Results of TRIO-15, a multicenter, open-label, phase II study of the efficacy and safety of ganitumab in patients with recurrent platinum-sensitive ovarian cancer.

BACKGROUND: IGF signaling has been implicated in the pathogenesis and progression of ovarian carcinoma (OC). Single agent activity and safety of ganitumab (AMG 479), a fully human monoclonal antibody against IGF1R that blocks binding of IGF1 and IGF2, were evaluated in patients with platinum-sensitive recurrent OC. METHODS: Patients with CA125 progression (GCIG criteria) or measurable disease per RECIST following primary platinum-based therapy received 18 mg/kg of ganitumab q3w. The primary endpoint was objective response rate (ORR) assessed per RECIST 1.1 by an independent radiology review committee (IRC) and/or GCIG CA125 criteria. Secondary endpoints included clinical benefit rate (CBR), progression free survival (PFS) and overall survival (OS). RESULTS: 61 pts. were accrued. Objective responses were seen in 5/61 patients (ORR 8.2%, 95% CI, 3.1-18.8) with 1 partial response (PR) by RECIST and 2 complete responses (CR) as well as 2 PR by CA125 criteria. CBR was 80.3% (95% CI, 67.8-89.0%). The median PFS according to RECIST by IRC was 2.1 months (95% CI, 2.0-3.1). The median PFS per RECIST IRC and/or CA125 was 2.0 months (95% CI, 1.8-2.2). The median OS was 21 months (95% CI, 19.5-NA). The most common overall adverse events were fatigue (36.1%) and hypertension (34.4%). Grade 1/2 hyperglycemia occurred in 30.4% of patients. Hypertension (11.5%) and hypersensitivity (8.2%) were the most frequent grade 3 adverse events. CONCLUSIONS: IGF1R inhibition with ganitumab was well-tolerated, however, our results do not support further study of ganitumab as a single agent in unselected OC patients.

Constant Isothiocyanate-Release Potentials across Biofumigant Seeding Rates.

Biofumigation is an integrated pest-management method involving the mulching of a glucosinolate-containing cover crop into a field in order to generate toxic isothiocyanates (ITCs), which are effective soil-borne-pest-control compounds. Variation in biofumigation efficacy demonstrates a need to better understand the factors affecting pest-control outcomes and develop best practices for choosing biofumigants, growth conditions, and mulching methods that allow the greatest potential isothiocyanate release. We measured the glucosinolate concentrations of six different commercial varieties of three biofumigant plant species: Brassica juncea (ISCI99, Vitasso, and Scala) Raphanus sativus (Diablo and Bento), and Sinapis alba (Ida Gold). The plants were grown in the range of commercially appropriate seeding rates and sampled at three growth stages (early development, mature, and 50% flowering). Within biofumigant species, the highest ITC-release potentials were achieved with B. juncea cv. ISCI99 and R. sativus cv. Bento. The highest ITC-release potential occurred at the 50% flowering growth stage across the species. The seeding rate had a minor impact on the ITC-release potential of R. sativus but had no significant effects on the ITC-release potentials of the B. juncea or S. alba cultivars.

QPCR analysis and RNAi define pharyngeal gland cell-expressed genes of Heterodera glycines required for initial interactions with the host.

Changes in transcript abundance of genes expressed in the three pharyngeal gland cells of Heterodera glycines after host invasion were monitored by quantitative polymerase chain reaction (qPCR) and the consequences of disrupting their expression studied by RNAi treatment prior to invasion. Two transcripts were known to be expressed in the two subventral gland cells (hg-pel and hg-eng-1), a further two in the single dorsal gland cell only (hg-gp and hg-syv46), and a fifth transcript (hg-cm) was expressed by both gland cell types. The qPCR study established that transcripts of hg-syv46 and hg-gp increased in abundance by 2 days postinfection (dpi), with the former remaining the most abundant. The hg-cm transcript level showed minor changes from 0 to 14 dpi but did fall by 21 dpi. In contrast, hg-eng-1 and hg-eng-2 messenger (m)RNA declined by 7 dpi and hg-pel by 14 dpi before it increased at 21 dpi. RNAi-targeting of hg-eng-1 reduced the number of females present on the plants at 10 days. Targeting of hg-gp, hg-cm, and hg-pel caused a change in sexual fate favoring male development on roots. Both effects were evident after targeting hg-syv46. Suppression of hg-eng-1 mRNA levels in second-stage juveniles (J2i) by RNAi was transient, with a recovery by 15 days of incubation in water after treatment. Presoaking H. glycines J2 with double-stranded RNA has value for studying gene function during the nematode's early interaction with a plant.

