodistribution of ¹²⁵I-ch14.18/CHO in mice with neuroblastoma was identical to ¹²⁵I-ch14.18/SP2/0, indicating GD₂ targeting activity in vivo.

Ch14.18 produced in CHO cells showed an unchanged toxicity profile and pharmacokinetics in neuroblastoma patients compared with ch14.18 produced in SP2/0 cells, and evidence of clinical activity was observed. In mice, analysis of pharmacokinetics and biodistribution showed comparable results between ch14.18/CHO and ch14.18/SP2/0. Based on these results, ch14.18/CHO was accepted for prospective clinical evaluation.     

Phase I trial of combined treatment with ch14.18 and R24 monoclonal antibodies and interleukin-2 for patients with melanoma or sarcoma

Purpose: We conducted a phase I trial of interleukin 2 (IL-2) in combination with chimeric 14.18 (ch14.18) and murine R24 antibodies to determine the maximal tolerated dose (MTD), immunological effects, and toxicity of this treatment combination. Experimental Design: Twenty-seven patients with either melanoma (23 patients) or sarcoma (4 patients) were enrolled to receive a combination therapy with ch14.18 and R24 antibodies together with continuous infusion of Roche IL-2 (1.5×10⁶ U/m²/day, 26 patients) or Chiron IL-2 (4.5×10⁶ U/m²/day, 1 patient) given 4 days/week for 3 weeks. The antibodies ch14.18 (2–7.5 mg/m²/day) and R24 (1–10 mg/m²/day) were scheduled to be administered for 5 days during the second week of IL-2 therapy. Results: When given in combination in this study, the MTD for ch14.18 was 5 mg/m²/day and the MTD for R24 was 5 mg/m²/day. Dose-limiting toxicities were severe allergic reactions to both ch14.18 and R24 as well as pain related to ch14.18. This ch14.18 MTD was lower than the 7.5 mg/m²/day MTD previously determined for ch14.18 given alone with the same dose and schedule of IL-2. Immunological effects included the induction of lymphokine-activated killer (LAK) activity and antibody-dependent cell-mediated cytoxicity (ADCC). Anti-idiotype response to ch14.18 was seen in six patients, including two melanoma patients who had a partial response to treatment. In addition to two partial responses, four patients had a stable disease and one patient remained without any evidence of disease. Conclusions: Immunotherapy with IL-2 in combination with ch14.18 and R24 antibodies augments LAK function and ADCC measured in vitro in all patients. While there exist theoretical advantages of combining these two antibodies, the MTD of ch14.18 and of R24 were lower than the MTD of each antibody in prior studies evaluating single antibody therapy with IL-2. As such, the combination of these two antibodies together with IL-2 therapy appeared to influence the MTD and toxicity of each of the administered antibodies. This work is supported by NIH grants M01-RR03186, R01-CA32685, and P30-CA14520     

Functional Bioassays for Immune Monitoring of High-Risk Neuroblastoma Patients Treated with ch14.18/CHO Anti-GD2 Antibody

Effective treatment of high-risk neuroblastoma (NB) remains a major challenge in pediatric oncology. Human/mouse chimeric monoclonal anti-GD₂ antibody (mAb) ch14.18 is emerging as a treatment option to improve outcome. After establishing a production process in Chinese hamster ovary (CHO) cells, ch14.18/CHO was made available in Europe for clinical trials. Here, we describe validated functional bioassays for the purpose of immune monitoring of these trials and demonstrate GD₂-specific immune effector functions of ch14.18/CHO in treated patients. Two calcein-based bioassays for complement-dependent- (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were set up based on patient serum and immune cells tested against NB cells. For this purpose, we identified LA-N-1 NB cells as best suited within a panel of cell lines. Assay conditions were first established using serum and cells of healthy donors. We found an effector-to-target (E:T) cell ratio of 20∶1 for PBMC preparations as best suited for GD₂-specific ADCC analysis. A simplified method of effector cell preparation by lysis of erythrocytes was evaluated revealing equivalent results at an E:T ratio of 40∶1. Optimal results for CDC were found with a serum dilution at 1∶8. For validation, both within-assay and inter-assay precision were determined and coefficients of variation (CV) were below 20%. Sample quality following storage at room temperature (RT) showed that sodium-heparin-anticoagulated blood and serum are stable for 48 h and 96 h, respectively. Application of these bioassays to blood samples of three selected high-risk NB patients treated with ch14.18/CHO (100 mg/m²) revealed GD₂-specific increases in CDC (4.5–9.4 fold) and ADCC (4.6–6.0 fold) on day 8 compared to baseline, indicating assay applicability for the monitoring of multicenter clinical trials requiring sample shipment at RT for central lab analysis.     

