Selection, characterization and in vivo evaluation of novel CD44v6-targeting antibodies for targeted molecular radiotherapy.

Molecular radiotherapy combines the advantages of systemic administration of highly specific antibodies or peptides and the localized potency of ionizing radiation. A potential target for molecular radiotherapy is the cell surface antigen CD44v6, which is overexpressed in numerous cancers, with limited expression in normal tissues. The aim of the present study was to generate and characterize a panel of human anti-CD44v6 antibodies and identify a suitable candidate for future use in molecular radiotherapy of CD44v6-expressing cancers. Binders were first isolated from large synthetic phage display libraries containing human scFv and Fab antibody fragments. The antibodies were extensively analyzed through in vitro investigations of binding kinetics, affinity, off-target binding, and cell binding. Lead candidates were further subjected to in vivo biodistribution studies in mice bearing anaplastic thyroid cancer xenografts that express high levels of CD44v6. Additionally, antigen-dependent tumor uptake of the lead candidate was verified in additional xenograft models with varying levels of target expression. Interestingly, although only small differences were observed among the top antibody candidates in vitro, significant differences in tumor uptake and retention were uncovered in in vivo experiments. A high-affinity anti-CD44v6 lead drug candidate was identified, mAb UU-40, which exhibited favorable target binding properties and in vivo distribution. In conclusion, a panel of human anti-CD44v6 antibodies was successfully generated and characterized in this study. Through comprehensive evaluation, mAb UU-40 was identified as a promising lead candidate for future molecular radiotherapy of CD44v6-expressing cancers due to its high affinity, excellent target binding properties, and desirable in vivo distribution characteristics.

Therapeutic approaches in clear cell and non-clear cell renal cell carcinoma.

Renal cell carcinoma (RCC) accounts for 2.4% of all malignancies worldwide diagnosed with 338,000 estimated new cases globally in 2012. In the last decade, the therapeutic landscape for RCC patients has changed tremendously. In this review, we will summarize the treatment options currently available for clear-cell localized, advanced and metastatic RCC (mRCC); as stated in the ESMO clinical practice guidelines, the EAU guidelines and the NCCN guidelines. Furthermore, we will discuss the recommended therapies in patients diagnosed with non-clear cell tumours.

[Cost evaluation of a model for integrated care of seriously mentally ill patients]. / Kostenevaluation eines Modells der Integrierten Versorgung für schwer psychisch Kranke.

BACKGROUND: The model for integrated care (IC) of those seriously mentally ill patients insured with the DAK-Gesundheit health insurance and various Betriebskrankenkassen (members of the VAG Mitte) from the regions Berlin, Brandenburg, Lower Saxony and Bremen allows a complex treatment in the outpatient setting which consists of psychiatrists, general practitioners and clinicians, psychiatric nursing, sociotherapy (only in Berlin), internal medicine quality circles, orientation on treatment guidelines and conceptual consensus with the relevant care clinics. The aim of the evaluation is to illustrate the health economic effects of IC. METHODS: In the period from 2006 to 2010 insured members of the DAK-Gesundheit and other involved health insurance companies with a serious mental illness, a significant impairment of social functioning and the need to be treated to avoid or substitute an in-hospital stay were included in the integrated care. The cost perspective was that of the statutory health insurance companies. For the health economic evaluation, the utilisation of continuous IC over 18 months was compared to the last 18 months prior to the inclusion in IC. The clinical findings were gathered quarterly during the IC using CGI (Clinical Global Impressions) and GAF (Global Assessment of Functioning Scale). RESULTS: A total of 1 364 patients receiving IC in 66 doctor's practices were documented (of those, 286 had diagnoses of ICD-10 F2, 724 ICD-10 F32-F39). The median age was 48.8 years, 69% were female. 24% had their own source of income, 40% were on the pension, and the rest of the patients were receiving transfer benefits in some form. In 54% of the cases IC was used to avoid an in-hospital stay, in 46% of the cases to substitute an in-hospital stay. The degree of the CGI was 5.5 on average at the time of inclusion and the GAF score was 36.5 on average. The 226 patients with continuous documentation over 18 months were included in the health economic analysis. The number of days spent in hospital was lower during the IC period as compared to the 18 months prior to IV (11.8 vs. 28.6 days, p<0.001), the inpatient costs were lower (5 929 ± 13 837 Euro vs. 2 458 ± 6 940 Euro, p<0.001), the total was not significantly changed (7 777 ± 14 263 Euro vs. 7 321 ± 7 910 Euro, p=0.65). The substantial reduction of inpatient costs was compensated by the additional costs for medication and the costs of the complex outpatient care. Results were comparable for the 2 subgroups of schizophrenic/schizoaffective (n=66, 40.9 vs. 17.9 days, p=0.03; inpatient cost 9 009 ± 15 677 Euro vs. 3 650 ± 8 486 Euro, p=0.02; total expenditures 11 789 ± 15 975 Euro vs. 9 623 ± 9 262 Euro, p=0.33) and unipolar depressive patients (n=90, 29.8 vs. 9.8 days, p=0.006; inpatient cost 5 664 ± 14 921 Euro vs. 1 967 ± 5 276 Euro, p=0.02; total expenditures 7 146 ± 15 164 Euro vs. 6 234 ± 6 292 Euro, p=0.57). CONCLUSION: The IC was able to considerably reduce the utilisation of inpatient treatment through offering a complex range of services in the outpatient setting and allowed for a weight-shift in a low-threshold comprehensive care structure without an increase in costs from the statutory health insurance companies' perspective. For a detailed description of clinical effects further studies are required.

