Randomised Introduction of 2-CDA as Intensification during Consolidation for Children with High-risk AML--results from Study AML-BFM 2004.

BACKGROUND: The outcome in children and adolescents with high-risk (HR) acute myeloid leukemia (AML) is still unsatisfactory. Therefore, in study AML-BFM 2004 we aimed to improve outcome of HR-patients by adding moderately dosed 2-Chloro-2-Deoxyadenosine (2-CDA) to the respective consolidation treatment backbone without increasing toxicity. The aim was to improve prognosis especially in FAB M4/M5/MLL patients, who represent the largest subgroup of HR patients. PATIENTS AND METHODS: In total, 343 children and adolescents with HR-AML were randomized to receive or not 2-CDA (6 mg/m²/d, days 1, 3) in combination with cytarabine/idarubicine (AI=500 mg/m² cytarabine 5 days continuous infusion plus 7 mg/m²/d idarubicin, days 3 and 5). RESULTS: RESULTS for patients of the AI/2-CDA arm (n=168) vs. the AI-arm (n=175) were similar: 5-year overall survival 68±4 vs. 72±4%, plogrank=0.38, event-free survival 53±4 vs. 49±4%, plogrank=0.77; cumulative incidence of relapse at 5 years: 35±4 vs. 37±4%, p(Gray)=0.89. RESULTS in patients with MLL rearrangement or FAB M4/M5 were also similar in the treatment groups. In addition, toxicities did not differ between the two arms. CONCLUSION: We conclude that additional, moderate dose 2-CDA does not improve prognosis in HR-patients when given during consolidation treatment. Its effect might be too low in this multidrug regimen, where the strongest effects are achieved during induction, or the chosen dose of 2-CDA might have been too low.

Outbreak of wound botulism in people who inject drugs, Norway, October to November 2013.

In October and November 2013, four cases of wound botulism were confirmed in people who inject drugs (PWID) in Norway. Two additional cases are suspected. Because of the international distribution pathways for heroin ­ the likely source of the outbreak ­ healthcare workers and public health authorities in other countries should remain vigilant for wound botulism in PWID. This outbreak serves as a reminder that countries should ensure access to botulinum antitoxin in case of outbreak situations.

Two outbreaks of diarrhoea in nurseries in Norway after farm visits, April to May 2009.

During a 2009 nationwide outbreak of sorbitolfermenting Escherichia coli O157 in Norway, the Norwegian Institute of Public Health was notified of diarrhoea outbreaks in two nurseries. A link to the nationwide outbreak was suspected and investigated, including retrospective cohort studies. Both nurseries had recently visited farms. Faecal specimens were obtained from symptomatic children as well as from the farm animals and tested for Campylobacter, Salmonella, Yersinia, Shigella and pathogenic E. coli, and isolates were further characterised. Nursery A had 12 symptomatic children, and we found the same strain of C. jejuni in faeces from children and lambs. Nursery B had nine symptomatic children, including one child with bloody diarrhoea carrying enterohaemorrhagic E. coli (EHEC) O26. EHEC O26 with a similar multiple-locus variable number tandem repeat analysis (MLVA)-profile was found in sheep. Five children had enteropathogenic E. coli (EPEC) O76. Animals were not tested for EPEC O76. We found no significant association between illness and risk factors for either nursery. The isolated pathogens differed from the one involved in the nationwide outbreak. In each nursery outbreak, the pathogens isolated from children matched those found in farm animals, implicating animal faeces as the source. Hygiene messages are important to prevent similar outbreaks.

Extraction of DNA from Forensic Biological Samples for Genotyping.