Pharmacokinetics of human-mouse chimeric anti-GD2 mAb ch14.18 in a phase I trial in neuroblastoma patients

A comprehensive analysis of the pharmacokinetics of human-mouse chimeric anti-ganglioside GD2 antibody mAb ch 14.18 was performed during a phase I clinical trial of ten children with neuroblastoma and one adult with osteosarcoma. The patients received a total of 20 courses of ch 14.18 at dose levels from 10 mg/m² to 200 mg/m². The plasma clearance of ch 14.18 was biphasic. Following the first course of treatmentt1/2,α was 3.4±3.1 h andt1/2,β 66.6±27.4 h in 9/10 children. Thet1/2,β values were significantly less than those of 181±73 h previously reported in adult melanoma patients (P<-0.001), and 147.5 h in the adult osteosarcoma patient in our trial. The latter suggests different pharmacokinetics of mAb ch 14.18 in children and adults. After a second course of treatment, administered to 5/10 children,t1/2,β decreased significantly from 72.9±19.8 h to 31.7±18.4 h (P=0.015). We there-fore conclude that the elimination kinetics of mAbs ch 14.18 in children and adults are different, and furthermore that repeated administration of mAb ch 14.18 to children with neuroblastoma leads to accelerated antibody clearance.     

Vaccination with anti-idiotype antibody ganglidiomab mediates a GD2-specific anti-neuroblastoma immune response

Purpose

Immunotherapy targeting disialoganglioside GD₂ emerges as an important treatment option for neuroblastoma, a pediatric malignancy characterized by poor outcome. Here, we report the induction of a GD₂-specific immune response with ganglidiomab, a new anti-idiotype antibody to anti-GD₂ antibodies of the 14.18 family.

Experimental design and results

Ganglidiomab was generated following immunization of Balb/c mice with 14G2a, and splenocytes were harvested to generate hybridoma cells. Clones were screened by ELISA for mouse antibody binding to hu14.18. One positive clone was selected to purify and characterize the secreted IgG protein (κ, IgG₁). This antibody bound to anti-GD₂ antibodies 14G2a, ch14.18/CHO, hu14.18, and to immunocytokines ch14.18-IL2 and hu14.18-IL2 as well as to NK-92 cells expressing scFv(ch14.18)-zeta receptor. Binding of these anti-GD₂ antibodies to the nominal antigen GD₂ as well as GD₂-specific lysis of neuroblastoma cells by NK-92-scFv(ch14.18)-zeta cells was competitively inhibited by ganglidiomab, proving GD₂ surrogate function and anti-idiotype characteristics. The dissociation constants of ganglidiomab from anti-GD₂ antibodies ranged from 10.8 ± 5.01 to 53.5 ± 1.92 nM as determined by Biacore analyses. The sequences of framework and complementarity-determining regions of ganglidiomab were identified. Finally, we demonstrated induction of a GD₂-specific humoral immune response after vaccination of mice with ganglidiomab effective in mediating GD₂-specific killing of neuroblastoma cells.

Conclusion

We generated and characterized a novel anti-idiotype antibody ganglidiomab and demonstrated activity against neuroblastoma.     

