Identification of serum angiopoietin-2 as a biomarker for clinical outcome of colorectal cancer patients treated with bevacizumab-containing therapy.

BACKGROUND: The combination of chemotherapy with the vascular endothelial growth factor (VEGF) antibody bevacizumab is a standard of care in advanced colorectal cancer (CRC). However, biomarkers predicting outcome of bevacizumab-containing treatment are lacking. As angiopoietin-2 (Ang-2) is a key regulator of vascular remodelling in concert with VEGF, we investigated its role as a biomarker in metastatic CRC. METHODS: Serum Ang-2 levels were measured in 33 healthy volunteers and 90 patients with CRC. Of these, 34 had metastatic disease and received bevacizumab-containing therapy. To determine the tissue of origin of Ang-2, quantitative real-time PCR was performed on microdissected cryosections of human CRC and in a murine xenograft model of CRC using species-specific amplification. RESULTS: Ang-2 originated from the stromal compartment of CRC tissues. Serum Ang-2 levels were significantly elevated in patients with metastatic CRC compared with healthy controls. Amongst patients receiving bevacizumab-containing treatment, low pre-therapeutic serum Ang-2 levels were associated with a significant better response rate (82 vs 31%; P<0.01), a prolonged median progression-free survival (14.1 vs 8.5 months; P<0.01) and a reduction of 91% in the hazard of death (P<0.05). CONCLUSION: Serum Ang-2 is a candidate biomarker for outcome of patients with metastatic CRC treated with bevacizumab-containing therapy, and it should be further validated to customise combined chemotherapeutic and anti-angiogenic treatment.

Changes in cross-sectional area of spinal canal and vertebral body under 2 years of teriparatide treatment: results from the EUROFORS study.

The treatment of osteoporotic patients with teriparatide is associated with a significant increase in bone formation and gain of bone mass. The purpose of this post hoc analysis was to determine if the cross-sectional area (CSA) of the spinal canal and the vertebral body is affected by teriparatide treatment. Narrowing of the spinal canal might represent a safety problem, while widening of the vertebral CSA might improve mechanical stability. High-resolution computed tomography (HRCT) scans of vertebra T12 were obtained at baseline and after 6, 12, and 24 months of teriparatide treatment (20 microg/day) from 44 postmenopausal women with established osteoporosis participating in the prospective, randomized EUROFORS study. The CSA of the spinal canal did not decrease but increased marginally by 0.9% (2.6 mm(2)) over 24 months (P < 0.001), with a range from -0.5% (-2 mm(2)) to 3.1% (+8 mm(2)). Even when analyzing the spinal CSA on a slice-by-slice basis, no clinically relevant narrowing of the spinal canal was observed. For vertebral bodies, the CSA increased by 0.7% (5.7 mm(2)) over 24 months (P < 0.001), with a range from -0.4% (-3 mm(2)) to 1.6% (+14 mm(2)). Our data do not provide evidence for safety concerns regarding spinal canal narrowing. On the other hand, the increases observed for vertebral CSA apparently also only minimally contribute to the mechanical strengthening of the vertebral body under teriparatide treatment.

Development of anti-CD30 radioimmunoconstructs (RICs) for treatment of Hodgkin's lymphoma. Studies with cell lines and animal studies.