The case for genetically modified crops with a poverty focus.

Recently seven National Academies of Science produced a report on transgenic plants and world agriculture. The report provides scientific perspectives to the ongoing public debate about the potential role for transgenic technology in world agriculture. In this article, we develop the themes of the report and emphasize the potential for future genetically modified (GM) crops with a poverty focus, emphasizing the potential of GM resistance to plant parasitic nematodes for subsistence potato farmers in Bolivia. We judge that a range of incremental gains to crop yields from many transgenes are valuable for future world security. We advocate the establishment of a standard that GM crops must achieve before they are both biosafe and appropriate for resource-poor farmers and we believe that the best interests of the poor require biotechnologists to work towards that objective.

Continual green-fluorescent protein monitoring of cauliflower mosaic virus 35S promoter activity in nematode-induced feeding cells in Arabidopsis thaliana.

The responsiveness of the cauliflower mosaic virus 35S promoter in feeding sites developed by both sexes of Heterodera schachtii and female Meloidogyne incognita has been studied. The objective was to establish the value of green-fluorescent protein (GFP) as a nondestructive reporter gene system for characterizing promoter activity at nematode feeding sites in vivo. Growth units were devised that allowed individual feeding sites in roots of Arabidopsis thaliana to be observed by both bright-field and epifluorescent illumination. Changes in GFP expression were visually observed under experimental conditions that resulted in chloroplast formation in syncytia but not other root cells. Changes in GFP levels altered the extent of quenching, by this protein, of red light emitted by chlorophyll within the chloroplasts under violet excitation. Image analysis provided a semiquantitative basis for simultaneous measurement of changes in GFP fluorescence and the unquenched emission by chlorophyll. GFP levels were constant in cells surrounding the syncytium induced by H. schachtii, but they fell progressive from 10 to 35 days postinfection within this structure. Significant reduction in GFP levels was not limited to the early part of the time course but also occurred between 27 and 35 days postinfection. GFP was detected by immunoblotting in females of M. incognita but not in H. schachtii parasitizing similar GFP-expressing roots.

Characterization of cDNAs encoding serine proteinases from the soybean cyst nematode Heterodera glycines.

Three cDNAs encoding serine proteinases (HGSPI-III) were isolated from a cDNA library constructed from feeding females of Heterodera glycines. The library was screened with three separate serine proteinase gene fragments amplified from cDNA of H. glycines using consensus oligonucleotide primers. Each predicted protein contains a secretion signal sequence, a propeptide and a mature protein of 226-296 amino acids. One of the predicted enzymes, HGSP-II has 41% identity to a chymotrypsin-like enzyme from the mollusc, Haliotis rufescens, and analysis of key residues involved in substrate binding also suggests a chymotrypsin-like specificity. HGSP-I and HGSP-III show greatest homology to kallikreins but sequence analysis does not allow prediction of their substrate preferences. Southern blot analysis suggests that HGSP-II and HGSP-III are encoded by single-copy genes in contrast to HGSP-I which may have two or more homologues. The regions encoding the mature proteinases were cloned into an expression vector and recombinant protein produced in Escherichia coli. Both HGSP-I and HGSP-II were shown, after refolding, to cleave the synthetic peptide N-CBZ-Phe-Arg-7-amido-4-methylcoumarin, and this activity could be inhibited by the cowpea trypsin inhibitor, CpTI. HGSP-III showed no activity against the synthetic substrates tested. The information gained from these studies indicates that serine proteinases are an important group of enzymes in H. glycines and further characterization will aid the development of a proteinase inhibitor-based approach for transgenic plant resistance to plant parasitic nematodes.

