Removal of EpCAM-positive tumor cells from blood collected during major oncological surgery using the Catuvab device- a pilot study.

BACKGROUND: Intraoperative blood salvage (IBS) is regarded as an alternative to allogeneic blood transfusion excluding the risks associated with allogeneic blood. Currently, IBS is generally avoided in tumor surgeries due to concern for potential metastasis caused by residual tumor cells in the erythrocyte concentrate. METHODS: The feasibility, efficacy and safety aspects of the new developed Catuvab procedure using the bispecific trifunctional antibody Catumaxomab was investigated in an ex-vivo pilot study in order to remove residual EpCAM positive tumor cells from the autologous erythrocyte concentrates (EC) from various cancer patients, generated by a IBS device. RESULTS: Tumor cells in intraoperative blood were detected in 10 of 16 patient samples in the range of 69-2.6 × 105 but no residual malignant cells in the final erythrocyte concentrates after Catuvab procedure. IL-6 and IL-8 as pro-inflammatory cytokines released during surgery, were lowered in mean 28-fold and 52-fold during the Catuvab procedure, respectively, whereas Catumaxomab antibody was detected in 8 of 16 of the final EC products at a considerable decreased and uncritical residual amount (37 ng in mean). CONCLUSION: The preliminary study results indicate efficacy and feasibility of the new medical device Catuvab allowing potentially the reinfusion of autologous erythrocyte concentrates (EC) produced by IBS device during oncological high blood loss surgery. An open-label, multicenter clinical study on the removal of EpCAM-positive tumor cells from blood collected during tumor surgery using the Catuvab device is initiated to validate these encouraging results.

First time intravesically administered trifunctional antibody catumaxomab in patients with recurrent non-muscle invasive bladder cancer indicates high tolerability and local immunological activity.

Transurethral resection of the tumor (TUR-B) followed by adjuvant intravesical treatment with cytostatic drugs or Bacillus Calmette-Guérin (BCG) as standard therapy of non-muscle-invasive bladder cancer (NMIBC) is associated with a high recurrence rate of about 60-70%, considerable side effects and requires close monitoring. Alternative treatment options are warranted. Two patients with epithelial cell adhesion molecule (EpCAM)-positive recurrent non-muscle invasive bladder cancer were treated the first time by an intravesical administration of the trifunctional bispecific EpCAM targeting antibody catumaxomab (total dosage of 470 and 1120 µg, respectively). The binding and killing activity of catumaxomab in urine milieu was evaluated in vitro. In contrast to its previous systemic application catumaxomab was well tolerated without any obvious signs of toxicity. Relevant cytokine plasma levels were not detected and no significant systemic drug release was observed. The induction of a human anti-mouse-antibody (HAMA) reaction was either absent or untypically weak contrary to the high immunogenicity of intraperitoneal applied catumaxomab. Tumor cells that were detectable in urine patient samples disappeared after catumaxomab therapy. Endoscopically confirmed recurrence-free intervals were 32 and 25 months. Our data suggest that intravesical administration of catumaxomab in NMIBC is feasible, safe and efficacious, thus arguing for further clinical development of catumaxomab in this indication.

The D-A-(C) index: A practical approach towards the microbiological-ecological monitoring of groundwater ecosystems.

Groundwater is not only a vital resource, but also one of the largest terrestrial aquatic ecosystems on Earth. However, to date, ecological criteria are often not considered in routine groundwater monitoring, mainly because of the lack of suitable ecological assessment tools. Prokaryotic microorganisms are ubiquitous in groundwater ecosystems even under the harshest conditions, making them ideal bioindicators for ecological monitoring. We have developed a simple, inexpensive approach that enables ecological groundwater monitoring based on three microbiological parameters that can be easily integrated into existing routine monitoring practices: prokaryotic cell density (D) measured by flow cytometry; activity (A) measured as prokaryotic intracellular ATP concentrations using a simple cell-lysis-luminescence assay; and, as an optional parameter, the bioavailable carbon (C) measured as the concentration of assimilable organic carbon in a simple batch growth assay. We analyzed data for three case studies of different disturbances representing some of the main threats to groundwater ecosystems, i.e. organic contamination with hydrocarbons, surface water intrusion, and agricultural land use. For all three disturbances, disturbed samples could be reliably distinguished from undisturbed samples based on a single index value obtained from multivariate outlier analyses of the microbial variables. We could show that this multivariate approach allowed for a significantly more sensitive and reliable detection of disturbed samples compared to separate univariate outlier analyses of the measured variables. Furthermore, a comparison of non-contaminated aquifers from nine different regions across Germany revealed distinct multivariate signatures along the three microbial variables, which should be considered when applying our approach in practice. In essence, our approach offers a practical tool for the detection of disturbances of groundwater ecosystems based on microbial parameters which can be seamlessly extended in the future by additional parameters for higher sensitivity as well as flexibility.

