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Biomedical subjects

Raj K Puri

Publications and source records attributed to Raj K Puri.

At least 19 recordsLinked to original sources
PubMed2006

N-linked glycosylation of IL-13R alpha2 is essential for optimal IL-13 inhibitory activity.

A high-affinity receptor for interleukin (IL)-13 (interleukin-13R alpha 2) is over-expressed in disease-related fibroblasts and neoplastic cells and is involved in cancer, allergic, and inflammatory diseases. The extracellular domain of IL-13R alpha2 (ECD alpha2) could be cleaved, which serves as a decoy receptor. We have expressed and purified ECD alpha2 in both Escherichia coli (E. coli) and mammalian systems as a soluble fragment and studied its biological activities. Although both products of ECD alpha2 showed IL-13 inhibitory activities, mammalian cell-derived ECD alpha2 appeared to be superior compared with purified protein from E. coli. When expressed in E. coli, ECD alpha2 appeared to be a monomer of 42 but a 60 kDa protein when purified from mammalian cells due to heavy glycosylation. The purified glycosylated ECD alpha2 efficiently inhibited IL-13-induced STAT6 phosphorylation in immune and Hodgkin's lymphoma cell lines, IL-13 binding, and cytotoxicity of IL-13 cytotoxin in various cancer cell lines. The improved potency of mammalian cell-derived ECD alpha2 was shown over ECD alpha2/Fc fusion protein. The N-linked glycosylation of ECD alpha2 was found to be essential for optimal IL-13 inhibitory activity as deglycosylation by PNGase F showed lower activity. ECD alpha2 did not inhibit IL-4-induced STAT6 phosphorylation, indicating that inhibitory effects of ECD alpha2 are receptor specific. These results indicate that glycosylated ECD alpha2 can serve as a potent inhibitor of IL-13 in a variety of conditions in which IL-13 is a key mediator, e.g., pulmonary, allergic, fibrotic, and neoplastic diseases.

Animals↗
PubMed2006

Interleukin-13 receptor alpha2 chain: a potential biomarker and molecular target for ovarian cancer therapy.

BACKGROUND: Epithelial ovarian cancer demonstrates high mortality due to diagnosis at an advanced stage. In the search for a biomarker for early diagnosis and a target for therapy, the issue of whether interleukin-13 receptor (IL-13R), shown to be expressed on a variety of human cancers, is expressed in ovarian tumor samples was explored. In addition, whether this receptor serves as a biomarker and can be targeted by IL-13 cytotoxin was examined. METHODS: IL-13R expression in 15 normal and 68 ovarian tumor tissue samples was determined by immunohistochemistry. Correlation between clinicopathologic features and IL-13R expression was analyzed. The efficacy of IL-13R-directed cytotoxin was determined in mice with subcutaneous, orthotopic, and peritoneal metastatic ovarian cancer. RESULTS: Immunohistochemical analyses revealed that 83% of ovarian cancer specimens express IL-13Ralpha2, a high-affinity IL-13R subunit chain, whereas normal ovary samples expressed none or very low levels. The majority of clear cell ovarian carcinomas with the worst prognosis showed strong staining for IL-13Ralpha2. IL-13 cytotoxin was highly cytotoxic to the IGROV-1 ovarian cancer cell line in vitro, and it mediated significant antitumor activity against a xenografted tumor model. The antitumor effects were confirmed by treating orthotopically implanted or peritoneal metastatic ovarian tumors, which showed significant extension of survival in immunodeficient mice. IL-13 cytotoxin also prevented cachexia in treated mice. The soluble form of IL-13Ralpha2 was detected in the serum of mice with peritoneal metastasis, and the level decreased to baseline in the treated group. CONCLUSIONS: IL-13Ralpha2 is a promising target for ovarian cancer therapy, and the soluble form of IL-13R may be a possible surrogate marker for disease monitoring.

Adult↗
PubMed2006

Intratumoral therapy with IL13-PE38 results in effective CTL-mediated suppression of IL-13Ralpha2-expressing contralateral tumors.