Biological forensic samples constitute evidence with probative organic matter. Evidence believed to contain DNA is typically processed for extraction and purification of its nucleic acid content. Forensic DNA samples are composed of two things, a tissue and the substrate it resides on. Compositionally, a sample may contain almost anything and for each, the type, integrity, and content of both tissue and substrate will vary, as will the contaminant levels. This fact makes the success of extraction one of the most unpredictable steps in genotypic analysis. The development of robust genotyping systems and analysis platforms for short tandem repeat (STR) and mitochondrial DNA sequencing and the acceptance of results generated by these methods in the court system, resulted in a high demand for DNA testing. The increasing variety of sample submissions created a need to isolate DNA from forensic samples that may be compromised or contain low levels of biological material. In the past decade, several robust chemistries and isolation methods have been developed to safely and reliably recover DNA from a wide array of sample types in high yield and free of PCR inhibitors. In addition, high-throughput automated workflows have been developed to meet the demand for processing increasing numbers of samples. This review summarizes a number of the most widely adopted methods and the best practices for DNA isolation from forensic biological samples, including manual, semiautomated, and fully automated platforms.

Extraction of DNA from Human Remains.

Improvements to analytical methods have made it possible for highly discriminative genotypic information to be gleaned from smaller and smaller amounts of sample material. This fact makes it practical to genotype samples or remains consisting of bone and tooth-samples that likely would not have yielded interpretable genotypic results a short time ago. In parallel, there have been improvements to protocols specifically designed to recover DNA from very old calcified tissues, i.e., ancient or compromised nature. This review discusses the current best practices for isolating and purifying DNA from bones and teeth with a focus on the processes of lysis and DNA purification linked together to yield DNA from these challenging samples. The mitochondrial and genomic DNA recovered from more recently developed techniques for isolation from skeletal remains and teeth, even very old samples, is surprisingly amenable to genotypic analysis.

Engineering the isoelectric point of a renal cell carcinoma targeting antibody greatly enhances scFv solubility.

BACKGROUND: The murine A6H monoclonal antibody targets a cell surface antigen associated with renal cell carcinoma with high specificity and excellent biodistribution properties. Tumor to blood ratios of > 40:1 have been achieved in clinical studies. OBJECTIVES: In order to generate an antibody engineering system that would allow the construction of improved derivatives for diagnostics and therapeutics, a single-chain Fv antibody (scFv) derived from A6H was constructed. The initial single-chain Fv, constructed with a cysteine residue and hexa-histidine sequence at the C-terminus, displayed a limited solubility of 100 microg/ml at pH 7.4. The low solubility and refolding yield of the original single-chain Fv required that a more soluble variant be designed and constructed. STUDY DESIGN: We hypothesized that lowering the pI of the scFv antibody away from the physiological range would yield a more soluble antibody. A derivative was thus subsequently engineered with five glutamic acid residues followed by the cysteine and hexa-histidine residues. The cysteine was included to provide a conjugation site for future radiolabeling studies. RESULTS: The redesigned A6H single-chain Fv has a predicted pI of 6.1, relative to 7.5 for the native scFv. The redesigned A6H scFv displayed a greatly enhanced solubility of > 15 mg/ml at pH 7.4. Both the original scFv and the redesigned single-chain Fv exhibited a strong tendency to form dimers and soluble high molecular weight aggregates. The monomer and disulfide bonded dimer were separated from the aggregates and complete cell binding isotherms were obtained, demonstrating that the purified A6H scFv retains much of the activity of the parent monoclonal. CONCLUSION: The addition of glutamic acid to the C-terminus of poorly soluble scFv antibodies could provide a straightforward avenue for improving their solubility properties. The increased solubility of the A6H scFv allowed the purification of the monomeric and dimeric species from the soluble aggregated species.

Study of free and complexed prostate-specific antigen in mice bearing human prostate cancer xenografts.