Seasonal Changes in the Dorsal Pharyngeal Gland Nucleolus of Unhatched Second-Stage Juveniles of Globodera spp. in Bolivia.

Changes in the diameter of the nucleolus of the dorsal pharyngeal gland (DPGN) in unhatched second-stage juveniles (J2) of potato cyst nematodes, Globodera spp., were monitored for cysts recovered during two field experiments in the Bolivian Central Andes. In the first experiment, cysts were extracted from soil left fallow or supporting crops of potato, barley, lupin, or quinoa. The highest mean DPGN diameter for unhatched J2 occurred shortly after planting in January. The values were similar for individuals recovered from cysts associated with all cultivations. For cysts from potato plots, the lowest mean DPGN diameter of 2.26 +/- 0.05 mum occurred in March, but the value increased again by May to 2.53 +/- 0.05 mum. Similar seasonal changes were found for J2 under both nonhost crops and fallow with the smallest diameters recorded in May of 2.48 +/- 0.02 mum and 2.34 +/- 0.05 mum, respectively. Two possible factors might cause this significant seasonal change. First, some J2 may hatch early in the growing season, even in the absence of the host. This would enhance the proportion of dormant, unhatched J2 remaining in the cyst samples. Secondly, a seasonal change in the DPGN diameter may occur for most individuals with a transient fall value between January and March/May. A model defined by this study provides a good description of the observed effect, providing both factors are assumed to occur. The second experiment studied if changes in size of DPGN in response to a hatching stimulus are influenced by the cyst population age. The DPGN in unhatched J2 was measured for cysts recovered from soils that had supported potatoes that growing season or 2 or 4 years earlier. The unhatched J2 from the freshly cropped potato site showed the largest mean DPGN diameter of 3.66 +/- 0.05 mum after 7 days in potato root diffusate, whereas those from the 4-year sample had the smallest value of 3.20 +/- 0.05 mum. This significant difference may indicate a delayed response to the hatching stimulus with more prolonged J2 dormancy.

The Influence of Potato Cultivar on Lipid Content and Fecundity of Bolivian and British Populations of Globodera rostochiensis.

The influence of host cultivar on the lipid levels provided by a female to her progeny was investigated with Oil Red O stain and a quantitative image analyzer. A population of Globodera rostochiensis was multiplied at Toralapa Field Station in Bolivia on 25 different potato cultivars grown in that country. The mean neutral lipid content of newly formed second-stage juveniles varied significantly with cultivar over a 200% range. The corresponding range was only 18% and 28% for the same Bolivian and a UK population of G. rostochiensis, respectively, when both completed reproduction concurrently on 10 pot-grown European cultivars in the United Kingdom. Egg numbers per female varied with host for Bolivian cultivars that lack known partial resistance to Globodera spp. There was a 15-fold range between the most and least fecund nematode-host combinations (Kosi and Gendarme). The Bolivian G. rostochiensis population showed only a 2-fold range in mean eggs per cyst when grown on European cultivars in the UK. The fatty acid profiles of lipids from Bolivian G. rostochiensis cysts reared on Bolivian potato cultivars were dominated by C(20) (37-64%) and C(18) (28-46%) fatty acids and ranged from C(14) to C(22). The three major fatty acids detected were C(20:4:), C(20:1), and C(18:1). Few differences between cultivars were observed. For a UK population of G. rostochiensis reared on ssp. tuberosum, higher relative percentages of C(18) and monounsaturated fatty acids and lower relative percentages of C(20) and polyunsaturated fatty acids were found.

The Immunofluorescent Localization of Subventral Pharyngeal Gland Epitopes of Preparasitic Juveniles of Heterodera glycines Using Laser Scanning Confocal Microscopy.

