bluuebunny/crossref_metadata_2025 huggingface dataset:
Major determinants of fasting heat production and energetic cost of activity in growing pigs of different body weight and breed/castration combination
Authors: Milgen J. Van , Bernier J. F. , Lecozler Y. , Dubois S. , Noblet J.
Published: September 2005

A total of sixty-five observations on heat production during fasting and physical activity were obtained in four groups of pigs differing in breed and/or castration (Meishan (MC) and Large White (LWC) castrates and Large White (LWM) and Piétrain (PM) males) with body weight (BW) ranging between 25 and 60 kg. Pigs were fed ad libitum before fasting. Heat production was measured using indirect calorimetry. Fasting heat production (FHP) was proportional to the body weight raised to the power 0.55, but with group-specific proportionality parameters (810, 1200, 1220 and 1120kJ/kg BW0.55 per d for MC, LWC, LWM and PM respectively). Group effects could be removed by expressing FHP as a function of muscle, viscera and fat: FHP (kJ/d) = 457(muscle)0.81 + 1969(viscera)0.81 - 644(fat)0.81. It is hypothesized that different breeds with equal muscle and visceral mass, can have different FHP. The negative coefficient for fat would then be the result of a low FHP rather than a cause of it. Because a large part of the variation in tissue composition between groups was due to MC group, a separate equation for the lean groups was established. For lean pigs, FHP could be expressed as a function of muscle and viscera alone: FHP (kJ/d) = 508(muscle)0.66 + 2011(viscera)0.66. Both type of pig and BW affected the number of bouts of physical activities (i.e. standing or sitting) per day, the duration of activity and the total cost of activity. Energetic cost of activity was proportional to the muscle mass raised to the power 0.91 (FHPactivity (kJ/h activity) = 21.0(muscle)0.91). Physical activity represented less than 10% of the total heat production in fasting growing pigs housed alone in metabolic cages and kept in a quiet environment.
Importance of measuring CO2-production rate when using 13C-breath tests to measure fat digestion
Authors: Amarri S. , Coward W. A. , Harding M. , Weaver L. T.
Published: September 2005

Stable isotope breath tests offer a safe, repeatable non-invasive method of measuring fat digestion. They involve the ingestion of a substrate labelled with 13C followed by serial measurements of the 13C: 12C ratio in expired CO2, from which the percentage of the 13C dose recovered (PDR) can be calculated. However PDR depends on the CO2-production rate. Our aim was to compare results obtained using directly measured CO2-production rates with those calculated from two predicted values. Twelve normal healthy children and twenty-four children with cystic fibrosis (CF) (without or with the normal dose of enzyme supplementation) were studied with 1,3-distearyl, 2[carboxyl[13C]octanoyl glycerol. The volume of CO2 produced (litres/min) was measured at rest for 30 min approximately 3 h after substrate ingestion, and the results were converted to mmol/min. For each subject the expected BMR was calculated from the equation of Schofield (1985), based on sex, age, weight and height, and from these values, CO2-production rate was derived. Surface area was calculated and an estimated value of 5 mmol/m2 per min (Shreeve et al. 1970) was used. Using these three CO2-production rates, three different PDR were calculated and compared. In healthy children there was a close concordance between measured and predicted CO2-production rates, but children with CF had a mean measured CO2-production rate 39% higher than normal children. This use of normal data for predicted CO2-production rates in children with CF underestimates cumulative PDR. If direct measurements of CO2-production rate are not available or impossible to perform the Vco2 obtained from the BMR calculated using the equations of Schofield (1985) or Shreeve et al. (1970) can be used in normal children. However, if accurate results for PDR are to be obtained, CO2-production rates should be measured when performing breath tests in conditions where energy expenditure and/or CO2-production rate are not expected to be normal.
Galaxy: A platform for interactive large-scale genome analysis
Authors: Giardine Belinda , Riemer Cathy , Hardison Ross C. , Burhans Richard , Elnitski Laura , Shah Prachi , Zhang Yi , Blankenberg Daniel , Albert Istvan , Taylor James , Miller Webb , Kent W. James , Nekrutenko Anton
Published: September 2005

Accessing and analyzing the exponentially expanding genomic sequence and functional data pose a challenge for biomedical researchers. Here we describe an interactive system, Galaxy, that combines the power of existing genome annotation databases with a simple Web portal to enable users to search remote resources, combine data from independent queries, and visualize the results. The heart of Galaxy is a flexible history system that stores the queries from each user; performs operations such as intersections, unions, and subtractions; and links to other computational tools. Galaxy can be accessed at http://g2.bx.psu.edu.
Simple, robust methods for high-throughput nanoliter-scale DNA sequencing
Authors: Smailus Duane E. , Marziali Andre , Dextras Philip , Marra Marco A. , Holt Robert A.
Published: September 2005

We have developed high-throughput DNA sequencing methods that generate high quality data from reactions as small as 400 nL, providing an approximate order of magnitude reduction in reagent use relative to standard protocols. Sequencing of clones from plasmid, fosmid, and BAC libraries yielded read lengths (PHRED20 bases) of 765 ± 172 (n = 10,272), 621 ± 201 (n = 1824), and 647 ± 189 (n = 568), respectively. Implementation of these procedures at high-throughput genome centers could have a substantial impact on the amount of data that can be generated per unit cost.
Proanthocyanidin Synthesis and Expression of Genes Encoding Leucoanthocyanidin Reductase and Anthocyanidin Reductase in Developing Grape Berries and Grapevine Leaves
Authors: Bogs Jochen , Downey Mark O. , Harvey John S. , Ashton Anthony R. , Tanner Gregory J. , Robinson Simon P.
Published: September 2005