BACKGROUND: Our objective was to evaluate five preclinical prostate cancer (CaP) xenograft models to determine whether (1) prostate-specific antigen (PSA) formed complexes in murine serum, (2) the percentage of free PSA (f-PSA) was characteristic of a given xenograft line, and (3) the percentage of f-PSA was similar to that in the patient at time of tumor harvest. Our fourth objective was to identify which murine serpin(s) bind(s) to PSA in vivo. METHODS: Xenografts were established from metastatic foci. The percentage of f-PSA, and total PSA (t-PSA) in serum of animals bearing CaP xenografts was determined by immunoassay. Size exclusion high-performance liquid chromatography and Western blots were used to evaluate the presence of PSA complexes in murine serum. Edman degradation was used to determine the N-terminal sequence of complexed proteins. RESULTS: PSA was detected as both free and complexed forms in murine serum from all mice bearing the CaP xenografts. Three xenografts (related sublines) produced PSA that resulted in low mean percentages of f-PSA (1.9-6.4%). In sera from the other two xenografts, the mean percentages of f-PSA were high (>25%); patient sera, where available at time of tumor acquisition, were in agreement. Western blots showed that murine protease inhibitors formed complexes with PSA. Edman degradation yielded a sequence with 80% homology over 15 amino acids with that of murine alpha1-protease inhibitor (alpha1-PI). CONCLUSIONS: Our data have shown that the majority of PSA secreted by these CaP xenografts complexes in murine serum with a protease inhibitor with high homology to murine alpha1-PI and that the percentage of f-PSA is a characteristic of each xenograft line tested, which is in agreement with patient values at time of tumor harvest. These CaP xenografts offer opportunities for study of human PSA biology and phenomenology.

Streptavidin in antibody pretargeting. Comparison of a recombinant streptavidin with two streptavidin mutant proteins and two commercially available streptavidin proteins.

In this investigation, a comparison of wild type recombinant streptavidin (r-SAv) with two genetically engineered mutant r-SAv proteins was undertaken. The investigation also included a comparison of the r-SAv with two streptavidin (SAv) proteins from commercial sources. In vitro characterization of the SAv proteins was conducted by HPLC, SDS-PAGE, IEF, and electrospray mass spectral analyses. All SAv proteins studied appeared to be a single species by size exclusion chromatography (HPLC) and SDS-PAGE analyses, but multiple species were noted in the IEF and MS analyses. In vivo comparisons of the SAv proteins were accomplished with dual isotope-labeled SAv in athymic mice. In an initial experiment, tissue localization of r-[131I]SAv directly radiolabeled using chloramine-T was compared with r-SAv radiolabeled with the N-hydroxysuccinimidyl p-iodobenzoate conjugate ([125I]-PIB), a radioiodination reagent that has been shown to result in iodine-labeled proteins which are stable to in vivo deiodination. The data obtained indicated that there is little difference in the distribution (except kidney localization) when r-SAv labeled by the two methods. Data obtained from comparison of r-[131I]SAv with a disulfide-stabilized r-SAv mutant (r-SAv-H127C), a C-terminal cysteine-containing r-SAv mutant (r-[125I]SAv-S139C), and two 125I-labeled SAv proteins obtained from commercial sources indicated that their distributions were quite similar, except the kidney concentrations were generally lower than that of r-[131I]SAv. On the basis of the similar distributions of the SAv proteins studied, it appears that the r-SAv mutants may be interchanged for the (wild type) r-SAv in pretargeting studies. Further, the similarity of distributions with two commercially available SAv proteins suggests that the results obtained in our studies and those of other groups may be directly compared (with consideration of animal model, sacrifice time, etc.).

Characterization of 10 new monoclonal antibodies against prostate-specific antigen by analysis of affinity, specificity and function in sandwich assays.