Laser scanning confocal microscopy (LSCM) was used to localize the reactivity of a monoclonal antibody (Sv2) that binds to the subventral pharyngeal glands of preparasitic juveniles of Heterodera glycines. The greater resolution, magnification, and image analysis of LSCM compared with conventional epifluorescent microscopy enabled Sv2 binding to be localized much more precisely to the periphery of the secretory granules. A linear increase of about 55% in fluorescent intensity was found over a 23-mum length of subventral pharyngeal gland just distal to the terminal ampullae. LSCM is a rapid and effective technique for precise immunolocalization of epitopes.

Stage-specific Monoclonal Antibodies to the Potato Cyst Nematode Globodera pallida Stone (Behrens).

Using standard hybridoma technology and hierarchical screening, monoclonal antibodies (MAbs) were obtained with specific reactivity against two developmental stages of Globodera pallida. The procedure was based on enzyme-linked immunosorbent assay (ELISA) with homogenates prepared from second-stage juveniles, young adult females, and potato roots. Hybridomas were formed by fusing myelomas with splenocytes derived from mice immunized with either infective juveniles or females of G. pallida. About 600 hybridoma lines were screened from the fusion involving the mouse immunized with juveniles. Two MAbs (LJMAbl &2) were identified with high reactivity toward second-stage juveniles but no reactivity with either potato roots or females of G. pallida. A total of 630 cell lines was screened from the corresponding fusion involving the spleen of a mouse receiving immunogens from adult female nematodes. One MAb (LFMAbl) was obtained with the required specificity against only adult female G. pallida. This work extends the application of monoclonal antibodies in nematology from valuable probes for research and species identification to recognition of developmental stages. These specific MAbs have potential value in plant breeding programs for screening for resistant lines unable to support nematode development.

Major Sperm Protein Genes from Globodera rostochiensis.

Three genes in the major sperm protein (MSP) gene family from the potato cyst nematode Globodera rostochiensis were cloned and sequenced. In contrast to the absence of introns in Caenorhabditis elegans MSP genes, these genes in G. rostochiensis contained a 57 nucleotide intron, with normal exon-intron boundaries, in the same relative location as the intron in Onchocerca volvulus. The MSP genes of G. rostochiensis had putative CAAT, TATA, and polyadenylation signals. The predicted G. rostochiensis MSP gene product is 126 amino acids long, one residue shorter than the products in the other species. The comparison of MSP amino acid sequences from four diverse nematode species suggests that O. volvulus, Ascaris suum, and C. elegans may be more closely related to each other than they are to G. rostochiensis.

Image Analysis of the Growth of Globodera pallida and Meloidogyne incognita on Transgenic Tomato Roots Expressing Cystatins.

An approach based on image analysis that enables rapid collection and analysis of nematode size and shape during growth is reported. This technique has been applied to assess Meloidogyne incognita and Globodera pallida during their development over 35 and 42 days, respectively, on transgenic tomato roots expressing the wild-type rice cystatin Oc-I or an engineered variant, Oc-IAD86. Morphometric values were established that subdivided enlarged saccate females from other life stages. Analysis of this data subset indicates that the size of females and the frequency with which they parasitize roots expressing a cystatin are reduced. Results also demonstrate that cystatins can influence the growth of G. pallida prior to the adult stage. Similar image analysis procedures should be generally applicable to the study of host status or erivironmental factors that influence growth rates of plant-parasitic nematodes.

An Algorithm for Optimizing Rotational Control of Globodera rostochiensis on Potato Crops in Bolivia.