OBJECTIVES: Comparison of the binding affinity to a CD30-positive Hodgkin lymphoma (HL) cell line and biodistribution in HL bearing mice of new anti-CD30 radioimmunoconjugates (RICs) of varying structure and labelling nuclides. METHODS: The antibodies Ki-4 and 5F11 were radioiodinated by the chloramine T method or labelled with (111)In via p-NCS-Benzyl-DOTA. In addition, the Ki-4-dimer was investigated in the iodinated form. The RICs were analyzed for retained immunoreactivity by immunochromatography. In-vitro binding studies were performed on CD30-positive L540 cell lines. For in-vivo biodistribution studies, SCID mice bearing human HL xenografts were injected with the various radioimmunoconjugates. After 24 h, activities in the organs and tumour were measured for all 5 RICs. Tumour-free animals were studied in the same way with (131)I- Ki-4 24 h p. i. The three RICs with the highest tumour/background ratios 24 h p.i. ((131)I-Ki-4, (131)I-5F11, (111)In-bz-DOTA-Ki-4) were analysed further at 48 h and 72 h. RESULTS: All the RICs were successfully labelled with high specific activities (28-47 TBq/mmol) and sufficient radiochemical yields (>80%). Scatchard plot analysis proved high tumour affinity (KD = 20-220 nmol/l). In-vivo tumour accumulation in % of injected dose per g tissue (%ID/g) lay between 2.6 ((131)I-5F11) and 12.3 % ID/g ((131)I-Ki-4) with permanently high background in blood. Tumour/blood-ratios of all RICs were below one at all time points. CONCLUSIONS: In-vitro tumour cell affinities of all RICs were promising. However, in-vivo biokinetics tested in the mouse model did not meet expectations. This highlights the importance of developing and testing further new anti-CD30 conjugates.

[Performance of record linkage for cancer registry data linked with mammography screening data]. / Zur Leistungsfähigkeit des Record-Linkage zwischen epidemiologischen Krebsregistern und dem Mammographie-Screening.

The evaluation of the German Mammography Screening Program requires record linkage with data from cancer registries in order to measure the number of false-negative mammograms and interval cancers. This study aims at evaluating the performance of the established linkage method based on identifiers encrypted by the standard procedure of the German cancer registries. In addition, the results are compared with an alternative method based on plain text identifiers. A total of 16,572 records from the Bremen Mammography Screening Pilot Study were linked with data from the Bremen Cancer Registry. Based on a gold standard set of matching record pairs, homonym and synonym errors were determined. Given the customary threshold value in cancer registries, the plain text method showed a lower rate of synonym errors (2.1-5.1%) and a lower rate of homonym errors (0.01-0.15%). As 10.4 million women are invited to take part biennially in screening, the corresponding figures would be 3,237 homonym errors for the standard procedure and 294 using the plain text method provided equivalent conditions. The 11-fold increase in the homonym error rate documents the trade-off for better data protection using encrypted data.

Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase.

We have developed a nuclear transformation system for Chlamydomonas reinhardtii, using micro-projectile bombardment to introduce the gene encoding nitrate reductase into a nit1 mutant strain which lacks nitrate reductase activity. By using either supercoiled or linear plasmid DNA, transformants were recovered consistently at a low efficiency, on the order of 15 transformants per microgram of plasmid DNA. In all cases the transforming DNA was integrated into the nuclear genome, usually in multiple copies. Most of the introduced copies were genetically linked to each other, and they were unlinked to the original nit1 locus. The transforming DNA and nit+ phenotype were stable through mitosis and meiosis, even in the absence of selection. nit1 transcripts of various sizes were expressed at levels equal to or greater than those in wild-type nit+ strains. In most transformants, nitrate reductase enzyme activity was expressed at approximately wild-type levels. In all transformants, nit1 mRNA and nitrate reductase enzyme activity were repressed in cells grown on ammonium medium, showing that expression of the integrated nit1 genes was regulated normally. When a second plasmid with a nonselectable gene was bombarded into the cells along with the nit1 gene, transformants carrying DNA from both plasmids were recovered. In some cases, expression of the unselected gene could be detected. With the advent of nuclear transformation in Chlamydomonas, it becomes the first photosynthetic organism in which both the nuclear and chloroplast compartments can be transformed.

Parentage analysis and outcrossing patterns in cacao (Theobroma cacao L.) farms in Cameroon.