While prostate-specific antigen (PSA) is already an invaluable marker for prostate cancer, there is continuing demand for new anti-PSA antibodies with specific characteristics, e.g., high sensitivity and specificity and equimolar binding to free PSA (f-PSA) and the PSA-alpha-1-antichymotrypsin complex (PSA-ACT), as well as the ability to distinguish between these 2 immunoreactive forms of PSA. We have therefore generated and characterized 10 anti-PSA monoclonal antibodies (MAbs). Apparent dissociation constants (Kd) of MAbs were determined by direct ELISA yielding Kd-0.2-164.0 nM. Western blots suggested that 3 of the MAbs (60-1A2, 60-8A2 and 17-1A2) bind to linear epitopes. Sandwich assays identified 5 major antigenic regions as binding targets of the MAbs. Three combinations of MAbs recognize f-PSA and PSA-ACT in equimolar fashion with high sensitivity. Two of the MAb combinations are specific for f-PSA. Physical analysis of the new antibodies has allowed us to assign the MAbs to binding classes (based on their sandwiching capabilities) and to determine accurate apparent dissociation constants.

High-affinity peptide ligands to prostate-specific antigen identified by polysome selection.

We have used the polysome-selection method to isolate peptide ligands that bind with high affinity to Prostate-Specific Antigen (PSA), an important prostate-cancer marker. Two random libraries, each encoding approximately 10(12) random peptides, were transcribed into RNA and translated in vitro. Polysomes were panned by affinity selection of the nascent peptides against immobilized PSA. Over 30% of the selected species had significant affinity for PSA; the dissociation constant of the complex formed by the best isolate with PSA was < 10(-9) M. Formation of streptavidin conjugates of selected peptides improved their affinities and, in one case, virtually eliminated non-specific binding. The polysome-selection method can be used to produce high-affinity peptide ligands of potential use in diagnostic and therapeutic procedures.

Antibody fragments in tumor pretargeting. Evaluation of biotinylated Fab' colocalization with recombinant streptavidin and avidin.

An evaluation of the use of a biotinylated monoclonal antibody Fab' fragment in tumor pretargeting was conducted. As a model system, tumor colocalization of avidin or recombinant streptavidin (r-streptavidin) and the biotinylated Fab' fragment (Fab'-S-biotin) of A6H, an antirenal cell carcinoma antibody, was evaluated in athymic mice bearing human renal cell carcinoma xenografts. A new water soluble sulfhydryl reactive biotinylation reagent, N-(13-N-maleimdo-4, 7,10-trioxatridecanyl)-biotinamide, was synthesized and used for biotinylation of Fab'. A biodistribution of ChT-labeled A6H Fab'-S-biotin was conducted. Data from that distribution indicated that the Fab'-S-biotin localized well (i.e. 28% ID/g at 24 h) to human tumor xenografts in athymic mice. Subsequently, a biodistribution study involving pretargeting radioiodinated A6H Fab'-S-biotin to tumor xenografts, followed by administration of r-streptavidin at 4 or 20 h, was conducted. Specific colocalization of r-streptavidin to tumors containing the A6H Fab'-S-biotin was evident from the data obtained. In a similar biodistribution study, specific colocalization of avidin to tumors pretargeted with A6H Fab'-S-biotin was also observed. The avidin used in the study was radioiodinated with the N-hydroxysuccinimidyl ester of p-[125I]iodobenzoate ([125I]PIB-NHS). Very low concentrations (e.g. 0.35% ID/g) of avidin colocalized at the tumor. To further show that specific colocalization within the tumor xenografts had occurred with biotinylated A6H Fab', radioiodinated avidin and r-streptavidin were co-injected into athymic mice bearing tumor xenografts to obtain their distributions without having biotinylated Fab' present. At 20 h postinjection, only small differences in the blood and tumor concentrations of either protein were observed, indicating that the specific tumor colocalization seen in the previous two biodistributions must have been due to the presence of Fab'-S-biotin. Calculations were conducted to estimate how much r-streptavidin (as a molar ratio) was colocalized. From the data obtained it was estimated that 36-61% of the tumor-localized Fab'-S-biotin molecules were bound with r-streptavidin and 4-23% bound with avidin, under the conditions studied.

Synthesis and nca-radioiodination of arylstannyl-cobalamin conjugates. Evaluation of aryliodo-cobalamin conjugate binding to transcobalamin II and biodistribution in mice.