Proanthocyanidins (PAs), also called condensed tannins, can protect plants against herbivores and are important quality components of many fruits. Two enzymes, leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), can produce the flavan-3-ol monomers required for formation of PA polymers. We isolated and functionally characterized genes encoding both enzymes from grapevine (Vitis vinifera L. cv Shiraz). ANR was encoded by a single gene, but we found two highly related genes encoding LAR. We measured PA content and expression of genes encoding ANR, LAR, and leucoanthocyanidin dioxygenase in grape berries during development and in grapevine leaves, which accumulated PA throughout leaf expansion. Grape flowers had high levels of PA, and accumulation continued in skin and seeds from fruit set until the onset of ripening. VvANR was expressed throughout early flower and berry development, with expression increasing after fertilization. It was expressed in berry skin and seeds until the onset of ripening, and in expanding leaves. The genes encoding LAR were expressed in developing fruit, particularly in seeds, but had low expression in leaves. The two LAR genes had different patterns of expression in skin and seeds. During grape ripening, PA levels decreased in both skin and seeds, and expression of genes encoding ANR and LAR were no longer detected. The results indicate that PA accumulation occurs early in grape development and is completed when ripening starts. Both ANR and LAR contribute to PA synthesis in fruit, and the tissue and temporal-specific regulation of the genes encoding ANR and LAR determines PA accumulation and composition during grape berry development.
In Vivo Regulatory Phosphorylation of Novel Phosphoenolpyruvate Carboxylase Isoforms in Endosperm of Developing Castor Oil Seeds
Authors: Tripodi Karina E. , Turner William L. , Gennidakis Sam , Plaxton William C.
Published: September 2005

Our previous research characterized two phosphoenolpyruvate (PEP) carboxylase (PEPC) isoforms (PEPC1 and PEPC2) from developing castor oil seeds (COS). The association of a shared 107-kD subunit (p107) with an immunologically unrelated bacterial PEPC-type 64-kD polypeptide (p64) leads to marked physical and kinetic differences between the PEPC1 p107 homotetramer and PEPC2 p107/p64 heterooctamer. Here, we describe the production of antiphosphorylation site-specific antibodies to the conserved p107 N-terminal serine-6 phosphorylation site. Immunoblotting established that the serine-6 of p107 is phosphorylated in COS PEPC1 and PEPC2. This phosphorylation was reversed in vitro following incubation of clarified COS extracts or purified PEPC1 or PEPC2 with mammalian protein phosphatase type 2A and is not involved in a potential PEPC1 and PEPC2 interconversion. Similar to other plant PEPCs examined to date, p107 phosphorylation increased PEPC1 activity at pH 7.3 by decreasing its Km(PEP) and sensitivity to l-malate inhibition, while enhancing glucose-6-P activation. By contrast, p107 phosphorylation increased PEPC2's Km(PEP) and sensitivity to malate, glutamic acid, and aspartic acid inhibition. Phosphorylation of p107 was promoted during COS development (coincident with a >5-fold increase in the I50 [malate] value for total PEPC activity in desalted extracts) but disappeared during COS desiccation. The p107 of stage VII COS became fully dephosphorylated in planta 48 h following excision of COS pods or following 72 h of dark treatment of intact plants. The in vivo phosphorylation status of p107 appears to be modulated by photosynthate recently translocated from source leaves into developing COS.
An Indole-3-Acetic Acid Carboxyl Methyltransferase RegulatesArabidopsisLeaf Development
Authors: Qin Genji , Gu Hongya , Zhao Yunde , Ma Zhiqiang , Shi Guanglu , Yang Yue , Pichersky Eran , Chen Haodong , Liu Meihua , Chen Zhangliang , Qu Li-Jia
Published: September 2005

Auxin is central to many aspects of plant development; accordingly, plants have evolved several mechanisms to regulate auxin levels, including de novo auxin biosynthesis, degradation, and conjugation to sugars and amino acids. Here, we report the characterization of an Arabidopsis thaliana mutant, IAA carboxyl methyltransferase1-dominant (iamt1-D), which displayed dramatic hyponastic leaf phenotypes caused by increased expression levels of the IAMT1 gene. IAMT1 encodes an indole-3-acetic acid (IAA) carboxyl methyltransferase that converts IAA to methyl-IAA ester (MeIAA) in vitro, suggesting that methylation of IAA plays an important role in regulating plant development and auxin homeostasis. Whereas both exogenous IAA and MeIAA inhibited primary root and hypocotyl elongation, MeIAA was much more potent than IAA in a hypocotyl elongation assay, indicating that IAA activities could be effectively regulated by methylation. IAMT1 was spatially and temporally regulated during the development of both rosette and cauline leaves. Changing expression patterns and/or levels of IAMT1 often led to dramatic leaf curvature phenotypes. In iamt1-D, the decreased expression levels of TCP genes, which are known to regulate leaf curvature, may partially account for the curly leaf phenotype. The identification of IAMT1 and the elucidation of its role in Arabidopsis leaf development have broad implications for auxin-regulated developmental process.
Radiotelemetry reveals differences in individual movement patterns between outbreak and non‐outbreak Mormon cricket populations
Authors: Lorch Patrick D. , Sword Gregory A. , Gwynne Darryl T. , Anderson Gerald L.
Published: September 2005

Abstract. 1. Outbreaks of insect pest populations are common and can have devastating effects on natural communities and on agriculture. Little is known about the causes of these outbreaks or the causes ofen massemigrations during outbreaks.

2. Flightless Mormon crickets (Anabrus simplex) were the focus of this study. They are a katydid species that forms large, dense, mobile groups (migratory bands) during outbreak periods, eating vegetation in their path.

3. Radiotelemetric methods were used to measure differences in movement rate and directionality in outbreak and non‐outbreak populations, testing the hypothesis that these populations differ in their travel rate and consistency of direction.

4. Daily individual movement in outbreak populations differs substantially from non‐outbreak populations that are at much lower density. In addition to large differences in distances travelled (1.6 km as compared with 1 m) and rates of travel, there is evidence for collective movement among individual Mormon crickets travelling in migratory bands.