Soil N and C trace gas fluxes and microbial soil N turnover in a sessile oak (Quercus petraea (Matt.) Liebl.) forest in Hungary

During two intensive field campaigns in summer and autumn 2004 nitrogen (N₂O, NO/NO₂) and carbon (CO₂, CH₄) trace gas exchange between soil and the atmosphere was measured in a sessile oak (Quercus petraea (Matt.) Liebl.) forest in Hungary. The climate can be described as continental temperate. Fluxes were measured with a fully automatic measuring system allowing for high temporal resolution. Mean N₂O emission rates were 1.5 μg N m⁻² h⁻¹ in summer and 3.4 μg N m⁻² h⁻¹ in autumn, respectively. Also mean NO emission rates were higher in autumn (8.4 μg N m⁻² h⁻¹) as compared to summer (6.0 μg N m⁻² h⁻¹). However, as NO₂ deposition rates continuously exceeded NO emission rates (−9.7 μg N m⁻² h⁻¹ in summer and −18.3 μg N m⁻² h⁻¹ in autumn), the forest soil always acted as a net NO _x sink. The mean value of CO₂ fluxes showed only little seasonal differences between summer (81.1 mg C m⁻² h⁻¹) and autumn (74.2 mg C m⁻² h⁻¹) measurements, likewise CH₄uptake (summer: −52.6 μg C m⁻² h⁻¹; autumn: −56.5 μg C m⁻² h⁻¹). In addition, the microbial soil processes net/gross N mineralization, net/gross nitrification and heterotrophic soil respiration as well as inorganic soil nitrogen concentrations and N₂O/CH₄ soil air concentrations in different soil depths were determined. The respiratory quotient (ΔCO_{2 resp} ΔO_{2 resp}⁻¹) for the uppermost mineral soil, which is needed for the calculation of gross nitrification via the Barometric Process Separation (BaPS) technique, was 0.8978 ± 0.008. The mean value of gross nitrification rates showed only little seasonal differences between summer (0.99 μg N kg⁻¹ SDW d⁻¹) and autumn measurements (0.89 μg N kg⁻¹ SDW d⁻¹). Gross rates of N mineralization were highest in the organic layer (20.1–137.9 μg N kg⁻¹ SDW d⁻¹) and significantly lower in the uppermost mineral layer (1.3–2.9 μg N kg⁻¹ SDW d⁻¹). Only for the organic layer seasonality in gross N mineralization rates could be demonstrated, with highest mean values in autumn, most likely caused by fresh litter decomposition. Gross mineralization rates of the organic layer were positively correlated with N₂O emissions and negatively correlated with CH₄ uptake, whereas soil CO₂ emissions were positively correlated with heterotrophic respiration in the uppermost mineral soil layer. The most important abiotic factor influencing C and N trace gas fluxes was soil moisture, while the influence of soil temperature on trace gas exchange rates was high only in autumn.     

Pharmacokinetics of human-mouse chimeric anti-GD2 mAb ch14.18 in a phase I trial in neuroblastoma patients

A comprehensive analysis of the pharmacokinetics of human-mouse chimeric anti-ganglioside GD2 antibody mAb ch 14.18 was performed during a phase I clinical trial of ten children with neuroblastoma and one adult with osteosarcoma. The patients received a total of 20 courses of ch 14.18 at dose levels from 10 mg/m² to 200 mg/m². The plasma clearance of ch 14.18 was biphasic. Following the first course of treatmentt1/2, was 3.4±3.1 h andt1/2, 66.6±27.4 h in 9/10 children. Thet1/2, values were significantly less than those of 181±73 h previously reported in adult melanoma patients (P<-0.001), and 147.5 h in the adult osteosarcoma patient in our trial. The latter suggests different pharmacokinetics of mAb ch 14.18 in children and adults. After a second course of treatment, administered to 5/10 children,t1/2, decreased significantly from 72.9±19.8 h to 31.7±18.4 h (P=0.015). We there-fore conclude that the elimination kinetics of mAbs ch 14.18 in children and adults are different, and furthermore that repeated administration of mAb ch 14.18 to children with neuroblastoma leads to accelerated antibody clearance.This work was supported by grants from FDA, FD-R-000377 and NIH U10 CA 28439, and in part by a grant from the General Clinical Research Center Program, MOI RR00827, of the National Center for Research Resources, National Institutes of Health. M.M.U.-F. and C.S.H. were in part supported by a grant from the Children's Cancer Research Foundation, and M.M.U.-F. was in part supported by a grant of the Kommission für Forschung und wissenschaftlichen Nachwuchs, Charité 95-010/9610766     

Dissolved Inorganic Carbon Inventory and CO2Sequestration in Ureolytic Biocalcification Process with Bacillus Pasteurii

The generation and depletion of dissolved inorganic carbon (DIC) in a close-environment ureolytic biocalcification process by Bacillus pasteurii was evaluated. Three experimental sets, each containing 50 mM urea, were amended with either 50 mM Ca²⁺ before incubation (set–I) or 100 mM Ca²⁺ after 24-h incubation (set-II) or no Ca²⁺ addition (urea control).