The outline area of new cysts of Globodera rostochiensis was measured by image analysis. A linear regression of this value against egg content provided a basis for adjusting the egg number for cyst size. This adjusted egg content provides an estimate of the relative fullness of a cyst with eggs. This value showed an exponential decline in eggs over 3.5 years since the last potato crop. It corresponds to an average loss in the dormant egg population of 32.8 +/- 5.6%/year for 26 fields at Toralapa, Bolivia. This value compared well with a mean decline of 40 +/- 4%/year for 42 fields after measuring viable eggs/100 g soil on two occasions one year apart. The new approach allows declines to be estimated at one time point. The decline in lipid content of the dormant, unhatched second-stage juveniles (J2) was 17 +/- 6% per annum as measured by image analysis after Oil red O staining. This may be sufficient to compromise infectivity after 3 to 4 years of dormancy. A standard model was modified to consider the effect of both lipid depletion during dormancy and choice of susceptible potato on the population dynamics of G. rostochiensis under rotational control. It is concluded that a few cultivars may impose lower populations on G. rostochiensis in 3 to 4-year rotations than the majority used in Bolivia.

Characterisation by RNAi of pioneer genes expressed in the dorsal pharyngeal gland cell of Heterodera glycines and the effects of combinatorial RNAi.

Changes in transcript abundance of 24 genes expressed in the dorsal pharyngeal gland cell of Heterodera glycines encoding for putative secretions of unknown function were monitored by quantitative PCR (qPCR) at 0, 2, 7, 14 and 21 days post-invasion (pi) of soybean plantlets. Five groups of temporal patterns (A, B1, B2, C and D) were defined for the 24 genes plus data for two previously studied genes expressed in the same cell. Group D (two genes) showed no significant increase between 0 and 2 days pi and were the least abundantly expressed at 7-21 days pi. Transcripts of group C (nine genes including one studied previously) increased in abundance from 0 to 2 days pi but were the second least expressed for 7-21 days pi. Groups A (three genes), B1 (seven genes) and B2 (five genes including one studied previously) were all abundant at 7-21 days pi. B1 and B2 were discriminated by their relative abundance at 0 and 2 days pi. RNA interference (RNAi) targeting two genes of group A and one each of B1 and B2 in nematodes prior to invasion resulted in phenotypic effects on total parasites per plant and sexual fate at 10 days pi. Phenotype penetrance was reduced for three genes showing such effects and one with a strong effect in earlier work when two genes rather than one were concurrently targeted for RNAi. One gene (dg13) was more abundantly expressed after combinatorial RNAi than for either control nematodes or when targeting singly by RNAi. This work reports the unexpected elevation in mRNA expression after combinatorial RNAi that requires understanding before combinatorial RNAi can be advanced for highly effective cyst nematode control via plant biotechnology.

Meloidogyne incognita: molecular and biochemical characterisation of a cathepsin L cysteine proteinase and the effect on parasitism following RNAi.

RNA interference has been used to investigate the function of a cathepsin L cysteine proteinase Mi-cpl-1, in the plant-parasitic nematode Meloidogyne incognita. A reduction in gene transcript was observed and the number of nematodes infecting plants was reduced by almost 60% as was the number of established females producing eggs at 21 days post-infection. The cysteine proteinase activity of M. incognita, reported by the substrate GLUpNA, was inhibited by the cysteine proteinase inhibitor Oc-IDeltaD86. A reduction in cysteine proteinase activity was also seen following RNAi of Mi-cpl-1 in J2 stage nematodes. In situ hybridization analysis in young and mature female nematodes has shown that Mi-cpl-1 is expressed in the intestine, which suggests that its product is a digestive enzyme. The effects of knocking-out Mi-cpl-1gene function were consistent with a reduction in feeding efficiency. Here, we have shown a correlation between transcript abundance proteinase activity and parasitic success of M. incognita.

The functional significance of the haemoglobin in a marine nematode, Enoplus brevis (Bastian).