5. These data suggest that the direction of group movement may be influenced by local environmental conditions such as wind direction and movement of nearby band members. This work forms the basis for ongoing work testing hypotheses about mass migrations in outbreak populations.
Metacommunity patterns of highly diverse stream midges: gradients, chequerboards, and nestedness, or is there only randomness?
Authors: Heino Jani
Published: September 2005

Abstract. 1. Several non‐random patterns in the distribution of species have been observed, including Clementsian gradients, Gleasonian gradients, nestedness, chequerboards, and evenly spaced gradients. Few studies have examined these patterns simultaneously, although they have often been studied in isolation and contrasted with random distribution of species across sites.

2. This study examined whether assemblages of chironomid midges exhibit any of the idealised distribution patterns as opposed to random distribution of species across sites within the metacommunity context in a boreal drainage system. Analyses were based on stream surveys conducted during three consecutive years. Analytical approaches included ordinations, cluster analysis, null models, and associated randomisation methods.

3. Midge assemblages did not conform to Clementsian gradients, which was evidenced by the absence of clearly definable assemblage types with numerous species exclusive to each assemblage type. Rather, there were signs of continuous Gleasonian variability of assemblage composition, as well as significant nested subset patterns of species distribution.

4. Midge assemblages showed only weak relationships with any of the measured environmental variables, and even these weak environmental relationships varied among years.

5. Midge assemblages did not appear to be structured by competition. This finding was somewhat problematic, however, because the two indices measuring co‐occurrence provided rather different signs of distribution patterns. This was probably a consequence of how they actually measure co‐occurrence.

6. Although midge assemblages did not show a perfect match with any of the idealised distribution patterns, they nevertheless showed a resemblance to the empirical patterns found previously for several plant and animal groups.
The Baroque, Modernity, and Postmodernity in Latin American Political Culture
Authors: Bilbao Luis Ochoa , Frankel Paul
Published: September 2005

This article offers a general view about the contemporary theoretical debate in Latin‐American concerning the idea of modernity. For some authors, modernity is part of Latin‐American history, and its ruts can be found in the baroque experience of the continent, for others, the consequences of modernity such as urbanization, industralization and hope in democracy shows the Latin America is in essence a modern territory, despite the academic difficulties to understand and visualize Latin‐American identity as an integral element of the concept of modernity.
A Metalloproteinase of
Coccidioides posadasii
Contributes to Evasion of Host Detection
Authors: Hung Chiung-Yu , Seshan Kalpathi R. , Yu Jieh-Juen , Schaller Ruth , Xue Jianmin , Basrur Venkatesha , Gardner Malcolm J. , Cole Garry T.
Published: September 2005

Coccidioides posadasii
is a fungal respiratory pathogen of humans that can cause disease in immunocompetent individuals. Coccidioidomycosis ranges from a mild to a severe infection. It is frequently characterized either as a persistent disease that requires months to resolve or as an essentially asymptomatic infection that can reactivate several years after the original insult. In this report we describe a mechanism by which the pathogen evades host detection during the pivotal reproductive (endosporulation) phase of the parasitic cycle. A metalloproteinase (Mep1) secreted during endospore differentiation digests an immunodominant cell surface antigen (SOWgp) and prevents host recognition of endospores during the phase of development when these fungal cells are most vulnerable to phagocytic cell defenses. C57BL/6 mice were immunized with recombinant SOWgp and then challenged with a mutant strain of
C. posadasii
in which the
MEP1
gene was disrupted. The animals showed a significant increase in percent survival compared to SOWgp-immune mice challenged with the parental strain. To explain these results, we proposed that retention of SOWgp on the surfaces of endospores of the mutant strain in the presence of high titers of antibody to the immunodominant antigen contributes to opsonization, increased phagocytosis, and killing of the fungal cells. In vitro studies of the interaction between a murine alveolar macrophage cell line and parasitic cells coated with SOWgp showed that the addition of anti-SOWgp antibody could enhance phagocytosis and killing of
Coccidioides
. We suggest that Mep1 plays a pivotal role as a pathogenicity determinant during coccidioidal infections and contributes to the ability of the pathogen to persist within the mammalian host.
Streptococcus iniae
Phosphoglucomutase Is a Virulence Factor and a Target for Vaccine Development
Authors: Buchanan John T. , Stannard Jason A. , Lauth Xavier , Ostland Vaughn E. , Powell Henry C. , Westerman Mark E. , Nizet Victor
Published: September 2005

Streptococcus iniae
represents a major health and economic problem in fish species worldwide. Random Tn
917
mutagenesis and high-throughput screening in a hybrid striped bass (HSB) model of meningoencephalitis identified attenuated
S. iniae
mutants. The Tn
917
insertion in one mutant disrupted an
S. iniae
homologue of a phosphoglucomutase (
pgm
) gene. Electron microscopy revealed a decrease in capsule thickness and cell wall rigidity, with ΔPGM mutant cells reaching sizes ∼3-fold larger than those of the wild type (WT). The ΔPGM mutant was cleared more rapidly in HSB blood and was more sensitive to killing by cationic antimicrobial peptides including moronecidin from HSB. In vivo, the ΔPGM mutant was severely attenuated in HSB, as intraperitoneal challenge with 1,000 times the WT lethal dose produced only 2.5% mortality. Reintroduction of an intact copy of the
S. iniae pgm
gene on a plasmid vector restored antimicrobial peptide resistance and virulence to the ΔPGM mutant. In analysis of the aborted infectious process, we found that ΔPGM mutant organisms initially disseminated to the blood, brain, and spleen but were eliminated by 24 h without end organ damage. Ninety to 100% of fish injected with the ΔPGM mutant and later challenged with a lethal dose of WT
S. iniae
survived. We conclude that the
pgm
gene is required for virulence in
S. iniae
, playing a role in normal cell wall morphology, surface capsule expression, and resistance to innate immune clearance mechanisms. An
S. iniae
ΔPGM mutant is able to stimulate a protective immune response and may have value as a live attenuated vaccine for aquaculture.
ENERGY DECAY OF ELECTROMAGNETIC SYSTEMS WITH MEMORY
Authors: GIORGI CLAUDIO , NASO MARIA GRAZIA , PATA VITTORINO
Published: September 2005