PURPOSE: IL13-PE38, a targeted cytotoxin comprised of interleukin 13 (IL-13) and a mutated form of Pseudomonas exotoxin, induces specific killing of tumor cells expressing abundant levels of the IL-13Ralpha2 chain. We hypothesized that tumor cells killed by the cytotoxin may release antigens and/or apoptotic bodies when cells are dying, which then induce adoptive immunity, and that the PE38 portion of IL13-PE38 may act as a stimulant for the induction of a CTL response. EXPERIMENTAL DESIGN: To test this hypothesis, we established D5 melanoma tumors with or without expression of the IL-13Ralpha2 chain in both flanks of C57BL/6 mice, and then IL13-PE38 was injected in the right flank tumors only. RESULTS AND CONCLUSIONS: When animals with IL-13Ralpha2-expressing D5 tumor (right) were injected with IL13-PE38, right flank tumors expressing the IL-13Ralpha2 chain not only showed dramatic regression but contralateral tumors (left flank) also showed tumor regression. Cell depletion experiments in tumor-bearing animals indicated that both CD8(+) and CD4(+) T cells contribute to the regression of contralateral tumors through CTL activation in the periphery and cellular infiltration into tumors. In addition, intratumoral treatment into s.c. tumors of mice bearing metastatic lung tumors with IL13-PE38 showed not only the reduction of treated s.c. tumor but also the reduction of lung metastasis. Thus, IL13-PE38 mediates an antitumor effect not only directly but also indirectly by inducing a host CD8(+) T cell immune response. Accordingly, targeted cytotoxins may be used to treat local disease even if they cannot be administered systemically, and yet may still induce a reasonable systemic antitumor response.

Animals↗
PubMed2006

Characterization of a novel human tumor antigen interleukin-13 receptor alpha2 chain.

The interleukin (IL)-13 receptor alpha2 (IL-13Ralpha2) chain is a primary binding and internalization subunit for a Th2-derived immune regulatory cytokine, IL-13. Although extremely high levels of IL-13Ralpha2 chain are expressed on a variety of human tumor cells and specimens, its precise role in tumor immunology has not been defined. To investigate the role of IL-13Ralpha2 in tumor immunity, we used D5 melanoma cells stably transfected with the human IL-13Ralpha2 gene (D5alpha2) to assess the effect of an IL-13Ralpha2 DNA vaccine in immunocompetent animals. Prophylactic immunization of mice with the IL-13Ralpha2 DNA vaccine resulted in protection against D5alpha2 tumor development. In vivo depletion experiments in C57BL/6 and RAG-2 knockout mice indicated that both T and B cells, but not natural killer cells, were required for the tumor protection. In addition, antibody induced by the IL-13Ralpha2 DNA vaccine showed a modest but significant inhibitory effect on D5alpha2 cells in vitro, suggesting that the antibody is biologically functional. The IL-13Ralpha2 DNA vaccine also exhibited antitumor activity against established D5alpha2 tumors in mice. Histologic analysis of regressing tumors identified infiltration of CD4+ and CD8+ T cells and the expression of CXCL9 chemokine in tumors. Taken together, our results identify the human IL-13Ralpha2 chain as a novel tumor rejection antigen.

Animals↗
PubMed2006

The IL-4 and IL-13 pseudomonas exotoxins: new hope for brain tumor therapy.