Analysis of laser-produced aerosols by inductively coupled plasma mass spectrometry: transport phenomena and elemental fractionation.

The transport phenomena of laser-produced aerosols prior to analysis by inductively coupled plasma mass spectrometry (ICPMS) were examined. Aerosol particles were visualized over the cross section of a transport tube attached to the outlet of a conventional ablation cell by light scattering using a pulsed laser source. Experiments were carried out under laminar or turbulent in-cell flow conditions applying throughputs of up to 2.0 L/min and reveal the nature of aerosol transportation to strongly depend on both flow rate and carrier gas chosen. For instance, laser ablation (LA) using laminar in-cell flow and helium as aerosol carrier resulted in stationary but inhomogeneous dispersion patterns. In addition, aerosols appear to be separated into two coexisting phases consisting of (i) dispersed particles that accumulate at the boundary layer of several vortex channel flows randomly arranged along the tube axis and (ii) larger fragments moving inside. The occurrence of these fragments was found to affect the accuracy of Si-, Zn-, and Cd-specific ICPMS analyses of aerosols released by LA of silicate glass (SRM NIST610). Accuracy drifts of more than 10% were observed for helium flow rates of >1 L/min, most probably, due to preferential evaporation and diffusion losses of volatile constituents inside the ICP. The utilization of turbulent in-cell flow made the vortex channels collapse and resulted in an almost complete aerosol homogenization. In contrast, LA using argon as aerosol carrier generally yielded a higher degree of dispersion, which was nearly independent of the flow conditions applied. To illustrate the differences among laminar and turbulent in-cell flow, furthermore, the velocity field inside the ablation cell was simulated by computational fluid dynamics.

Capabilities of femtosecond laser ablation inductively coupled plasma mass spectrometry for depth profiling of thin metal coatings.

The capabilities of ultraviolet femtosecond laser ablation inductively coupled plasma mass spectrometry (UV-fs-LA-ICPMS) for depth profile analysis of thin metal coatings were evaluated. A standard sample consisting of a single Cr thin layer of 500 nm +/- 5% on a Ni substrate was used. A fast washout was obtained by a high-efficiency aerosol dispersion ablation cell (V approximately 1 cm3), which allowed single-shot analysis with increased depth resolution. Laser ablation was performed in helium at atmospheric pressure conditions. A laser repetition rate of 1 Hz and low laser fluence (<0.5 J/cm2) were used. Very low ablation rates (<10 nm/pulse) were determined by atomic force microscopy (AFM). Information about the crater geometry and morphology was investigated using scanning electron microscopy and AFM. The depth resolution, calculated via the maximum slope of the tangent in the layer interface region, was smaller than 300 nm. Our data indicate that UV-fs-LA-ICPMS represents a powerful combination of high lateral and depth resolution for the analysis of thin metal coatings. Moreover, an overall ion yield, defined as the ratio of detected ions and ablated atoms, of approximately 5 x 10-5 was estimated for the chromium layer under the operating conditions chosen. The absolute amount of ablated material per laser pulse was approximately 1 pg, which corresponds to a detection limit of 180 microg/g.

Proposed mechanism of acetate accumulation in two recombinant Escherichia coli strains during high density fermentation.