We consider a linear evolution problem with memory arising in the theory of hereditary electromagnetism. Under general assumptions on the memory kernel, all single trajectories are proved to decay to zero, but the decay rate is not uniform in dependence of the initial data, so that the system is not exponentially stable. Nonetheless, if the kernel is rapidly fading and close to the Dirac mass at zero, then the solutions are close to exponentially stable trajectories.
Combination Phase I Trial of a Novel Oral Fluorouracil Derivative S-1 with Low-Dose Cisplatin for Unresectable and Recurrent Gastric Cancer (JFMC27–9902)
Authors: Nakata Bunzo , Mitachi Yasushi , Tsuji Akihito , Yamamitsu Susumu , Hirata Koichi , Shirasaka Tetsuhiko , Hirakawa Kosei
Published: September 2005

Purpose: The Japanese Foundation for Multidisciplinary Treatment of Cancer conducted a Phase I study of a novel oral fluorouracil derivative, S-1, combined with a low dose of cisplatin in unresectable and recurrent gastric cancer.

Experimental Design: S-1 was administered orally at 80–120 mg/body/day, depending on body surface area. One course consisted of consecutive administration for 28 days followed by a rest of 14 days. Low-dose cisplatin was given i.v. on days 1–5, 8–12, 15–19, and 22–26 of each course. The dose escalation of cisplatin began with an initial dose of 1 mg/m2/day as level 1 and was stepped up to 2, 3, 4, and 6 mg/m2/day as level 2, 3, 4, and 5, respectively. The regimen was repeated for at least two courses.

Results: A total of 24 patients was entered in the study. There was no treatment-related death. At level 5, consisting of 5 evaluable patients, dose-limiting toxicity was experienced as grade 3 appetite loss in 2 patients and grade 4 neutropenia in 1 patient. The maximum-tolerated dose of cisplatin was estimated to be 6 mg/m2/day. We decided on a recommended dose of cisplatin of 4 mg/m2/day because the dosage was one level under the maximum-tolerated dose. All 3 patients at level 4 showed partial response, suggesting promising clinical efficacy with this dosage. The serum concentration of cisplatin at level 4 was 918 ± 92 ng/ml on day 26 of the first course.

Conclusions: S-1 with low-dose cisplatin may become an effective regimen with acceptable toxicity for gastric cancer.
QuantitativeRARβ2Hypermethylation
Authors: Jerónimo Carmen , Henrique Rui , Hoque Mohammad O. , Ribeiro Franclim R. , Oliveira Jorge , Fonseca Daniel , Teixeira Manuel R. , Lopes Carlos , Sidransky David
Published: September 2005

Retinoic acid receptor β2 (RARβ2) is a tumor suppressor gene frequently hypermethylated in several human neoplasms. To further characterize this epigenetic alteration in prostate cancer progression, we examined tumor tissue from 118 patients with prostate carcinoma (PCa), 38 paired high-grade prostatic intraepithelial neoplasias (HGPIN), and non-neoplastic prostate tissue from 30 patients with benign prostate hyperplasia (BPH), using quantitative methylation-specific PCR. We found RARβ2 hypermethylation in 97.5% of PCa, 94.7% of HGPIN, and 23.3% of BPH. Methylation levels were significantly higher in PCa compared with HGPIN and BPH (P < 0.00001). By establishing an empiric cutoff value, we were able to discriminate between neoplastic and non-neoplastic tissue, with 94.9% sensitivity and 100% specificity. Moreover, RARβ2 methylation levels correlated with higher pathological stage (r = 0.30, P = 0.0009). This quantitative assay represents a novel and promising molecular marker that may augment current approaches for prostate cancer detection.
Estrogen Receptor Status in
BRCA1
- and
BRCA2
-Related Breast Cancer
Authors: Foulkes William D. , Metcalfe Kelly , Sun Ping , Hanna Wedad M. , Lynch Henry T. , Ghadirian Parviz , Tung Nadine , Olopade Olufunmilayo I. , Weber Barbara L. , McLennan Jane , Olivotto Ivo A. , Bégin Louis R. , Narod Steven A.
Published: September 2005

Purpose: BRCA1-related breast cancers are more frequently estrogen receptor (ER) negative than are either BRCA2-related or nonhereditary breast cancers. The relationship between ER status and other clinical features of hereditary breast cancers has not been well studied.

Experimental Design: ER status, grade, and histological tumor type were evaluated in 1131 women with invasive breast cancer, ascertained at 10 centers in North America. There were 208 BRCA1 mutation carriers, 88 BRCA2 carriers, and 804 women without a known mutation. We stratified the patients by mutation status, grade, age, and histological type and calculated the percentage of ER-positive tumors within each stratum.

Results: BRCA1 mutation carriers were more likely to have ER-negative breast cancers than were women in other groups, after adjustment for age, grade, and histological subtype (P < 0.001). Only 3.9% of BRCA1-related breast cancers were ER-positive cancers occurring in women in their postmenopausal years. The direction and magnitude of the change in ER status with increasing age at diagnosis in BRCA1 carriers was significantly different from in BRCA2 carriers (Pintercept = 0.0002, Pslope = 0.04). Notably, changes in ER status with age at diagnosis for BRCA1 carriers and noncarriers were almost identical (Pslope = 0.98).