Targeting cell surface receptors with cytotoxins or immunotoxins provides a unique opportunity for brain tumor therapy. The authors have discovered that receptors for two cytokines, interleukin (IL)-4 and IL-13, are overexpressed on tumor biopsy samples and on cell lines derived from a variety of human tumors, including brain tumors. These investigators have demonstrated that the structure of these cytokine receptors on tumor cells is different from that found on normal immune cells. In human solid tumor cells, IL-4 binds to two chains (IL-4Ra and IL-13Ra1), whereas IL- 13 binds to three chains in many solid tumor cells, including glioma cells (to IL-4Ra, IL-13Ra1, and IL-13Ra2). To target IL-4Rs and IL-13Rs, the authors generated two recombinant fusion cytotoxins composed of IL-4 or IL-13 and a mutated form of pseudomonas exotoxin (PE), which for simplicity are called IL4-PE and IL13-PE in this paper. These chimeric cytotoxins are highly toxic in vitro to human tumor cell lines and primary cell cultures, including glioma cells, and in vivo to animal models of human tumors, including gliomas. In contrast, normal cells, including immune, endothelial, and brain cells, are spared from their cytotoxic effects. Based on numerous preclinical studies, IL13-PE (also known as IL13-PE38QQR or cintredekin besudotox) has been tested in four Phase I/II clinical trials. The agent IL13-PE was administered intracranially by using convection-enhanced delivery (CED). The drug was delivered through catheters placed either directly into the tumor bed or in the peritumoral region after resection of the lesion. The CED of IL13-PE was fairly well tolerated, with a reasonable benefit/risk profile for treatment of patients with glioma. Based on Phase I/II clinical trials, the Phase III Randomized Evaluation of CED of IL13-PE Compared to Gliadel Wafer with Survival Endpoint Trial (also known as the PRECISE Trial) in patients with initial recurrence of glioblastoma multiforme has recently been completed. Patients are being monitored for safety of the agents, duration of overall survival, and quality of life.

Antineoplastic Agents↗
PubMed2006

Safety of intraparenchymal convection-enhanced delivery of cintredekin besudotox in early-phase studies.

OBJECT: Convection-enhanced delivery (CED) is an increasingly used novel local/regional delivery method targeted directly to tissue. It relies on a continuous pressure gradient for distribution of therapeutic agents into the interstitial space, with administration of the infusate over a few days. Cintredekin besudotox (also known as IL13- PE38QQR) is a recombinant chimeric cytotoxin consisting of interleukin-13 and a truncated exotoxin produced by the Pseudomonas aeruginosa bacterium, which targets malignant glioma cells. METHODS: Cintredekin besudotox was administered via intraparenchymal CED after resection of supratentorial recurrent malignant glioma. The safety and toxicity profile was reviewed for 53 patients in whom infusion catheters had been placed; 51 of them received CED of the study drug. Adverse events were categorized based on time of onset in relation to CED, and the causal relationship with catheter placement or delivery of cintredekin besudotox. Catheters were placed in 53 patients, although only 51 of them received cintredekin besudotox. Most adverse events related to catheter placement or the study drug originated from the central nervous system. Three symptomatic windows were defined: the first one was between surgical procedure and CED; the second was during CED and up to 1 week after its completion; and the third window was 2 to 10 weeks after treatment. Those windows generally reflected adverse events related to surgical procedures, mass effect from infusate, and drug effect on tumor-infiltrated and normal brain parenchyma, respectively. CONCLUSIONS: The symptomatic windows identified in this study apply to any CED clinical trials, particularly those in which chimeric cytotoxins are used, and will help to determine the most likely underlying pathophysiological process causing symptoms. This information, in turn, will help to prevent adverse events or minimize their severity. Those events also have implications for dose escalation and outcome measures.

Adult↗
PubMed2006

Quality prediction of cell substrate using gene expression profiling.

Changes in cell culture conditions influence the metabolism of cells, which consequently affects the quality of the products that they produce, such as viral vectors, recombinant proteins, or vaccines. Currently there is no effective technique available to monitor global quality of cells in cell culture. Here we describe a new method using gene expression profiling by microarray to predict the quality of cell substrates. Human embryonic kidney 293 cells are a commonly used cell substrate in the production of biological products. We demonstrate that the yield of adenoviral vectors was lower in over-confluent 293 cells, compared to 40 or 90% confluent cells. Total RNA derived from these cells of different confluence states was reverse transcribed, labeled, and used to hybridize 10K cDNA arrays to determine biomarkers for confluence states. Phenotype scatter-plot analysis and cluster analysis were used for class discovery. Based on this approach, we identified genes that were either up-regulated or down-modulated in response to different cell confluence states. By multivariate predictive models we identified a set of 37 genes that were either down-regulated or up-regulated compared to 90% confluent cells as a predictor of cell confluence and quality of 293 cell cultures. The predictive accuracy of these models was assessed by the leave-one-out cross-validation method. The expression of selected gene predictors was validated by quantitative PCR analysis. Our results demonstrate that gene expression profiling can assess the quality of cell substrates prior to large-scale production of a biological product.