A new method of preparing radiolabeled cobalamin derivatives has been developed. The method involves the use of cobalamin-tri-n-butylstannyl hippurate conjugates as intermediates to obtain radioiodinated cobalamin-iodohippurate conjugates. The arylstannyl functionality was used as an exchangeable group to obtain high specific activity radioiodinations and to circumvent some deleterious side reactions common to cobalamins under electrophilic iodination conditions. The first step in the synthesis of tri-n--butylstannyl hippurate conjugates was to obtain free carboxylate groups on the cobalamin moiety. This was accomplished by mild acid hydrolysis of the b-, d-, or e-propionamide side chains on the corrin ring, followed by careful separation of the isomeric products. The second step was to couple a linking molecule (diaminododecane) to the carboxylate. The final step was to conjugate p-tri-n-butylstannyl hippurate to the cobalamin-diaminododecane adduct. All three isomeric cobalamin-p-tri-n-butylstannyl hippurate conjugates were prepared, as were the corresponding cobalamin-p-iodohippurate conjugates (HPLC standards). Radioiodination reactions were conducted with N-chlorosuccinimide and Na[*I]I in Me OH using conditions previously developed for arylstannylations. However, unlike the previous reactions, a key factor in obtaining the desired radioiodinated cobalamins was that the reaction be conducted under neutral conditions. Isolated yields of 40-65% were obtained for all three cobalamin isomers. Specific activities of 10-33% theoretical were obtained for the radioiodinated cobalamins. Evaluation of competitive binding of (nonradioactive) cobalamin-iodohippurate conjugates with recombinant human transcobalamin II showed that the e-isomer bound nearly as well as [57Co]cyanocobalamin (50%), whereas the b-isomer had decreased binding (6%) and the d-isomer was significantly decreased in its binding (0.7%). Two biodistributions of the radioiodinated e-isomer were conducted in athymic mice. One biodistribution investigated tissue localization in mice bearing a renal cell carcinoma xenograft, and the other biodistribution investigated tissue localization when the radioiodinated cyanocobalamin was mixed with 1% BSA prior to injection. A comparison of the results of the two biodistributions and a discussion of how they relate to previous [57/60Co]cyanocobalamin biodistributions are provided.

Monoclonal antibody Fab' fragment cross-linking using equilibrium transfer alkylation reagents. A strategy for site-specific conjugation of diagnostic and therapeutic agents with F(ab')2 fragments.

An investigation was conducted to evaluate the feasibility of site-selective addition of diagnostic and therapeutic agents to monoclonal antibody F(ab')2 fragments through cross-linking of antibody Fab' fragments. In the investigation, trifunctional equilibrium transfer alkylation cross-link (ETAC) reagents, 4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]benzoic acid, 1a, N-[4-[2,2bis[(p-tolylsulfonyl)methyl]acetyl]-benzoyl]-4- (tri-n-butylstannyl)phenethylamine, 3a, and N-[4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]- benzoyl]-4-[125,131I]iodophenethylamine, 3b, were synthesized. The ETAC derivatives were reacted with Fab' fragments of an antirenal cell carcinoma antibody (A6H) produced from reduction of F(ab')2 using 1,4-dithiothreitol. Cross-linking of Fab' was obtained to yield a radioiodinated modified F(ab')2, [mF(ab')2], fragment. The cross-linking reaction produced mixed addition products, requiring the desired mF(ab')2 to be separated from radioiodinated Fab' by size exclusion HPLC. Tumor cell binding immunoreactivities varied (60-90%) for five isolated mF(ab')2 preparations but were consistent with other radiolabeled antibody preparations tested on the same day. In vitro stability testing indicated that the mF(ab')2 was reasonably stable toward loss of the ETAC cross-linking reagent, except under strongly basic conditions. Under reducing sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, protein bands believed to be cross-linked heavy chain dimers were observed. Biodistribution of purified radioiodinated A6H mF(ab')2 was conducted in athymic mice bearing a renal cell carcinoma xenograft (TK-82). A nonmodified control A6H F(ab')2, radioiodinated as a p-[125,131I]-iodobenzoyl conjugate, was coinjected for comparison. The radioiodionated mF(ab')2 had a similar distribution to the radioiodinated control at 3.5, 19, and 43 h postinjection. In another study, the distribution of radioiodinated A6H Fab' was evaluated at 4 and 24 h to establish clearance and pharmacokinetics for comparison with the data obtained from the mF(ab')2. The biodistribution data indicated that A6H mF(ab')2 was quite different from that of A6H Fab'. The results from this preliminary study suggest that it may be possible to attach (large polymeric) diagnostic or therapeutic agents to monoclonal antibody F(ab')2 fragments through the use of ETAC reagents.