Conclusions: The strong relationship between the presence of a BRCA1 mutation and the ER-negative status of the breast cancers is neither a consequence of the young age at onset nor the high grade but is an intrinsic property of BRCA1-related cancers. The ER-negative status of these cancers may reflect the cell of origin of BRCA1-related cancers.
Meta-Analysis Suggests Association of L-
myc Eco
RI Polymorphism with Cancer Prognosis
Authors: Spinola Monica , Pedotti Paola , Dragani Tommaso A. , Taioli Emanuela
Published: September 2005

The L-myc EcoRI polymorphism is a noncoding variation in the second intron of the L-myc gene, resulting in S and L alleles. Individuals carrying the S allele tend to have poor prognosis and increased risk of several tumor types, although controversial results have been reported. A meta-analysis of 36 studies on L-myc EcoRI genotyping, including 3563 patients with different types of cancer and 2953 controls, was performed. In lung cancer patients the S/S genotype was significantly associated with lymph node metastasis [odds ratio (OR), 2.8; 95% confidence interval (CI), 1.8–4.3], distant metastasis (OR, 4.7; 95% CI, 2.4–9.2), and stage (OR, 2.3; 95% CI, 1.2–4.4). No association was observed between the S/S genotype and cancer (OR, 1.1; 95% CI, 0.8–1.4). In patients with other cancers, the S/S genotype was significantly associated with tumor recurrence (OR, 2.8; 95% CI, 1.4–6.0), whereas no significant association was seen for the other prognostic parameters. When all types of cancer were examined together, the S/S genotype was associated with lymph node metastasis (OR, 2.3; 95% CI, 1.6–3.3), distant metastasis (OR, 2.9; 95% CI, 1.8–4.6), clinical stage (OR, 1.8; 95% CI, 1.2–2.9), and cancer risk (OR, 1.25; 95% CI, 1.07–1.45). The meta-analysis suggests that the L-myc EcoRI polymorphism is a marker of tumor prognosis in lung cancer and possibly in other types of cancer.
Imiquimod Treatment Induces Expression of Opioid Growth Factor Receptor
Authors: Urosevic Mirjana , Oberholzer Patrick A. , Maier Tanja , Hafner Jürg , Laine Elisabeth , Slade Herbert , Benninghoff Bernd , Burg Günter , Dummer Reinhard
Published: September 2005

Purpose: Imiquimod represents a synthetic local immune response modifier that has demonstrated efficacy in clearing basal cell carcinoma. Via interaction with Toll-like receptor 7 on immune cells, imiquimod induces local production of cytokines, such as interferon (IFN)-α.

Experimental Design: To more closely define and elucidate mechanisms leading to basal cell carcinoma clearance in vivo, we examined gene expression profiles of skin basal cell carcinoma before and after treatment with 5% imiquimod cream (Aldara) by using high-density oligonucleotide arrays.

Results: We show that imiquimod predominantly induces genes involved in different aspects of immune response. In addition to effects on immunity, imiquimod treatment modulates the expression of genes involved in the control of apoptosis and oncogenesis. Array data indicated that imiquimod treatment induces expression of opioid growth factor receptor, a molecule recently reported to be a target for antitumor antibody responses. Immunohistochemistry revealed in vivo up-regulation of opioid growth factor receptor protein on tumor and on infiltrating cells after treatment. By using basal cell carcinoma cell lines treated with IFN-α or imiquimod, we show that opioid growth factor receptor up-regulation is IFN-α-mediated, rather then directly imiquimod-mediated. By using tissue microarray containing 52 basal cell carcinomas, we demonstrate opioid growth factor receptor expression in almost half of the cases. Expression of opioid growth factor receptor correlated with a longer recurrence-free period in basal cell carcinoma that recurred after radiotherapy (Kaplan-Meier analysis, P = 0.041).

Conclusions: In addition to its immunomodulatory and antiproliferative activity, opioid growth factor receptor seems to have a prognostic significance in basal cell carcinoma patients. Our data add to the growing list of basal cell carcinoma-associated tumor antigens.
Expression of ΔNp73 Predicts Poor Prognosis in Lung Cancer
Authors: Uramoto Hidetaka , Sugio Kenji , Oyama Tsunehiro , Nakata Shoji , Ono Kenji , Morita Masaru , Funa Keiko , Yasumoto Kosei
Published: September 2005

Purpose: ΔNp73 is an isoform of the p53 homologue p73, which lacks an NH2-terminal transactivation domain and antagonizes the induction of gene expression by p53/p73. The aim of this study was to detect ΔNp73 expression in lung cancer and to evaluate the relationship between the ΔNp73 expression level and the prognosis of patients with resected lung cancer.

Experimental Design: We used immunohistochemistry to analyze the protein expression of ΔNp73 in paraffin-embedded tumor samples from 132 well-characterized lung cancer patients and compared the expression level of ΔNp73, clinical variables, and survival outcome.

Results: Positive expression of ΔNp73 was detected mainly in the cytoplasm of tumor cells in 77 of 132 patients (58.3%) with lung cancer. The incidence of positive expression of ΔNp73 was 52.2, 50.0, and 70.2% in patients with stage I, II, and III, respectively (P = 0.04). Positive expression of ΔNp73 was associated with gender but not associated with age, histologic type, pathological stage, pathological T status, and pathological N status. Lung cancer patients with positive ΔNp73 expression had a poorer prognosis than those with negative ΔNp73 expression. In addition, multivariate analysis of the clinicopathological characteristics of lung cancer indicated that positive expression of ΔNp73 was a significant independent factor for predicting poor prognosis (P < 0.0001, risk ratio = 3.39).