The present study investigates the parentage of farm accessions in Cameroon using data from 12 microsatellite loci. Bayesian analysis suggests that 25.5% of the 400 farm accessions studied is still closely related to the traditional Amelonado variety called 'German Cocoa' by the farmers. Another 46.3% of the farm accessions were found to be direct descendants (20.8% first-generation (F1) hybrids and 25.5% selfed genotypes) from 24 parental clones used in biclonal seed gardens (BSGs) established in the 1970s in southern and western Cameroon. Furthermore, 28.3% of farm accessions appeared to descent from uncontrolled pollination events in cacao farms, which could be related to a common practice of cacao growers to use seeds collected in their own farm for new plantings. All farm accessions descending from BSG could be individually related through parentage analysis to the 24 progenitors of the BSG. Only 25% of progenies distributed from BSG corresponded to F1 hybrids combinations originally planned to be released. Significant biparental inbreeding estimates were observed for all 'traditional' farms and for most 'F1 hybrids' farms due to presence of a high proportion of selfed accessions. Biparental inbreeding occurs when plants receive pollen from genetically related neighbors. High levels of outcrossing observed in 'mixed' farms might be explained by the admixture of traditional varieties and BSG progenies. The implications of our finding for management of seed gardens and for further breeding using farm accessions in Cameroon are discussed.

A position effect on the expression of a tRNA gene mediated by the SIR genes in Saccharomyces cerevisiae.

The SIR genes of Saccharomyces cerevisiae are responsible for the position-dependent regulation of the a and alpha mating-type genes. Previous work by others has shown that the products of the SIR genes prevent the accumulation of stable transcripts of the a and alpha genes at HML and HMR. Results of this study establish that this regulation is a region-specific effect rather than a gene-specific effect since expression of a tRNA gene placed at HMR is repressed by the products of the SIR genes.

Development of a marker assisted selection program for cacao.

ABSTRACT Production of cacao in tropical America has been severely affected by fungal pathogens causing diseases known as witches' broom (WB, caused by Moniliophthora perniciosa), frosty pod (FP, caused by M. roreri) and black pod (BP, caused by Phytophthora spp.). BP is pan-tropical and causes losses in all producing areas. WB is found in South America and parts of the Caribbean, while FP is found in Central America and parts of South America. Together, these diseases were responsible for over 700 million US dollars in losses in 2001 (4). Commercial cacao production in West Africa and South Asia are not yet affected by WB and FP, but cacao grown in these regions is susceptible to both. With the goal of providing new disease resistant cultivars the USDA-ARS and Mars, Inc. have developed a marker assisted selection (MAS) program. Quantitative trait loci have been identified for resistance to WB, FP, and BP. The potential usefulness of these markers in identifying resistant individuals has been confirmed in an experimental F(1) family in Ecuador.

Ribosomes with large synthetic N-terminal extensions of protein S15 are active in vivo.

The genes for ribosomal proteins S4, S13 or S15 were fused with the gene for staphylococcal protein A, or derivatives thereof (2A'-7A'). The gene fusions were introduced into Escherichia coli strains, mutated in the corresponding ribosomal protein gene, by transformation. These mutated ribosomal proteins cause a phenotype that can be complemented. Thus, the phenotype of the transformants was tested and the ribosomal proteins were analyzed. The S4 N-terminal fusion protein severely disturbed growth of both the mutant and the wild-type strains. The S13 C-terminal fusion protein was proteolyzed close to the fusion point, giving a ribosomal protein moiety that could assemble into the ribosome normally. S15 N-terminal fusion proteins complemented a cold-sensitive strain lacking protein S15 in its ribosomes. These fused proteins were assembled into active ribosomes. The position of S15 in the 30S ribosomal subunit is well known. Therefore, in structural studies of the ribosome in vivo, the S15 fusion proteins can be used as a physical reporter for S15.

Isolation of the Chlamydomonas regulatory gene NIT2 by transposon tagging.