Extent of ureolysis was maximum in urea control set (88%), followed by set–II (66.4%) and set–I (35.2%). Out of total DIC generated from microbial metabolism and ureolysis in set–I (277.6 mg/l) and set–II (464.9 mg/l), only about 54.1 mg/l and 180.1 mg/l was precipitated as CaCO₃, whereas 189.3 mg/l and 231.3 mg/l DIC escaped into headspace, respectively. Increased time separation between ureolysis and calcification steps in set–II and higher dosage of Ca²⁺ resulted in synergistic improvement in DIC capture. In a reusability test, the spent supernatant from set–II could precipitate additional amount CaCO₃from CO₂saturated water_,which was twice as much as that of the fresh media control.     

Investigation of the antibacterial activity of a short cationic peptide against multidrug-resistant Klebsiella pneumoniae and Salmonella typhimurium strains and its cytotoxicity on eukaryotic cells

With the growing microbial resistance to conventional antimicrobial agents, the development of novel and alternative therapeutic strategies are vital. During recent years novel peptide antibiotics with broad spectrum activity against many Gram-positive and Gram-negative bacteria have been developed. In this study, antibacterial activity of CM11 peptide (WKLFKKILKVL-NH2), a short cecropin–melittin hybrid peptide, is evaluated against antibiotic-resistant strains of Klebsiella pneumoniae and Salmonella typhimurium as two important pathogenic bacteria. To appraise the antibacterial activity, minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and bactericidal killing assay were utilized with different concentrations (2–128 mg/L) of peptide. To evaluate cytotoxic effect of peptide, viability of RAJI, Hela, SP2/0, CHO, LNCAP cell lines and primary murine macrophage cells were also investigated with MTT assay in different concentrations (3–24 and 0.5–16 mg/L, respectively). MICs of K. pneumoniae and S. typhimurium isolates were in range of 8–16 and 4–16 mg/L, respectively. In bactericidal killing assay no colonies were observed at 2X MIC for K. pneumoniae and S. typhimurium isolates after 80–90 min, respectively. Despite the fact that CM11 reveals no significant cytotoxicity on RAJI, Hela, SP2/0, and CHO cell lines beneath 6 mg/L at first 24 and 48 h, the viability of LNCAP cells are about 50 % at 3 mg/L, which indicates strong cytotoxicity of the peptide. In addition, macrophage toxicity by MTT assay showed that LD₅₀ of CM11 peptide is 12 μM (16 mg/L) after 48 h while in this concentration after 24 h macrophage viability was about 70 %.     

Depositional fluxes and sources of particulate carbon and nitrogen in natural lakes and a young boreal reservoir in Northern Québec

We investigated the depositional trends of total particles, carbon and nitrogen in a newly created, 600-km² hydroelectric reservoir in Northern Québec, and compared the results with those observed in lakes of the surrounding region. We show that particulate fluxes exhibit a large degree of spatial heterogeneity in both the reservoir (68–548 mg POC m^?2 d^?1 and 5–33 mg PN m^?2 d^?1) and the natural lakes (30–150 mg POC m^?2 d^?1 and 3–12 mg PN m^?2 d^?1) and that on average, settling fluxes of the reservoir (211 ± 46 mg POC m^?2 d^?1 and 14 ± 3 mg PN m^?2 d^?1) exceeded lake deposition (79 ± 13 mg POC m^?2 d^?1 and 7 ± 1 mg PN m^?2 d^?1) by approximately two-fold. Our results also show that the nature of the organic matter reaching the sediments was significantly different between lakes and the reservoir, which can have consequences for benthic metabolism and the long-term storage. We found that sinking fluxes in the reservoir were mostly regulated by local morphological and hydrological conditions, with higher fluxes along or in the vicinity of the old riverbed (average 400 ± 73 mg POC m^?2 d^?1 and 24 ± 5 mg PN m^?2 d^?1) and lower fluxes in calmer zones such as side bays (average 106 ± 10 mg POC m^?2 d^?1 and 8 ± 1 mg PN m^?2 d^?1). In lakes, where settling fluxes were not linked to the trophy, or dissolved organic carbon, the actual nature of the sedimenting organic material was influenced by lake morphometry and the relative contribution of algal versus terrestrial sources. We conclude that re-suspension and erosion play a major role in shaping the reservoir sinking fluxes which explain both, the higher reservoir deposition and also some of the qualitative differences between the two systems. Despite all these differences, sinking particulate organic carbon fluxes were small and surprisingly similar relative to the surface carbon dioxide emissions in both the reservoir and lakes, representing approximately 16–17 % of the carbon efflux estimated for these same systems in 2008.     