Adenoviridae↗
PubMed2006

Role of interleukin-13 in cancer, pulmonary fibrosis, and other T(H)2-type diseases.

Interleukin (IL)-13 plays a major role in various inflammatory diseases including cancer, asthma, and allergy. It mediates a variety of different effects on various cell types including B cells, monocytes, natural killer cells, endothelial cells, and fibroblasts. IL-13 binds to two primary receptor chains IL-13Ralpha1 and IL-13Ralpha2. The IL-13Ralpha2 but not IL-13Ralpha1 chain binds IL-13 with high affinity and is overexpressed in a variety of human cancer cells derived from glioma, squamous cell carcinoma of head and neck, and AIDS-associated Kaposi's sarcoma. We have also demonstrated that IL-13Ralpha2 expression is greatly increased in lung cells when mice were challenged intranasally with bleomycin or Aspergillus fumigatus. In addition, IL-13Ralpha2 increased in surgical lung biopsies from patients with usual interstitial pneumonia, nonspecific interstitial pneumonia, and respiratory bronchiolitic interstitial pneumonia of unknown origin. Based on various studies, it is concluded that IL-13Ralpha2-expressing cells are involved in various pulmonary pathological conditions. In contrast, normal tissues such as brain, lung, endothelial cells, and head and neck tissues express IL-13Ralpha1 chain, but show only marginal expression of IL-13Ralpha2 chain. Thus, IL-13Ralpha2 chain may serve as a novel biomarker for diseased cells such as cancer or fibrosis and a target for receptor-directed therapeutic agents. To target IL-13R, a recombinant fusion protein composed of IL-13 and a derivative of Pseudomonas exotoxin (PE) has been produced. This cytotoxin termed as IL-13PE38QQR or IL-13PE38, or IL-13PE is highly and specifically cytotoxic to a variety of human tumor cell lines. In preclinical models of human glioblastoma, head and neck and AIDS-associated Kaposi's cancer, IL-13PE has been found to have significant antitumor activity at a tolerated dose. Several phase I clinical trials have been completed in patients with recurrent malignant glioma. Recently a phase III clinical trial (PRECISE) in patients with recurrent malignant glioma has been completed recruiting a total of 294 patients. IL-13PE cytotoxin has also shown a significant therapeutic effect in preclinical bleomycin or A. fumigatus or Schistosoma mansoni-induced pulmonary pathology including granulomatous fibrosis in mouse models. A clinical study in these diseases has yet to be initiated.

Animals↗
PubMed2006

The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements.

Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings.

Equipment Design↗
PubMed2006

Comparisons between transcriptional regulation and RNA expression in human embryonic stem cell lines.

Recent studies have focused on transcriptional regulation and gene expression profiling of human embryonic stem cells (hESCs). However, little information is available regarding the relationship between RNA expression and transcriptional regulation, which is critical in the complete understanding of pluripotency and differentiation of hESCs. In the current study, we determined RNA expression of three different hESC lines compared to Human universal reference RNA expression (HuU-RNA) using a full genome expression microarray, and compared our results to target genes previously identified using ChIP-on-chip analysis. The objective was to identify genes common between the two methods, and generate a more reliable list of embryonic signature genes. Even though hESCs were obtained from different sources and maintained under different conditions, a considerable number of genes could be identified as common between RNA expression and transcriptional regulation analyses. As an example, results from ChIP-on-chip studies show that OCT4, SOX2, and NANOG co-occupy SOX2, OCT4, TDGF1, GJA1, SET, and DPPA4 genes. The results are consistent with RNA expression analyses that demonstrate these genes as differently expressed in our hESC lines, further substantiating their role across cell types and confirming their importance as embryonic signatures. In addition, we report the differential expression of growth arrest-specific (GAS) family of genes in hESC. GAS2L1 and GAS3 members of this family appear to be transcriptionally regulated by OCT4, SOX2, or NANOG, whereas GAS5 and GAS6 are not; all of the genes are differentially expressed, as determined by microarray and validated via quantitative (Q)- PCR. Collectively, these data provide insight into the relationship between gene expression and transcriptional regulation, resulting in a reliable list of genes associated with hESCs.