The productivity of Escherichia coli as a producer of recombinant proteins is affected by its metabolic properties, especially by acetate production. Two commercially used E. coli strains, BL21 (lambdaDE3) and JM109, differ significantly in their acetate production during batch fermentation at high initial glucose concentrations. E. coli BL21 grows to an optical density (OD, 600 nm) of 100 and produces no more than 2 g/L acetate, while E. coli JM109 grows to an OD (600 nm) of 80 and produces up to 14 g/L acetate. Even in fed-batch fermentation, when glucose concentration is maintained between 0.5 and 1.0 g/L, JM109 accumulates 4 times more acetate than BL21. To investigate the difference between the two strains, metabolites and enzymes involved in carbon utilization and acetate production were analyzed (isocitrate, ATP, phosphoenolpyruvate, pyruvate, isocitrate lyase, and isocitrate dehydrogenase). The results showed that during batch fermentation isocitrate lyase activity and isocitrate concentration were higher in BL21 than in JM109, while pyruvate concentration was higher in JM109. The activation of the glyoxylate shunt pathway at high glucose concentrations is suggested as a possible explanation for the lower acetate accumulation in E. coli BL21. Metabolic flux analysis of the batch cultures supports the activity of the glyoxylate shunt in E. coli BL21.

The Influence of Operative Laparoscopy on the General Operative Concept in Gynecology

At Caritas-Klinik St. Theresia, Saarbrucken, Germany, 1663 patients underwent gynecologic surgery between 1990 and 1993. In the same period therapy via laparotomy was replaced stepwise by operative laparoscopy. While operative laparoscopy amounted to 70% (n=316) of all operations in 1990, it was 87% (n=515) in 1993. Additionally, the positive experience with minimally invasive surgery permitted us to enlarge the spectrum of indications for operative laparoscopy considerably, so that extensive adhesiolysis within the entire abdominal cavity (n=78 in 1993), adnexal surgery in pre- and postmenopausal women (n=254 in 1993), and laparoscopic hysterectomy (n=71 in 1993) have become routine today. Morbidity of patients after operative laparoscopy was significantly lower than after laparotomy. Our experiences support the view that operative laparoscopy is not only a benefit to selected subgroups of patients but may be applied successfully to the majority of women requiring gynecologic surgery.

Use of high density cultures of Escherichia coli for high level production of recombinant Pseudomonas aeruginosa exotoxin A.

An efficient fermentation method for the production of two modified recombinant Pseudomonas aeruginosa exotoxin As cloned in Escherichia coli BL21(lambda DE3) was developed. Cell densities of 16-30 g dry weight/1 were found to be most suitable for the induction of protein synthesis, which was under the isopropyl beta-D-thiogalactopyranoside (IPTG)-inducible T7 expression system. A concentration of 0.6 mM IPTG and induction time of 90 min were found to give the best results for production of the modified toxins. Using this procedure, gram amounts of the proteins were obtained in a 3-1 bench-top fermentor. The high density growth of the bacteria did not impair the integrity of the proteins and did not interfere with the purification procedure.

Interaction of ganglioside GM1 with the B subunit of cholera toxin modulates growth and differentiation of neuroblastoma N18 cells.

The present study uses the B subunit of cholera toxin, a protein that binds specifically to ganglioside GM1, to examine the role of endogenous GM1 in the process of growth and differentiation of mouse neuroblastoma N18 cells. Binding of the B subunit to neuroblastoma N18 cells inhibited DNA synthesis with concomitant induction of differentiation. The B subunit induced pronounced morphological changes: an increase in neurite outgrowth with branched neurites and spinelike processes. The distinct morphological alterations and neuritogenesis in response to the B subunit were also revealed by immunofluorescence with fluorescein-labeled B subunit. The mechanism of the B subunit-induced differentiation is different than that of spontaneous differentiation. Thrombin, a serine protease present in normal serum, inhibits neurite outgrowth induced by the removal of serum from the medium. In contrast, thrombin did not cause retraction of the neurites induced by the B subunit. Thus, thrombin or a thrombin-like protease is not involved in the process of neurite outgrowth mediated through endogenous GM1. The biological effects of the B subunit are due to the binding of the B subunit to ganglioside GM1 and not due to changes in cAMP levels resulting from contaminating A subunit. We used highly purified cloned B subunit that cannot contain any A subunit because it was isolated from a Vibrio cholerae mutant that only expresses the B subunit. Neither the cloned nor commercial preparations of the B subunit induced increases of cAMP in these cells. There was a good correlation between the amount of B subunit bound to the cells and the biological effect. Finally, treatment with neuraminidase, which caused a fourfold increase in the level of membrane GM1 as determined by iodinated cholera toxin binding, enhanced the biological effect of the B subunit. However, neuraminidase treatment alone did not have significant effects, either on DNA synthesis or on morphology of the cells, indicating that elevations in the level of GM1 per se are not sufficient by themselves to cause significant changes in cell growth or differentiation. It seems most likely that the aggregation of endogenous GM1 on the cell surface by the B subunit is responsible for these effects on mouse neuroblastoma N18 cells.