Conclusions: Expression of ΔNp73 may be a useful marker for predicting poor prognosis of patients who underwent resection of lung cancer.
Characterization of a Folate Transporter in HeLa Cells with a Low pH Optimum and High Affinity for Pemetrexed Distinct from the Reduced Folate Carrier
Authors: Wang Yanhua , Zhao Rongbao , Goldman I. David
Published: September 2005

Studies were undertaken to characterize a low pH transport activity in a reduced folate carrier (RFC)-null HeLa-derived cell line (R5). This transport activity has a 20-fold higher affinity for pemetrexed (PMX; Kt, ∼45 nmol/L) than methotrexate (MTX; Kt, ∼1 μmol/L) with comparable Vmax values. The Ki values for folic acid, ZD9331, and ZD1694 were ∼ 400–600 nmol/L, and the Ki values for PT523, PT632, and trimetrexate were >50 μmol/L. The transporter is stereospecific and has a 7-fold higher affinity for the 6S isomer than the 6R isomer of 5-formyltetrahydrofolate but a 4-fold higher affinity for the 6R isomer than the 6S isomer of dideazatetrahydrofolic acid. Properties of RFC-independent transport were compared with transport mediated by RFC at low pH using HepG2 cells, with minimal constitutive low pH transport activity, transfected to high levels of RFC. MTX influx Kt was comparable at pH 7.4 and pH 5.5 (1.7 versus 3.8 μmol/L), but Vmax was decreased 4.5-fold. There was no difference in the Kt for PMX (∼1.2 μmol/L) or the Ki for folic acid (∼130 μmol/L) or PT523 (∼ 0.2 μmol/L) at pH 7.4 and pH 5.5. MTX influx in R5 and HepG2 transfectants at pH 5.5 was trans-stimulated in cells loaded with 5-formyltetrahydrofolate, inhibited by Cl− (HepG2-B > R5), Na+ independent, and uninhibited by energy depletion. Hence, RFC-independent low pH transport activity in HeLa R5 cells is consistent with a carrier-mediated process with high affinity for PMX. Potential alterations in protonation of RFC or the folate molecule as a function of pH do not result in changes in affinity constants for antifolates. Whereas both activities at low pH have similarities, they can be distinguished by folic acid and PT523, agents for which they have very different structural specificities.
Effect of Rapamycin Alone and in Combination with Antiangiogenesis Therapy in an Orthotopic Model of Human Pancreatic Cancer
Authors: Stephan Susann , Datta Kaustubh , Wang Enfeng , Li Jinping , Brekken Rolf A. , Parangi Sareh , Thorpe Philip E. , Mukhopadhyay Debabrata
Published: September 2005

Purpose: The overall 5-year survival of patients with pancreatic cancer remains <5%. Novel therapeutic strategies are needed. We examined the effect of rapamycin, alone and in combination with antiangiogenesis therapy, on pancreatic cancer in vivo.

Experimental Design: Human pancreatic cancer AsPC-1 cells were orthotopically injected into severe combined immunodeficient/beige mice to evaluate primary tumor growth and liver metastasis after treatment with rapamycin alone or in combination with anti-vascular endothelial growth factor antibody 2C3. Tumor cell proliferation was determined by bromodeoxyuridine incorporation. To detect tumor cell apoptosis, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was used. Tumor angiogenesis was investigated by using a monoclonal anti-CD31 antibody. All statistical tests were two-sided.

Results: Rapamycin, alone and in combination with 2C3, strongly inhibited primary and metastatic tumor growth in an orthotopic pancreatic cancer animal model. Furthermore, the combination therapy significantly improved the effect on liver metastasis compared with single treatment with either rapamycin (P = 0.0128) or 2C3 (P = 0.0099). Rapamycin alone inhibited pancreatic tumor cell proliferation, induced apoptosis, and decreased tumor angiogenesis. Nevertheless, the combination therapy showed a significant, stronger inhibition of tumor cell proliferation (P = 0.0002 versus rapamycin alone and P < 0.0001 versus 2C3 alone). The induction of apoptosis was significantly higher than in the rapamycin-treated group (P = 0.0039). Additionally, the combination therapy further improved suppression of tumor cell angiogenesis compared with rapamycin treatment (P = 0.029)

Conclusions: Our studies propose new therapeutic strategies to inhibit both primary and metastatic tumor growth in pancreatic cancer. Considering the fact that liver metastasis is a crucial problem in advanced stages of pancreatic cancer, the combination therapy of rapamycin plus anti-vascular endothelial growth factor antibody 2C3 is a significant advantage compared with single treatment with rapamycin.
NY-ESO-1 Expression and Immunogenicity in Esophageal Cancer
Authors: Fujita Shoichiro , Wada Hisashi , Jungbluth Achim A. , Sato Shuichiro , Nakata Tetsuya , Noguchi Yuji , Doki Yuichiro , Yasui Masayoshi , Sugita Yurika , Yasuda Takushi , Yano Masahiko , Ono Toshiro , Chen Yao-Tseng , Higashiyama Masahiko , Gnjatic Sacha , Old Lloyd J. , Nakayama Eiichi , Monden Morito
Published: September 2005

Purpose: Although NY-ESO-1 was isolated from an esophageal carcinoma patient, its expression in this type of cancer and its immunogenicity in esophageal cancer patients have not yet been fully elucidated. We report here the frequency of NY-ESO-1 mRNA and protein expression in esophageal cancer and the presence of NY-ESO-1-specific immune response in patients.

Experimental Design: One hundred twenty three esophageal squamous cell carcinoma specimens were analyzed for the expression of NY-ESO-1 mRNA by conventional and real-time reverse transcription-PCR and the expression of protein by immunohistochemistry and Western blot. Sera and peripheral blood lymphocytes from 51 patients were analyzed for the NY-ESO-1 antibody production by enzyme-linked immunosorbent assay and NY-ESO-1 T cell response by enzyme-linked immunospot assay. Survival analyses were also performed.