Genetic evidence suggests that the NIT2 gene of Chlamydomonas reinhardtii encodes a positive regulator of the nitrate-assimilation pathway. To learn more about the function of the NIT2 gene product, we isolated the gene using a transposon-tagging strategy. A nit2 mutation caused by the insertion of a transposon was identified by testing spontaneous nit2 mutants for the presence of new copies of Gulliver or TOC1, transposable elements that have been identified in Chlamydomonas. In 2 of the 14 different mutants that were analyzed, a Gulliver element was found to be genetically and phenotypically associated with the nit2 mutation. Using the Gulliver element as a probe, one of the transposon-induced nit2 alleles was isolated, and a sequence adjoining the transposon was used to isolate the corresponding wild-type locus. The NIT2 gene was delimited by mapping DNA rearrangements associated with nit2 mutations and mutant rescue by genetic transformation. The NIT2 gene encodes a 6-kb transcript that was not detected in cells grown in the presence of ammonium. Likewise, NIT2-dependent genes are repressed in ammonium-grown cells. These results suggest that repression of the NIT2 gene may mediate metabolite repression of the nitrate assimilation pathway in Chlamydomonas.

Genetic and molecular characterization of suppressors of SIR4 mutations in Saccharomyces cerevisiae.

In order to learn more about other proteins that may be involved in repression of HML and HMR in Saccharomyces cerevisiae, extragenic suppressor mutations were identified that could restore repression in cells defective in SIR4, a gene required for function of the silencer elements flanking HML and HMR. These suppressor mutations, which define at least three new genes, SAN1, SAN2 and SAN3, arose at the frequency expected for loss-of-function mutations following mutagenesis. All san mutations were recessive. Suppression by san1 was allele-nonspecific, since san1 could suppress two very different alleles of SIR4, and was locus-specific since san1 was unable to suppress a SIR3 mutation or a variety of mutations conferring auxotrophies. The SAN1 gene was cloned, sequenced, and used to construct a null allele. The null allele had the same phenotype as the EMS-induced mutations and exhibited no pleiotropies of its own. Thus, the SAN1 gene was not essential. SAN1-mediated suppression was neither due to compensatory mutations in interacting proteins, nor to translational missense suppression. SAN1 may act posttranslationally to control the stability or activity of the SIR4 protein.

A phase I/II study of idarubicin, dexamethasone and interferon-alpha (I-Dexa) in patients with relapsed or refractory multiple myeloma.

To improve the outcome for patients with relapsed or refractory multiple myeloma (MM), we combined idarubicin (Ida), dexamethasone and interferon (IFN) in a new regimen, 'I-Dexa'. Here we report our results of a phase I/II study using the I-Dexa protocol in an outpatient setting. A total of 31 patients were enrolled. Most patients were heavily pretreated with a median of two different chemotherapy regimen (range, 1-4). All but four patients had advanced disease (stage III). The dose of Ida was escalated to define the maximal tolerated dose (MTD) in this regimen. Four patients were treated with 5 mg/m2 and 27 patients with 7.5 mg/m2 Ida (day 1, i.v.). Dexamethasone was given at a fixed dose of 20 mg per os daily on days 2-5 and 11-14. Treatment was repeated at day 21 and consisted of up to six cycles. Patients who achieved a response or stable disease received IFN maintenance. IFN was administered three times weekly at a dose of 3 x 10(6) U/day s.c. until relapse. At the time of evaluation, 125 courses of I-Dexa were analyzed. The MTD of Ida in this regimen was 7.5 mg/m2. Hematological toxicity was mild including 5% leukocytopenia, 3% thrombocytopenia and 1% anemia (WHO grade III) at dose level 2. The MTD was defined by nonhematological toxicities including two WHO grade IV infections and one renal failure. The overall response rate including stable disease was 62.5% (PR: nine patients, 37.5%). So far, nine patients have been treated with IFN maintenance therapy with a median duration of 7 months (range, 1-16). In conclusion, I-Dexa can be given safely in an outpatient setting and is effective for the treatment of relapsed and refractory MM.

Treatment of refractory Hodgkin's lymphoma patients with an anti-CD25 ricin A-chain immunotoxin.