Non-random processes determine the colonization of groundwater sediments by microbial communities in a pristine porous aquifer.

Sediments accommodate the dominating share of groundwater microbiomes, however the processes that govern the assembly and succession of sediment-attached microbial communities in groundwater aquifers are not well understood. To elucidate these processes, we followed the microbial colonization of sterile sediments in in situ microcosms that were exposed to groundwater for almost 1 year at two distant but hydrologically connected sites of a pristine, shallow, porous aquifer. Our results revealed intriguing similarities between the community succession on the newly-colonized sediments and succession patterns previously observed for biofilms in other more dynamic aquatic environments, indicating that the assembly of microbial communities on surfaces may be governed by similar underlying mechanisms across a wide range of different habitats. Null model simulations on spatiotemporally resolved 16S rRNA amplicon sequencing data further indicated selection of specific OTUs rather than random colonization as the main driver of community assembly. A small fraction of persistent OTUs that had established on the sediments during the first 115 days dominated the final communities (68%-85%), suggesting a key role of these early-colonizing organisms, in particular specific genera within the Comamonadaceae and Oxalobacteraceae, for community assembly and succession during the colonization of the sediments. Overall, our study suggests that differences between planktonic and sediment-attached communities often reported for groundwater environments are not the result of purely stochastic events, but that sediment surfaces select for specific groups of microorganisms that assemble over time in a reproducible, non-random way.

Evaluating the performance of water purification in a vegetated groundwater recharge basin maintained by short-term pulsed infiltration events.

Infiltration of surface water constitutes an important pillar in artificial groundwater recharge. However, insufficient transformation of organic carbon and nutrients, as well as clogging of sediments often cause major problems. The attenuation efficiency of dissolved organic carbon (DOC), nutrients and pathogens versus the risk of bioclogging for intermittent recharge were studied in an infiltration basin covered with different kinds of macrovegetation. The quality and concentration of organic carbon, major nutrients, as well as bacterial biomass, activity and diversity in the surface water, the porewater, and the sediment matrix were monitored over one recharge period. Additionally, the numbers of viral particles and Escherichia coli were assessed. Our study showed a fast establishment of high microbial activity. DOC and nutrients have sustainably been reduced within 1.2 m of sediment passage. Numbers of E. coli, which were high in the topmost centimetres of sediment porewater, dropped below the detection limit. Reed cover was found to be advantageous over bushes and trees, since it supported higher microbial activities along with a good infiltration and purification performance. Short-term infiltration periods of several days followed by a break of similar time were found suitable for providing high recharge rates, and good water purification without the risk of bioclogging.

Spatio-temporal patterns of microbial communities in a hydrologically dynamic pristine aquifer.

Seasonal patterns of groundwater and sediment microbial communities were explored in a hydrologically dynamic alpine oligotrophic porous aquifer, characterized by pronounced groundwater table fluctuations. Rising of the groundwater level in consequence of snow melting water recharge was accompanied by a dramatic drop of bacterial Shannon diversity in groundwater from H' = 3.22 ± 0.28 in autumn and winter to H' = 1.31 ± 0.35 in spring and summer, evaluated based on T-RFLP community fingerprinting. Elevated numbers of bacteria in groundwater in autumn followed nutrient inputs via recharge from summer rains and correlated well with highest concentrations of assimilable organic carbon. Sterile sediments incubated to groundwater in monitoring wells were readily colonized reaching maximum cell densities within 2 months, followed by a consecutive but delayed increase and leveling-off of bacterial diversity. After 1 year of incubation, the initially sterile sediments exhibited a similar number of bacteria and Shannon diversity when compared to vital sediment from a nearby river incubated in parallel. The river bed sediment microbial communities hardly changed in composition, diversity, and cell numbers during 1 year of exposure to groundwater. Summing up, the seasonal hydrological dynamics were found to induce considerable dynamics of microbial communities suspended in groundwater, while sediment communities seem unaffected and stable in terms of biomass and diversity.