Factors affecting the performance of horizontal flow constructed treatment wetland vegetated with Cyperus papyrus for municipal wastewater treatment

The effect of hydraulic loading rate (HLR) and hydraulic retention time (HRT) on the bioremediation of municipal wastewater using a pilot scale subsurface horizontal flow constructed treatment wetland (HFCTW) vegetated with Cyprus papyrus was investigated. Different HLRs were applied to the treatment system namely 0.18, 0.10, and 0.07 m³/m². d with corresponding HRTs of 1.8, 3.2, and 4.7 days, respectively. The flow rate was 8 m³/d, and the average organic loading rate (OLR) was 0.037 kg BOD/m³/d. Results showed that the performance of the HFCTW was linearly affected by decreasing the HLR and increasing the HRT. The highest treatment efficiency was achieved at HRT (4.7 days) and HLR (0.07 m³/m². d). The percentage reductions of chemical oxygen demand (COD), biochemical oxygen demand (BOD), and total suspended solids (TSS) were 86%, 87%, and 80%, respectively. Satisfactory nutrient removal was obtained. Also, removal of 2–3 logs of bacterial indicators of pollution was achieved. The dry biomass of Cyperus was 7.7 kg/m² and proved to be very efficient in nitrification processes due to high diversity of the roots that increase the treatment surface area.     

Seasonal nitrous oxide and methane emissions across a subtropical estuarine salinity gradient

Currently, there is a lack of knowledge about GHG emissions, specifically N₂O and CH₄, in subtropical coastal freshwater wetland and mangroves in the southern hemisphere. In this study, we quantified the gas fluxes and substrate availability in a subtropical coastal wetland off the coast of southeast Queensland, Australia over a complete wet-dry seasonal cycle. Sites were selected along a salinity gradient ranging from marine (34 psu) in a mangrove forest to freshwater (0.05 psu) wetland, encompassing the range of tidal influence. Fluxes were quantified for CH₄ (range ?0.4–483 mg C–CH₄ h^?1 m^?2) and N₂O (?5.5–126.4 μg N–N₂O h^?1 m^?2), with the system acting as an overall source for CH₄ and N₂O (mean N₂O and CH₄ fluxes: 52.8 μg N–N₂O h^?1 m^?2 and 48.7 mg C–CH₄ h^?1 m^?2, respectively). Significantly higher N₂O fluxes were measured during the summer months (summer mean 64.2 ± 22.2 μg N–N₂O h^?1 m^?2; winter mean 33.1 ± 24.4 µg N–N₂O h^–1 m^?2) but not CH₄ fluxes (summer mean 30.2 ± 81.1 mg C–CH₄ h^?1 m^?2; winter mean 37.4 ± 79.6 mg C–CH₄ h^?1 m^?2). The changes with season are primarily driven by temperature and precipitation controls on the dissolved inorganic nitrogen (DIN) concentration. A significant spatial pattern was observed based on location within the study site, with highest fluxes observed in the freshwater tidal wetland and decreasing through the mangrove forest. The dissolved organic carbon (DOC) varied throughout the landscape and was correlated with higher CH₄ fluxes, but this was a nonlinear trend. DIN availability was dominated by N–NH₄ and correlated to changes in N₂O fluxes throughout the landscape. Overall, we did not observe linear relationships between CH₄ and N₂O fluxes and salinity, oxygen or substrate availability along the fresh-marine continuum, suggesting that this ecosystem is a mosaic of processes and responses to environmental changes.     