Preparation and evaluation of para-[211At]astatobenzoyl labeled anti-renal cell carcinoma antibody A6H F(ab')2. In vivo distribution comparison with para-[125I]iodobenzoyl labeled A6H F(ab')2.

A preliminary investigation of an 211At labeled anti-renal cell carcinoma antibody fragment, A6H F(ab')2, was conducted. In the investigation, A6H F(ab')2 was labeled by conjugation with N-succinimidyl p-[211At]astatobenzoate, and the in vivo biodistribution was evaluated in athymic mice bearing TK-82 renal cell carcinoma xenografts. As a control, p-[125I]iodobenzoyl labeled A6H F(ab')2 was coinjected with the astatinated F(ab')2. The data obtained demonstrated that the two radiolabels (211At and 125I) had quite similar distributions, providing evidence that the 211At remained attached to the A6H F(ab')2 in vivo. Further, the astatinated antibody attained a 2:1 tumor-to-blood ratio, and greater than 35:1 tumor-to-muscle ratio, at 4h post-injection, suggesting that this antibody conjugate could be used to evaluate treatment of metastatic renal cell carcinoma in a mouse model.

The immunodominant outer membrane antigen of Actinobacillus actinomycetemcomitans is located in the serotype-specific high-molecular-mass carbohydrate moiety of lipopolysaccharide.

Most patients with juvenile periodontitis manifest serum antibodies, sometimes at very high titers, to antigens of Actinobacillus actinomycetemcomitans, but the antigens inducing the immune response have been only partly characterized. We separated A. actinomycetemcomitans serotype b cells into protein, lipopolysaccharide (LPS), and soluble polysaccharide fractions and characterized them. Coomassie blue- and silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels were used to detect protein and LPS components, and gas-liquid chromatography was used to determine their carbohydrate and fatty acid composition. Western blots, dot blots, and enzyme-linked immunosorbent assay inhibition with high-titer sera from juvenile periodontitis patients revealed which components were highest in antibody binding activity. These results showed that the major portion of the immunoglobulin G binding activity resides in the purified mannan-free LPS, with lesser amounts in the total protein fraction. Using Sephacryl S-300 chromatography, we separated LPS into high-molecular-mass components with high carbohydrate contents by gas-liquid chromatography and a low-molecular-mass component consisting mainly of lipid A and the inner core sugar heptulose. The results of quantitative dot blot assays and enzyme-linked immunosorbent assay inhibition show that the serotype-specific antibody binding activity is highly concentrated in the high-molecular-mass carbohydrate-rich LPS fraction and is almost completely absent in the low-molecular-weight lipid-rich fraction. Our observations contrast with previous reports that the predominant serotype antigen of A. actinomycetemcomitans resides in a mannan-rich polysaccharide isolated from spent culture medium. These observations support the conclusion that the immunodominant antigen of the outer membrane is the O antigen of the LPS.

Toxicity of Bacillus sphaericus crystal toxin to adult mosquitoes.

Adult Culex quinquefasciatus mosquitoes were killed by alkaline-solubilized Bacillus sphaericus toxin when it was introduced by enema into the midgut of the insect but not when it was administered orally. Adult Aedes aegypti mosquitoes were not affected by the toxin.