Microbial CO2 fixation potential in a tar-oil-contaminated porous aquifer.

CO(2) fixation is one of the most important processes on the Earth's surface, but our current understanding of the occurrence and importance of chemolithoautotrophy in the terrestrial subsurface is poor. Groundwater ecosystems, especially at organically polluted sites, have all the requirements for autotrophic growth processes, and CO(2) fixation is thus suggested to contribute significantly to carbon flux in these environments. We explored the potential for autotrophic CO(2) fixation in microbial communities of a petroleum hydrocarbon-contaminated aquifer by detection of functional marker genes (cbbL, cbbM), encoding different forms of the key enzyme RubisCO of the Calvin-Benson-Bassham cycle. Quantification of (red-like) cbbL genes revealed highest numbers at the upper fringe of the contaminant plume and the capillary fringe where reduced sulphur and iron species are regularly oxidized in the course of groundwater table changes. Functional gene sequences retrieved from this area were most closely related to sequences of different thiobacilli. Moreover, several cultures could be enriched from fresh aquifer material, all of which are able to grow under chemolithoautotrophic conditions. A novel, nitrate-reducing, thiosulfate-oxidizing bacterial strain, recently described as Thiobacillus thiophilus D24TN(T) sp. nov., was shown to carry and transcribe RubisCO large-subunit genes of form I and II. Enzyme tests proved the actual activity of RubisCO in this strain.

The potential use of fauna and bacteria as ecological indicators for the assessment of groundwater quality.

The use of ecological criteria for the assessment of aquatic ecosystem status is routine for surface waters. So far no ecological parameters are considered for the assessment and monitoring of groundwater quality. It has been well known for decades that aquifers are ecosystems harbouring a vast diversity of invertebrates and microorganisms. The growing knowledge on groundwater microbial and faunal communities as well as the molecular and statistical tools available form a solid ground for the development of first ecologically sound assessment schemes. The sensitivity of groundwater communities towards impacts from land use and surface waters is exemplarily demonstrated by a data set of two geologically similar but hydrologically partially separated aquifer systems. Subgroups of the fauna in groundwater (stygobites vs. stygophiles and stygoxenes) successfully indicated elevated nitrate impacts linked to land use activities. Within the microbial communities, impacts from land use are mirrored by high bacterial biodiversity values atypical for pristine groundwater of comparable systems. The data show that there is legitimate hope for the application of ecological criteria for groundwater quality assessment in the future.

Thiobacillus thiophilus sp. nov., a chemolithoautotrophic, thiosulfate-oxidizing bacterium isolated from contaminated aquifer sediments.

Strain D24TN(T) was enriched and isolated from sediment collected from a tar oil-contaminated aquifer at a former gasworks site located in Duesseldorf-Flingern, Germany. Cells of strain D24TN(T) were rod-shaped, non-spore-forming and stained Gram-negative. Thiosulfate was used as an electron donor. The organism was obligately chemolithoautotrophic and facultatively anaerobic, and grew with either oxygen or nitrate as electron acceptor. Growth was observed at pH values between 6.3 and 8.7 and at temperatures of -2 to 30 degrees C; optimum growth occurred at pH 7.5-8.3 and 25-30 degrees C. The DNA G+C content was 61.5 mol%. On the basis of the 16S rRNA gene sequence analysis, strain D24TN(T) clustered in the Betaproteobacteria and was most closely related to Thiobacillus denitrificans (97.6 %) and Thiobacillus thioparus (97.5 %). Based on the phenotypic, chemotaxonomic and phylogenetic data, strain D24TN(T) represents a novel species of the genus Thiobacillus, for which the name Thiobacillus thiophilus sp. nov. is proposed. The type strain is D24TN(T) (=DSM 19892(T)=JCM 15047(T)).