Measurement of net ecosystem exchange,productivity and respiration in three spruce forests in Sweden shows unexpectedly large soil carbon losses

Measurement of net ecosystem exchange was made using the eddy covariance method above three forests along a north-south climatic gradient in Sweden: Flakaliden in the north, Knottåsen in central and Asa in south Sweden. Data were obtained for 2 years at Flakaliden and Knottåsen and for one year at Asa. The net fluxes (N_ep) were separated into their main components, total ecosystem respiration (R_t) and gross primary productivity (P_g). The maximum half-hourly net uptake during the heart of the growing season was highest in the southernmost site with ?0.787 mg CO₂ m^?2 s^?1 followed by Knottåsen with ?0.631 mg CO₂ m^?2 s^?1 and Flakaliden with ?0.429 mg CO₂ m^?2 s^?1. The maximum respiration rates during the summer were highest in Knottåsen with 0.245 mg CO₂ m^?2 s^?1 while it was similar at the two other sites with 0.183 mg CO₂ m^?2 s^?1. The annual N_ep ranged between uptake of ?304 g C m^?2 year^?1 (Asa) and emission of 84 g C m^?2 year^?1 (Knottåsen). The annual R_t and P_g ranged between 793 to 1253 g C m^?2 year^?1 and ?875 to ?1317 g C m^?2 year^?1, respectively. Biomass increment measurements in the footprint area of the towers in combination with the measured net ecosystem productivity were used to estimate the changes in soil carbon and it was found that the soils were losing on average 96–125 g C m^?2 year^?1. The most plausible explanation for these losses was that the studied years were much warmer than normal causing larger respiratory losses. The comparison of net primary productivity and P_g showed that ca 60% of P_g was utilized for autotrophic respiration.     

Stackable and submergible microbial fuel cell modules for wastewater treatment

The stackable and submergible microbial fuel cell (SS-MFC) system was fabricated consisting of three MFC modules (#1, #2 and #3) that were immersed in an anaerobic tank as a 30 L anode compartment. Each module consisted of the anion exchange membrane–membrane electrode assembly (A-MEA) and cation exchange membrane-MEA (C-MEA). Two MEAs shared a cathode compartment in the module and the three modules shared a anode compartment The SS-MFC system was operated with two phase. After batch feeding (phase I), the system was operated under continuous mode (phase II) with different organic concentrations (from 50 to 1000 mg/L) and different hydraulic retention times (HRT; from 3.4 to 7.2 h). The SS-MFC system successfully produced a stable voltage. A-MEA generated a lower power density than the C-MEA because of the former’s high activation and resistance loss. C-MEA showed a higher average maximum power density (3.16 W/m³) than A-MEA (2.82 W/m³) at 70 mL/min (HRT of 7.2 h). The current density increased as the organic concentration was increased from 70 to 1000 mg/L in a manner consistent with Monod kinetics. When the HRT was increased from 3.4 to 7.2 h, the power densities of the C-MEAs increased from 34.3–40.9 to 40.7–45.7 mW/m², but those of the A-MEAs decreased from 25.3–48.0 to 27.7–40.9 mW/m². Although power generation was affected by HRT, organic concentrations, and separator types, the proposed SS-MFC modules can be applied to existing wastewater treatment plants.     

Generation and Characterization of a Human/Mouse Chimeric GD2-Mimicking Anti-Idiotype Antibody Ganglidiximab for Active Immunotherapy against Neuroblastoma