DNA-Binding Proteins↗
PubMed2006

Convection-enhanced delivery of interleukin-13 receptor-directed cytotoxin for malignant glioma therapy.

The treatment of patients with malignant brain tumors, in particular glioblastoma multiforme (GBM) is very challenging because of their diffuse infiltrative nature and the cytological heterogeneity. The median survival of patients with newly diagnosed GBM is only 12-15 months, and only 8-12% of them survive for two years. Novel approaches for brain tumor therapy are needed. Recently, targeted therapies have emerged as promising modality for cancer targeting. We have discovered that high affinity plasma membrane receptor for interleukin-13 (IL-13), an immune regulatory cytokine, is over-expressed in 60-80% of malignant brain tumors. To target these IL-13R, we generated a chimeric fusion protein, composed of human IL-13 and mutated Pseudomonas exotoxin (PE), termed IL-13 cytotoxin (IL13-PE), and tested its cytotoxicity to IL-13R-expressing GBM cells. IL-13 cytotoxin was highly potent and selective in killing IL-13R-expressing GBM cells. In contrast, normal cells including brain, immune, and endothelial cells were generally not affected by this cytotoxin due to no or low expression of IL-13R. In vivo pre-clinical studies for safety and toxicity were also performed in mice, rats, and monkeys, and IL-13 cytotoxin was found to be well tolerated by both systemic and intracerebral administrations. IL-13 cytotoxin was found to mediate remarkable efficacy in animal models of human brain tumors. Encouraged by these pre-clinical studies, four Phase 1/2 clinical trials in adult patients with recurrent malignant glioma have been completed. These clinical trials involved convection-enhanced delivery (CED) of IL-13 cytotoxin either intratumoral or intraparenchymal after resection of tumor. CED is a novel loco-regional drug delivery method for intracranial tumors that relies on a continuous pressure gradient to distribute drug into interstitial space. This route of IL-13 cytotoxin administration appears to be very well tolerated and have a good risk-benefit profile. Most recently, a randomized controlled Phase 3 clinical trial (PRECISE) with intraparenchymal IL-13 cytotoxin administration was completed and subjects are being monitored for safety and survival.

Animals↗
PubMed2005

IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis.

Interleukin (IL)-13 is a major inducer of fibrosis in many chronic infectious and autoimmune diseases. In studies of the mechanisms underlying such induction, we found that IL-13 induces transforming growth factor (TGF)-beta(1) in macrophages through a two-stage process involving, first, the induction of a receptor formerly considered to function only as a decoy receptor, IL-13Ralpha(2). Such induction requires IL-13 (or IL-4) and tumor necrosis factor (TNF)-alpha. Second, it involves IL-13 signaling through IL-13Ralpha(2) to activate an AP-1 variant containing c-jun and Fra-2, which then activates the TGFB1 promoter. In vivo, we found that prevention of IL-13Ralpha(2) expression reduced production of TGF-beta(1) in oxazolone-induced colitis and that prevention of IL-13Ralpha(2) expression, Il13ra2 gene silencing or blockade of IL-13Ralpha(2) signaling led to marked downregulation of TGF-beta(1) production and collagen deposition in bleomycin-induced lung fibrosis. These data suggest that IL-13Ralpha(2) signaling during prolonged inflammation is an important therapeutic target for the prevention of TGF-beta(1)-mediated fibrosis.

Animals↗
PubMed2005

Comparison of the gene expression profile of undifferentiated human embryonic stem cell lines and differentiating embryoid bodies.