Production of cholera toxin subunit B by a mutant strain of Vibrio cholerae.

The B subunit (CTB) of cholera toxin (CT) can be used as a carrier protein for conjugate vaccines designed to elicit antipolysaccharide antibodies. A defined medium, AGM4, was designed to grow a high-producing mutant of Vibrio cholerae expressing only the B subunit of CT: V. cholerae 0395-NI. AGM4 contains four amino acids, asparagine, glutamic acid, arginine and serine, salts and a trace element solution. The carbon source is glucose. The fermentations performed in AGM4 indicated that CTB production paralleled the growth of the organism but that there was a maximal release of CTB during the stationary phase. There was a clear optimum of productivity at pH 8.0 and 30 degrees C. The pH had an influence on CTB production and not only on its release. Analysis of the amino acids present in the medium showed a correlation between their consumption rates and CTB productivity.

Unequal partitioning of bovine mitochondrial genotypes among siblings.

Two polymorphic mitochondrial DNA genomes, differing by a single Hpa II restriction site, are present at significantly different levels in tissue of three sibling dairy cows. The relative ratio of the two heteroplasmic molecules varies 3-fold among these three animals and documents a rapid segregation of mitochondrial genotypes in mammals. DNA sequencing shows the difference is due to a single guanine at position 364 in bovine mitochondrial DNA. A model involving unequal partitioning of the two amplified mitochondrial DNA species during the early cell divisions of the embryo can explain the appearance of such variation in heteroplasmic sibling animals. The model provides a basis for understanding the rapid DNA sequence variation observed in vertebrate mitochondrial DNA despite its high copy number and strict maternal inheritance.

Partial protection of lambs against Haemonchus contortus by vaccination with a fractionated preparation of the parasite.

Florida Native lambs, less than 6 months of age, were successfully vaccinated against Haemonchus contortus with a high mol. wt fraction (greater than 30,000 daltons) derived from a somatic extract of H. contortus larvae (SEL) and excretions and secretions (ES) of larvae isolated during in vitro development from the infective 3rd to 4th stage. A 59% reduction in adult worm numbers was obtained in vaccinates compared to naive lambs following challenge. The protection in vaccinated lambs was similar to that seen in lambs exposed to a primary infection of H. contortus larvae which had been cleared with anthelmintic prior to the challenge infection. The unfractionated SEL/ES preparation and a low mol. wt fraction gave no significant protection against challenge infection.

Heterogeneous mitochondrial DNA D-loop sequences in bovine tissue.

Mitochondrial DNA from bovine tissue contains heterogeneous sequences located within an evolutionary conserved cytosine homopolymer sequence near the 5' end of the D-loop region. This part of the mammalian mitochondrial genome is known to contain the origin of heavy strand DNA synthesis and the major transcriptional promoter for each strand. Nucleotide sequence analysis of cloned DNA and electrophoretic analysis of appropriate small fragments from animal tissue reveal a population of length polymorphs containing from nine to 19 cytosine residues. No individual length species represents more than 40% of the population. These data imply a state of significant intraanimal mtDNA sequence heterogeneity, which most likely occurs intracellularly as well. The localization of variability to a homopolymer run suggests that replication slip-page generated the sequence population. We also report that when recombinant clones containing this region are repeatedly passaged in E. coli, they begin to regenerate length variation similar to that seen in animal mtDNA.

Nucleotide sequence evidence for rapid genotypic shifts in the bovine mitochondrial DNA D-loop.

Mitochondrial DNA (mtDNA) is unusual in its rapid rate of evolution and high level of intraspecies sequence variation. The latter is thought to be related to the strict maternal inheritance of mtDNA, which effectively isolates within a species mitochondrial gene pools that accumulate mutations and vary independently. A fundamental and as yet unexplained aspect of this process is how, in the face of somatic and germ-line mtDNA ploidy of 10(3) to 10(5) (refs 4, 5), individual variant mtDNA molecules resulting from mutational events can come to dominate the large intracellular mtDNA population so rapidly. To help answer this question, we have determined here the nucleotide sequence of all or part of the D-loop region in 14 maternally related Holstein cows. Four different D-loop sequences can be distinguished in the mtDNA of these animals. One explanation is that multiple mitochondrial genotypes existed in the maternal germ line and that expansion or segregation of one of these genotypes during oogenesis or early development led to the rapid genotypic shifts observed.