Vaccination with proteins mimicking GD₂ that is highly expressed on neuroblastoma (NB) cells is a promising strategy in treatment of NB, a pediatric malignancy with poor prognosis. We previously showed efficacy of ganglidiomab in vivo, a murine anti-idiotype (anti-Id) IgG1. In order to tailor immune responses to variable regions, we generated a new human/mouse chimeric anti-Id antibody (Ab) ganglidiximab by replacing murine constant fragments with corresponding human IgG1 regions. DNA sequences encoding for variable regions of heavy (VH) and light chains (VL) were synthesized by RT-PCR from total RNA of ganglidiomab-producing hybridoma cells and further ligated into mammalian expression plasmids with coding sequences for constant regions of human IgG1 heavy and light chains, respectively. We established a stable production cell line using Chinese hamster ovarian (CHO) cells co-transfected with two expression plasmids driving the expression of either ganglidiximab heavy or light chain. After purification from supernatants, anti-idiotypic characteristics of ganglidiximab were demonstrated. Binding of ganglidiximab to anti-GD₂ Abs of the 14.18 family as well as to NK-92tr cells expressing a GD₂-specific chimeric antigen receptor (scFv(ch14.18)-zeta) was shown using standard ELISA and flow cytometry analysis, respectively. Ganglidiximab binding affinities to anti-GD₂ Abs were further determined by surface plasmon resonance technique. Moreover, binding of anti-GD₂ Abs to the nominal antigen GD₂ as well as GD₂-specific Ab-mediated cytotoxicity (ADCC, CDC) was competitively inhibited by ganglidiximab. Finally, ganglidiximab was successfully used as a protein vaccine in vivo to induce a GD₂-specific humoral immune response. In summary, we report generation and characterization of a new human/mouse chimeric anti-Id Ab ganglidiximab for active immunotherapy against NB. This Ab may be useful to tailor immune responses to the paratope regions mimicking GD₂ overexpressed in NB.     

The benthos off King George Island (South Shetland Islands,Antarctica): further evidence for a lack of a latitudinal biomass cline in the Southern Ocean

The benthic fauna off King George Island (South Shetland Islands, Antarctica) was investigated during "Polarstern" expedition ANT XV/3 in March 1998. Samples were taken along two cross-shelf/slope transects both north (Drake Passage) and south of the island (Bransfield Strait, off Potter Cove) at water depths ranging from 130 m to 2,000 m. For a quantitative inventory, a multibox corer was used at nine stations to collect mostly infaunal macrobenthos; at seven stations, seabed photography was employed concomitantly to survey the epibenthic megafauna. Macrofauna abundances ranged from 730 ind. m^–2 at 2,000 m to >14,000 ind. m^–2 at 100 m; biomass values varied between about 50 g wet mass m^–2 (6 g ash-free dry mass m^–2) at 2,000 m and about 950 g wet mass m^–2 (about 90 g ash-free dry mass m^–2) at 200 m. Densities were dominated everywhere by polychaetes, followed by bivalves, crustaceans and ophiuroids; in terms of biomass, krill and holothurians surpassed polychaetes at some stations. No significant differences between the northern and the southern transects in total abundance and biomass were obvious. Megafauna abundances were clearly higher south of King George Island, totalling about 110–150 ind. m^–2 on the shelf (235–330 m) and about 50 ind. m^–2 at the continental slope (750 m), whereas along the northern transect they reached values of only 21–31 ind. m^–2 on the shelf (130–430 m) and decreased at the continental slope (950 m) to about 5 ind. m^–2. A brittle star, Ophionotus victoriae, strongly dominated the southern-shelf epibenthos, with relative abundances of 70–95% and a biomass of about 40–80 g wet mass m^–2 (about 4–7 g ash-free dry mass m^–2), but was numerically less important at the slope (5%) where ammotheid pycnogonids prevailed (80%). Macro- and megabenthos distribution patterns were characterized by a pronounced shelf-slope gradient – in standing stock as well as in faunistic composition – but this resemblance was statistically not significant. This finding indicates that the spatial distributions of macrobenthos and megabenthos are primarily determined by a depth-dependent factor, most probably food supply, but apparently respond differently to secondary driving forces, possibly seabed features. Our results provide further evidence for the notion that there is no distinct latitudinal gradient in benthic abundance and biomass in the Southern Ocean between the South American Magellan region and high-Antarctic waters of the Weddell Sea.     

Efficacy of Nimotuzumab Combined with Docetaxel–Cisplatin–Fluorouracil Regimen in Treatment of Advanced Oral Carcinoma