Results: NY-ESO-1 mRNA was expressed in 41 of 123 (33%) esophageal squamous cell carcinoma specimens, and its expression was found at higher frequency in well-differentiated and moderately differentiated type of cancer. No mRNA copy was detected in any of the adjacent normal tissues. Twenty-one of 24 (87.5%) NY-ESO-1 mRNA-positive tumors were stained positively by immunohistochemistry. Correlation between the level of NY-ESO-1 mRNA expression and the degree of immunohistochemistry positivity was observed. Antibody production was observed in 2 patients with tumors that showed protein expression. Furthermore, a CD8 T-cell response against NY-ESO-1 was observed in 1 of the 2 seropositive patients.

Conclusions: The high expression frequency of NY-ESO-1 mRNA and protein indicates NY-ESO-1 as a feasible vaccine target in esophageal cancer.
Cellular Uptake and Intracellular Levels of the Bcl-2 Antisense G3139 in Cultured Cells and Treated Patients with Acute Myeloid Leukemia
Authors: Dai Guowei , Chan Kenneth K. , Liu Shujun , Hoyt Dale , Whitman Susan , Klisovic Marko , Shen TianSheng , Caligiuri Michael A. , Byrd John , Grever Michael , Marcucci Guido
Published: September 2005

Purpose: Down-regulation of Bcl-2 by the antisense G3139, currently under clinical evaluations, could restore chemosensitivity in otherwise resistant malignant cells. To date, the mechanism of intracellular accumulation of G3139 following in vivo administration remains to be elucidated. This study aimed to assess whether detectable intracellular concentrations of G3139 are achievable in vivo and how these relate to Bcl-2 down-regulation.

Experimental Design: Cellular uptake of G3139 was studied in leukemia myeloid cell lines and blasts collected from treated patients using a newly developed, novel, and highly sensitive ELISA-based assay. Real-time reverse transcription-PCR was used to quantify Bcl-2 mRNA changes in treated cells.

Results: The assay was fully validated and showed a limit of quantification of 50 pmol/L. When exposed to 0.33 to 10 μmol/L G3139, K562 cells exhibited intracellular concentrations in the range of 2.1 to 11.4 pmol/mg protein. When G3139 was delivered with cationic lipids, a 10- to 25-fold increase of the intracellular concentrations was observed. There was an accumulation of G3139 in the nuclei, and the ratio of nucleus to cytoplasm was increased 7-fold by cationic lipids. Intracellular concentrations of G3139 were correlated with Bcl-2 mRNA down-regulation. Robust intracellular concentrations of G3139 were achieved in vivo in bone marrow (range, 3.4-40.6 pmol/mg protein) and peripheral blood mononuclear cells (range, 0.47-19.4 pmol/mg protein) from acute myeloid leukemia patients treated with G3139.

Conclusions: This is the first evidence that measurable intracellular levels of G3139 are achievable in vivo in acute myeloid leukemia patients and that Bcl-2 down-regulation is likely to depend on the achievable intracellular concentrations rather than on plasma concentrations.
MEN4901/T-0128, a New Camptothecin Derivative–Carboxymethyldextran Conjugate, Has Potent Antitumor Activities in a Panel of Human Tumor Xenografts in Nude Mice
Authors: Fujita Fumiko , Koike Masako , Fujita Masahide , Sakamoto Yasuo , Okuno Satoshi , Kawaguchi Takayuki , Yano Shigeru , Yano Toshiro , Kiuchi Satoko , Fujiwara Toshihisa , Kudoh Shinzoh , Kakushima Masatoshi
Published: September 2005

Purpose: The purpose of the present study was to evaluate the antitumor activity and pharmacokinetic profile of MEN4901/T-0128 in nude mice bearing human tumor xenografts in comparison with irinotecan (CPT-11) and T-2513.

Experimental Design: We have determined the antitumor activity of MEN4901/T-0128, CPT-11, and T-2513 in BALB/cA Jcl nude mice bearing human gastric (H-81), colon (H-110), lung (Mqnu-1, H-74), esophageal (H-204), liver (H-181), and pancreatic (H-48) cancer lines, which had been serially transplanted s.c. and maintained in nude mice, and characterized the pharmacokinetic profile of MEN4901/T-0128 in nude mice bearing human gastric carcinoma St-4.

Results: MEN4901/T-0128 administered i.v. showed a marked antitumor activity in each of these tumor models, producing tumor shrinkage in the models of H-204 and H-181 carcinomas at its maximum tolerated dose of 80 mg/kg (expressed as T-2513) weekly for 4 weeks (q7d × 4) and tumor-shrinking or marked growth-inhibitory effects in the models of H-81, H-110, Mqnu-1, H-74, and H-48 carcinomas at 1/3 of its maximum tolerated dose (q7d × 4). Pharmacokinetic analysis showed that MEN4901/T-0128 had an extended plasma half-life with sustained tumor levels of T-2513, which may explain the superior activity of MEN4901/T-0128 in vivo.

Conclusions: Because the efficacies of some drugs in this human cancer-nude mouse panel correlated well with their clinical outcomes in patients with the same type of cancers, the findings provide direct support that MEN4901/T-0128 is more efficacious than CPT-11 and is an excellent candidate for clinical trials for the treatment of solid tumors.
A Phase II, Pharmacokinetic, and Biological Correlative Study of Oblimersen Sodium and Docetaxel in Patients with Hormone-Refractory Prostate Cancer
Authors: Tolcher Anthony W. , Chi Kim , Kuhn John , Gleave Martin , Patnaik Amita , Takimoto Chris , Schwartz Garry , Thompson Ian , Berg Kristin , D'Aloisio Susan , Murray Nevin , Frankel Stanley R. , Izbicka Elzbieta , Rowinsky Eric
Published: September 2005

Purpose: To determine the antitumor activity and safety of oblimersen sodium, a phosphorothioate antisense oligonucleotide directed to the bcl-2 mRNA, with docetaxel in patients with hormone-refractory prostate cancer (HRPC) and to determine if relevant pharmacokinetic and pharmacodynamic variables of oblimersen or docetaxel influence response to this therapy.