The anti-CD25 immunotoxin RFT5.dgA was constructed by coupling the monoclonal antibody RFT5 via a sterically hindered disulfide linker to deglycosylated ricin A-chain and was administered to patients with relapsed Hodgkin's lymphoma in four bolus infusions over 7 days (day 1, 3, 5 and 7). The maximum tolerated dose in these patients as defined in a previous phase I study was 15 mg/m2. Subsequently, further patients were enrolled at the maximum tolerated dose and a total of 18 patients were treated at this level. All patients had signs of progressive disease and were heavily pretreated. Side-effects in this trial were moderate and related to vascular leak syndrome. Five of 18 patients experienced NCI grade III toxicities including weakness, edema, dyspnea, and myalgia. Eleven of 16 (69%) patients receiving two or more cycles produced human anti-ricin antibodies and human anti-mouse antibodies (>/=1.0 microg/ml). Seventeen of 18 patients were evaluable for clinical response. These included two partial remissions. One patient demonstrated minor response and five patients stable diseases. We conclude that RFT5.dgA is of moderate clinical efficacy in this group of heavily pretreated refractory patients. Leukemia (2000) 14, 129-135.

[Radioimmunotherapy with yttrium-90 ibritumomab tiuxetan. Clinical considerations, radiopharmacy, radiation protection, perspectives]. / Radioimmuntherapie mit 90Y-markiertem Ibritumomab-Tiuxetan: Klinik, Radiopharmazie, Strahlenschutz, Perspektiven.

90Y-ibritumomab tiuxetan (Zevalin) is currently approved for radioimmunotherapy of patients with relapsed or refractory follicular non-Hodgkin's lymphoma pretreated with rituximab. Future directions are the combined use of 90Y-ibritumomab tiuxetan as part of the initial treatment and as first-line multi-agent therapy of relapsed disease. Current studies investigate patients with other than follicular indolent histologies, e. g. diffuse large cell lymphoma. Labelling of 90Y ibritumomab tiuxetan is a safe procedure, the radiochemical purity is not disturbed by a higher room temperature or by metallic impurity. Quality control is recommended by thin layer chromatography (TLC), strips >15 cm are favourable. TLC cannot distinguish between the correctly radiolabelled antibodies and radiocolloid impurity. If necessary, additional HPLC should be performed. Radiocolloid impurities are absorbed to the solid phase and do not reach the eluate. If the radiochemical purity test is insufficient (<95%), the additional cleaning using EconoPac 10 DG columns (Biorad, Hercules, CA, USA) is a reliable procedure to reduce the percentage of free radionuclide. However, this procedure is not part of the approval.

Predictors of intelligence quotient and intelligence quotient change in persons treated for phenylketonuria early in life.

Ninety-one individuals with phenylketonuria who were treated early in life were followed for as many as 22 years. Regression analyses were used to determine the best predictors of IQ and IQ change. Among treatment-related variables, good dietary control of the blood phenylalanine level stood out as the best predictor of IQ. Diet discontinuation and the natural (off diet) blood phenylalanine level best predicted IQ loss, suggesting that diet continuation may be important for children with natural blood phenylalanine levels greater than 18 mg/dL.

The in vivo anti-tumor activity of immunotoxins containing two versus one deglycosylated ricin A chains.

We have compared the in vitro and in vivo activities of three deglycosylated ricin A chain (dgA)-containing immunotoxins (ITs) with one versus two molecules of dgA per molecule of IgG. Two of the ITs (anti-CD19-dgA2 and anti-CD25-dgA2) had 3-0-fold improvements over their dgA1 counterparts in vitro. However, the in vivo anti-tumor activity of both constructs of each IT was the same. In contrast, anti-CD22-dgA2 was 7-fold more potent in vitro and significantly more potent in vivo than its dgA1 counterpart. These studies suggest that monoclonal antibodies must be individually tested to determine whether the addition of two versus one dgA will improve cytotoxic activity in vivo.

Studies on cadmium-induced inhibition of hepatic microsomal drug biotransformation in the rat.