The present study aimed to evaluate efficacy and adverse effects of Nimotuzumab combined with docetaxel–cisplatin–fluorouracil regimen in the treatment of advanced oral carcinoma. Nine patients with advanced oral carcinoma were treated with Nimotuzumab combined with docetaxel–cisplatin–fluorouracil regimen (test group). The treatment was given as follows: Nimotuzumab 200 mg, given as intravenous infusion once a week for 6 weeks; docetaxel and cisplatin, 75 mg/m² each, on day 1 only; 5-fluorouracil, 750 mg/m² infused continually for 8 h, used from day 1 to 5; the total cycle was for 21 days. Another eight patients comprised control group (docetaxel–cisplatin–fluorouracil regimen alone). Study patients from both groups were evaluated for objective response. The response rate was significantly (p = 0.044) higher in test group (88.9 vs. 37.5 % in control group). The disease control rate also tended to be higher in test group (100 vs. 62.5 % in control group; p = 0.083). The major adverse effects were bone marrow suppression, nausea, vomiting, and alopecia. The incidence of adverse effects was similar between both study groups. In conclusion, Nimotuzumab combined with docetaxel–cisplatin–fluorouracil regimen is effective and safe in the treatment of advanced oral carcinoma.     

Fenofibrate Increases Serum Creatinine in a Patient with Familial Nephropathy Associated to Hyperuricemia

Background: Kidney function progressively deteriorates in patients with familial juvenile hyperuricemiac nephropathy (FJHN, OMIN 162000) and chronic renal disease is commonly associated to dyslipidemia. We report for the first time abrupt renal insufficiency in a patient with FJHN and hypertrygliceridemia following fenofibrate administration.Case report: A 53-year-old man was diagnosed clinically with FJHN at age 24 years which was subsequently confirmed by genotypic analysis of the UMOD gene at age 40 years. His mother and two brothers suffered the disease. At that time, renal size and function were normal, as was his blood pressure and serum lipids. At age 34 years, serum urate was 8.5 mg/dL and creatinine 1.7 mg/dL (GFR, 58 mL/min/1.73 m²). He was treated with allopurinol, losartan, and lovastatin. Serum TG levels ranged between 150 and 250 mg/dL. At age 52 years, serum urate was 4.1 mg/dL, creatinine 3.2 mg/dL, LDLc 99 mg/dL (atorvastatin 40 mg/d), and TG 275 mg/dL. Fenofibrate (160 mg/d) was added. One month later, serum creatinine increased to 4.2 mg/dL and TG decreased to 125 mg/dL. He did not complain of muscle pain, weakness, or changes in urinary frequency or color and rabdomyolysis was discarded. Fenofibrate was withheld and three months later serum creatinine decreased to baseline levels (3.2 mg/dL) and TG increased to 197 mg/dL.Conclusion: To our knowledge, this is the first patient with FJHN in whom fenofibrate administration was associated to a further impairment in renal function not attributable to rabdomyolysis.     

Continued Tumor Reduction of Metastatic Pheochromocytoma/Paraganglioma Harboring Succinate Dehydrogenase Subunit B Mutations with Cyclical Chemotherapy

Patients harboring germline mutations in the succinate dehydrogenase complex subunit B (SDHB) gene present with pheochromocytomas and paragangliomas (PPGL) that are more likely malignant and clinically aggressive. The combination chemotherapy cyclophosphamide, vincristine, and dacarbazine (CVD) was retrospectively evaluated in patients with SDHB-associated metastatic PPGL.Query Twelve metastatic PPGL patients harboring SDHB mutations/polymorphisms with undetectable SDHB immunostaining were treated with CVD. CVD therapy consisted of 750 mg/m² cyclophosphamide with 1.4 mg/m² vincristine on day 1 and 600 mg/m² dacarbazine on days 1 and 2, every 21–28 days. Treatment outcome was determined by RECIST criteria as well as determination of response duration and progression-free and overall survivals. A median of 20.5 cycles (range 4–41) was administered. All patients had tumor reduction (12–100% by RECIST). Complete response was seen in two patients, while partial response was observed in 8. The median number of cycles to response was 5.5. Median duration of response was 478 days, with progression-free and overall survivals of 930 and 1190 days, respectively. Serial [¹⁸F]-fluorodeoxyglucose positron emission tomography and computed tomography imaging demonstrated continued incremental reduction in maximal standardized uptake values (SUV_max) values in 26/30 lesions. During treatment administration, the median SUV decreased from?> 25 to?< 6, indicating the efficacy of chemotherapy over a prolonged period of time. Prolonged therapy results in continued incremental tumor reduction, and is consistent with persistent drug sensitivity. CVD chemotherapy is recommended to be considered part of the initial management in patients with metastatic SDHB-related PPGL.