BACKGROUND: The identification of molecular pathways of differentiation of embryonic stem cells (hESC) is critical for the development of stem cell based medical therapies. In order to identify biomarkers and potential regulators of the process of differentiation, a high quality microarray containing 16,659 seventy base pair oligonucleotides was used to compare gene expression profiles of undifferentiated hESC lines and differentiating embryoid bodies. RESULTS: Previously identified "stemness" genes in undifferentiated hESC lines showed down modulation in differentiated cells while expression of several genes was induced as cells differentiated. In addition, a subset of 194 genes showed overexpression of greater than > or = 3 folds in human embryoid bodies (hEB). These included 37 novel and 157 known genes. Gene expression was validated by a variety of techniques including another large scale array, reverse transcription polymerase chain reaction, focused cDNA microarrays, massively parallel signature sequencing (MPSS) analysis and immunocytochemisty. Several novel hEB specific expressed sequence tags (ESTs) were mapped to the human genome database and their expression profile characterized. A hierarchical clustering analysis clearly depicted a distinct difference in gene expression profile among undifferentiated and differentiated hESC and confirmed that microarray analysis could readily distinguish them. CONCLUSION: These results present a detailed characterization of a unique set of genes, which can be used to assess the hESC differentiation.

Biomarkers↗
PubMed2005

Expression and targeting of interleukin-4 receptor for primary and advanced ovarian cancer therapy.

Because the most characteristic property of ovarian cancer is i.p. spread, the majority of patients are diagnosed at an advanced stage, leading to limited availability of options for curative therapies. With an intent to identify targeted therapeutic approaches, we have observed that approximately 60% of 21 ovarian cancer tissue samples express a high density of interleukin-4 receptor (IL-4R), whereas normal ovarian tissues tested (n = 7) expressed no or low levels of IL-4R. To target IL-4R, we have developed IL-4 cytotoxin, in which circular-permuted IL-4 is fused to a mutated form of Pseudomonas exotoxin. This cytotoxin is specifically and highly cytotoxic to PA-1, IGROV-1, and SK-OV3 ovarian carcinoma cell lines in vitro. In addition, it shows remarkable antitumor activities against established s.c. ovarian tumors in immunodeficient animals. i.p. administration of IL-4 cytotoxin in mice with orthotopically implanted ovarian tumors caused regression of established tumors and prevented these animals from tumor metastasis. Continuous i.p. infusion of IL-4 cytotoxin prolonged survival of tumor-bearing mice even with bulky disease. These results indicate that IL-4R-targeted cytotoxin may be a useful agent for the management of patients with ovarian cancer, and further studies need to be done to evaluate its safety, tolerability, and efficacy.

Animals↗
PubMed2005

Combined effects of radiation and interleukin-13 receptor-targeted cytotoxin on glioblastoma cell lines.

PURPOSE: Interleukin-13 receptor-targeted cytotoxin (IL13-PE38) is highly cytotoxic to human glioblastoma (GBM) cells. Although this molecule is being tested in a multicenter Phase III clinical trial (PRECISE Study) in patients with recurrent disease, the activity of IL13-PE38 when combined with radiation therapy has not been investigated. METHODS AND MATERIALS: Cytotoxicity of IL13-PE38 to GBM cell lines was assessed by protein synthesis inhibition and clonogenic assays, and the growth of GBM cells receiving radiation was assessed by thymidine uptake assays. Expression of IL-13 receptor alpha2 (IL-13Ralpha2) messenger ribonucleic acid (mRNA) in GBM cells exposed to radiation was assessed by quantitative reverse transcriptase/polymerase chain reaction (RT-PCR) and IL-13R density by radiolabeled IL-13 binding assays. RESULTS: Prior irradiation of GBM cell lines followed by IL13-PE38 treatment did not enhance cytotoxicity; however, concomitant 5 Gy irradiation and IL13-PE38 treatment was highly cytotoxic to T98G, M059K, A172, and LN-229 cell lines as determined by cell viability assays. There was a statistically significant decrease in number of viable cells in IL13-PE38 and irradiated cells compared with irradiated cells alone (p < 0.05) or IL13-PE38 treated cells alone (p < 0.05). In contrast, U251, SN19, and U87MG cell lines did not show any combined effect. These results were confirmed by clonogenic assays. Although three GBM cell lines-U251, SN19, and A172-showed 2.8- to 13.9-fold upregulation of IL-13Ralpha2 mRNA expression at 6-24 h after exposure to 5 Gy radiation, specific binding of radiolabeled IL-13 to these cell lines did not improve. CONCLUSIONS: Our results suggest that concomitant radiation therapy and IL13-PE38 treatment may be beneficial for the treatment of patients with GBM. This strategy may be worth exploring in animal models of human glioma.