An animal chamber and a simple microspectrophotometer for investigating the in vivo oxygenation of the haemoglobin of E. brevis are described. The in vivo absorption peaks of this haemoglobin occur at similar wavelengths to those of other nematodes. Mean values, given with their corresponding standard errors, occur at 577.6 plus or minus 0.6 nm, 543.6 plus or minus 0.5 nm and 421.7 plus or minus 1.9 nm for oxyhaemoglobin, and 555.2 plus or minus 0.9 nm and 432.2 plus or minus 1.3 nm for the deoxygenated pigment. The percentage of oxyhaemoglobin in the pharynx of E. brevis decreased at external oxygen tensions of less than 20 Torr, and the pigment was completely deoxygenated at 5 Torr. Stimulation of individuals in aerated sea water for 1-2 min caused a partial deoxygenation of the haemoglobin; the pigment reloaded soon after this period of increased activity had ended. The functional significance of the haemoglobin of E. brevis is disucssed.

Nematodirus battus: development of cold hardiness in dormant eggs.

Thermistors and an amplified bridge were used to detect supercooling points of Nematodirus battus eggs weighing ca. 1 microgram wet weight. A cooling rate of about 1 C min-1 was achieved with a manually controlled cold stage using the Peltier effect. The supercooling point of eggs fell during chilling at 5 +/- 1 C for up to 8 weeks from -34.48 +/- 0.49 C to -37.17 +/- 0.76 C. Juveniles freed from these eggs were less cold hardy than intact eggs but chilling improved their supercooling to a greater extent from -19.33 +/- 1.38 C to -32.10 +/- 0.68 C. These results were obtained with eggs showing the characteristic hatching response for this species after transfer from chilling at 5 C to higher temperatures (5-37 +/- 1 C). The results indicate eggs of N. battus acclimate to chilling at a time when previous work had established an increase in their trehalose content.

An assay for detecting nanogram levels of proteolytic enzymes.

A rapid, cheap, and sensitive method has been developed for determining proteolytic activity of different classes of endoproteinases. The method is based on a solid-phase assay employing as substrate biotinylated gelatin adsorbed onto microtiter plates. Enzymatic activity is measured by incubating proteinase with the immobilized biotin-protein. Any remaining, undigested substrate bound to the microtiter plate is assayed with streptavidin-alkaline phosphatase. It was established that papain, pepsin, thermolysin, and trypsin all hydrolyzed the biotinylated substrate to varying degrees. Furthermore, the activity of these proteinases was blocked by their respective inhibitors. The assay presented is quick, highly reproducible, inexpensive, and useful for detecting all classes of endoproteolytic enzymes. By using different biotinylated proteins or peptides as substrates, and employing specific buffers and inhibitors, this assay may be utilized for detecting other and more specific endoproteinases.

Nematodirus battus: permeability changes, calcium binding, and phosphorylation of the eggshell during hatching.

Eggshells of Nematodirus battus leaked trehalose 4 hr after being stimulated to hatch, and became permeable to trypan blue at their poles; 80% of eggs were stained blue 24 hr later. Exogenous application of ruthenium red significantly inhibited chill- and sodium fluoride-stimulated hatching, 50% hatch inhibition occurring in 44.67 +/- 2.2 and 8.5 +/- 1.5 microM, respectively. Lanthanum chloride, however, was not as inhibitory as ruthenium red on fluoride-stimulated hatching, 50% occurring at 31.60 +/- 1.25 microM. A Scatchard plot of the competitive binding of ruthenium red to eggshells demonstrated a high-affinity binding site for calcium, KCa' = 1.92 microM and a second, low-affinity site, KCa" = 1169.60 microM. Ruthenium red binding was significantly reduced by several enzymes, e.g., EGTA-buffered trypsin reduced binding by 73%. Radioiodinated concanavalin A also bound competitively to the eggshells in the presence of alpha-D-glucosyl-alpha-D-glucopyranoside and alpha-methyl-D-mannopyranoside. Eggshells incorporated phosphorus-32 from ATP after chilling or on exposure to sodium fluoride; gel filtration of solubilized homogenates of these samples showed that two proteins were radiolabelled with molecular weights of 38 X 10(3) and 8 X 10(3) Da, respectively. This phosphorylation was inhibited by N-ethylmaleimide, which also prevented hatching.