Experimental Design: Patients with HRPC were treated with oblimersen sodium by continuous i.v. infusion on days 1 to 8 with docetaxel given i.v. over 1 hour on day 6 every 3 weeks. Plasma samples were analyzed to characterize the pharmacokinetic variables of both oblimersen and docetaxel, and paired collections of peripheral blood mononuclear cells were collected to determine Bcl-2 protein expression pretreatment and post-treatment.

Results: Twenty-eight patients received 173 courses of oblimersen (7 mg/kg/d continuous i.v. infusion on days 1-8) and docetaxel (75 mg/m2 i.v. on day 6). Prostate-specific antigen responses were observed in 14 of 27 (52%) patients, whereas 4 of 12 (33%) patients with bidimensionally measurable disease had objective responses. The mean oblimersen steady-state concentration (Css) was a significant determinant of antitumor activity; mean Css values were higher in responders compared with nonresponders (6.24 ± 1.68 versus 4.27 ± 1.22; P = 0.008). The median survival of all patients was 19.8 months. Bcl-2 protein expression decreased a median of 49.9% in peripheral blood mononuclear cells post-treatment, but the individual incremental change did not correlate with either oblimersen Css or response.

Conclusions: Oblimersen combined with docetaxel is an active combination in HRPC patients demonstrating both an encouraging response rate and an overall median survival. The absence of severe toxicities at this recommended dose, evidence of Bcl-2 protein inhibition, and encouraging antitumor activity in HPRC patients warrant further clinical evaluation of this combination, including studies to optimize oblimersen Css.
Tissue Inhibitor of Matrix Metalloproteinase-1 Is Prognostic in Head and Neck Squamous Cell Carcinoma: Comparison of the Circulating and Tissue Immunoreactive Protein
Authors: Ruokolainen Henni , Pääkkö Paavo , Turpeenniemi-Hujanen Taina
Published: September 2005

Purpose: Tissue inhibitors of metalloproteinases (TIMP) are capable of inhibiting the matrix metalloproteinases, but they also possess other biological functions. Little is known about the role of TIMP-1 in the progression and spreading of cancer cells among patients with head and neck squamous cell carcinoma (HNSCC). In this study, the pretreatment serum levels of TIMP-1 or the overexpression of TIMP-1 immunoreactive protein in the primary tumor was correlated to the clinical course in patients with HNSCC.

Experimental Design: The TIMP-1 immunoreactive protein was studied in 74 cases representing HNSCC. The tissue immunoreactive protein was evaluated from paraffin-embedded tumor sections in 68 cases using immunohistologic staining with a specific antibody, and in 68 cases the pretreatment serum levels of TIMP-1 were quantitatively measured by ELISA assay. The results were compared with the clinicopathologic factors of the disease and the patients' outcome.

Results: A positive correlation was found between the size of the primary tumor (T) and the circulating TIMP-1 level (P = 0.021) or the positive immunoreaction of TIMP-1 in tumor (P = 0.039). The 5-year cause-specific survival was significantly lower in patients presenting with a high serum TIMP-1 level than in those with a low level of TIMP-1 (38% versus 64%, P = 0.034). They also had an unfavorable 5-year relapse-free survival rate (37% versus 56%, respectively). Similarly, the expression of TIMP-1 in tumor was prognostic for shortened survival, the 5-year cumulative relapse-free survival being 42% in patients with a TIMP-1–positive tumor versus 75% in cases with a negative tumor (P = 0.035). Tissue TIMP-1 positivity also seemed associated to the cause-specific survival (P = 0.075) and to be connected with later lymph node or hematogenic relapses.

Conclusions: This study shows for the first time that both circulating and tissue TIMP-1 immunoreactive protein predicts the clinical course and dissemination in HNSCC, suggesting that TIMP-1 might be related to both tumor growth and metastasis in HNSCC.
Phase I Study of the Intratumoral Administration of Recombinant Canarypox Viruses Expressing B7.1 and Interleukin 12 in Patients with Metastatic Melanoma
Authors: Triozzi Pierre L. , Allen Karen O. , Carlisle Ronda R. , Craig Mark , LoBuglio Albert F. , Conry Robert M.
Published: September 2005

The objective of this study was to evaluate the safety and activity of the intratumoral administration of the immune costimulatory molecule, B7.1, encoded by a vector derived from the canarypox virus, ALVAC (ALVAC-B7.1), alone and with the intratumoral injection of ALVAC encoding the immune-stimulatory cytokine, interleukin 12 (ALVAC-IL-12). Fourteen patients with metastatic melanoma who had s.c. nodules received intratumoral injections on days 1, 4, 8, and 11. Nine patients were given escalating doses of up to 25 × 108 plaque-forming units of ALVAC-B7.1. Five patients were given 25 × 108 plaque-forming units of ALVAC-B7.1 combined with ALVAC-IL-12 50% tissue culture infective dose of 2 × 106. Toxicity was mild to moderate and consisted of inflammatory reactions at the injection site and fever, chills, myalgia, and fatigue. Higher levels of B7.1 mRNA were observed in ALVAC-B7.1–injected tumors compared with saline-injected control tumors. Higher levels of intratumoral vascular endothelial growth factor and IL-10, cytokines with immune suppressive activities, were also observed in ALVAC-B7.1– and ALVAC-IL-12–injected tumors compared with saline-injected controls. Serum levels of vascular endothelial growth factor increased at day 18 and returned to baseline at day 43. All patients developed antibody to ALVAC. Intratumoral IL-12 and IFN-γ mRNA decreased. Changes in serum IL-12 and IFN-γ levels were not observed. Tumor regressions were not observed. The intratumoral injections of ALVAC-B7.1 and ALVAC-IL-12 were well tolerated at these dose levels and at this schedule and resulted in measurable biological response. This response included the production of factors that may suppress the antitumor immunologic activity of these vectors.