Cadmium is a potent inhibitor of hepatic microsomal drug biotransformation in the rat. Male rats receiving a single intraperitoneal dose of cadmium exhibit significant decreases in hepatic microsomal metabolism of a variety of substrates. The threshold cadmium dose is 0.84 mg Cd/kg, and the effect lasts at least 28 days. Mechanistically, the inhibitory effect results from decreased cytochrome P-450 content since cadmium does not alter NADPH cytochrome c reductase activity. This effect is also observed following acute oral administration of cadmium in doses greater than 80 mg Cd/kg but is not observed following chronic administration of the metal via drinking water in concentrations of 5-200 ppm for periods ranging from 2 to 50 weeks. A tolerance to the inhibitory cadmium effect is observed if male rats are pretreated with subthreshold doses of the metal prior to the challenge cadmium dose. The degree of tolerance can be overcome by increasing the challenge dose of cadmium. Characterization of the tolerance phenomenon in terms of onset, duration, and intensity reveals a good correlation with the kinetics of metallothionein production, suggesting that the underlying basis for the tolerance phenomenon is likely the induction of metallothionein. A sex-related difference in the inhibitory effect of cadmium was observed. Cadmium did not inhibit the metabolism of hexobarbital or ethylmorphine in female rats but did inhibit that of aniline or zoxazolamine. Cadmium did not lower cytochrome P-450 content in female rats.

Barbital alteration of central nervous system sensitivity to hexobarbital in the rat.

Alteration in central nervous system (CNS) sensitivity to the hypnotic effect of the hexobarbital was assessed by analytical, electroencephalographic, and pharmacokinetic techniques in barbital-treated rats. Each of these conceptually diverse experimental methodologies yielded similar conclusions concerning the alteration of CNS sensitivity by barbital treatment.

Non-Hodgkin's Lymphoma: A Review of Immunotherapeutic Approaches to Treatment.

Non-Hodgkin's lymphomas (NHL) represent a heterogenous group of diseases including low, intermediate and high grade histological subtypes. Most entities are sensitive to chemotherapy and radiotherapy. However, most relapsed patients are incurable with conventional treatment. The major reason for unsatisfactory long-term results in NHL is tumour cells that persist after standard treatment. New sensitive techniques have been developed to detect occult lymphoma cells. These cells might be eradicated by new immunotherapeutic agents with different modes of action, such as cytokines or antibody-based agents. In NHL, most experience has been accumulated with interferon-alpha, which seems to be effective against minimal residual disease (MRD). The experience with interleukin-2 and interleukin-3 is less convincing. Monoclonal antibodies have been used in their native form, or conjugated with radioisotopes or toxins to selectively destroy lymphoma cells. Such immunotoxins and radioisotope-coupled antibodies have shown promising results in early clinical trials, and are now being evaluated in patients with smaller tumour burdens.

Studies relating morphine hypothermia with serotonin reuptake in the rat hypothalamus.

Experiments were conducted to determine if the acute hypothermia elicited by morphine in the rat resulted from morphine-induced inhibition of serotonin reuptake into hypothalamic nerve-endings. The acute hypothermia elicited by morphine (30 mg/kg, s.c.) was reversed by pretreatment with naloxone (10 mg/kg, s.c.), a narcotic antagonist, which alone did not alter body temperature. In vitro uptake of serotonin into synaptosomes prepared from rat hypothalamus was inhibited 53% by the addition of morphine (7 X 10(-4) M) and 53.9% by naloxone (1 X 10(-3) M). Simultaneous addition of both drugs in these same concentrations further inhibited synaptosomal serotonin uptake 77.8%. Uptake of serotonin uptake into synaptosomes isolated from rats injected with morphine (30 mg/kg, s.c.) was not altered as compared to controls. These data suggest that the acute hypothermic acition in morphine in the rat is not related to an inhibition of the serotonin reuptake mechanism in hypothalamic nerve-endings.