Bacterial Toxins↗
PubMed2005

Adenoviral vector-mediated gene transfer of IL-13Ralpha2 chain followed by IL-13 cytotoxin treatment offers potent targeted therapy for cytotoxin-resistant cancers.

Previous studies demonstrated that IL-13Ralpha2 chain-overexpressing cancer cells were highly sensitive to IL-13 cytotoxin (IL13-PE38QQR) and could be targeted by cytotoxin treatment. However, the majority of human tumors do not express high levels of IL-13Ralpha2 chain. To expand the IL-13 cytotoxin-mediated cancer targeting therapy, we combined cytotoxin treatment with gene transfer of IL-13Ralpha2 chain. We constructed a recombinant adenoviral vector carrying the human IL-13Ralpha2 gene (Ad-IL-13Ralpha2), which expresses high levels of IL-13Ralpha2 chain on infected cells. Human cancer cell lines A549 and HOS, which originally show no IL-13Ralpha2 expression and little sensitivity to IL-13 cytotoxin, were effectively converted to become sensitive to this cytotoxin after Ad-IL-13Ralpha2 infection. The CC(50) of IL-13 cytotoxin for Ad-IL-13Ralpha2-infected A549 cells was <10 ng/ml, whereas the CC(50) for uninfected or control vector-infected cells was >500 ng/ml. We also examined the antitumor activity of IL-13 cytotoxin in an established xenograft model of cytotoxin-resistant human lung tumor. Only a single i.t. injection of Ad-IL-13Ralpha2 markedly enhanced the sensitivity of established tumors to IL-13 cytotoxin treatment; furthermore, this antitumor effect was significantly sustained for more than 1 month after the last treatment with IL-13 cytotoxin. Taken together, these results suggest the combination of adenoviral vector-mediated IL-13Ralpha2 gene transfer and IL-13 cytotoxin administration can be an effective targeting approach for several types of IL-13 cytotoxin-resistant cancers which show no or little expression of IL-13Ralpha2 chain.

Adenoviridae↗
PubMed2005

Microarray scanner calibration curves: characteristics and implications.

BACKGROUND: Microarray-based measurement of mRNA abundance assumes a linear relationship between the fluorescence intensity and the dye concentration. In reality, however, the calibration curve can be nonlinear. RESULTS: By scanning a microarray scanner calibration slide containing known concentrations of fluorescent dyes under 18 PMT gains, we were able to evaluate the differences in calibration characteristics of Cy5 and Cy3. First, the calibration curve for the same dye under the same PMT gain is nonlinear at both the high and low intensity ends. Second, the degree of nonlinearity of the calibration curve depends on the PMT gain. Third, the two PMTs (for Cy5 and Cy3) behave differently even under the same gain. Fourth, the background intensity for the Cy3 channel is higher than that for the Cy5 channel. The impact of such characteristics on the accuracy and reproducibility of measured mRNA abundance and the calculated ratios was demonstrated. Combined with simulation results, we provided explanations to the existence of ratio underestimation, intensity-dependence of ratio bias, and anti-correlation of ratios in dye-swap replicates. We further demonstrated that although Lowess normalization effectively eliminates the intensity-dependence of ratio bias, the systematic deviation from true ratios largely remained. A method of calculating ratios based on concentrations estimated from the calibration curves was proposed for correcting ratio bias. CONCLUSION: It is preferable to scan microarray slides at fixed, optimal gain settings under which the linearity between concentration and intensity is maximized. Although normalization methods improve reproducibility of microarray measurements, they appear less effective in improving accuracy.

Calibration↗