Neil Hoa

Antigenic Profiling of Glioma Cells to Generate Allogeneic Vaccines or Dendritic Cell–Based Therapeutics

Purpose: Allogeneic glioma cell lines that are partially matched to the patient at class I human leukocyte antigen (HLA) loci and that display tumor-associated antigens (TAA) or antigenic precursors [tumor antigen precursor proteins (TAPP)] could be used for generating whole tumor cell vaccines or, alternatively, for extraction of TAA peptides to make autologous dendritic cell vaccines. Experimental Design: Twenty human glioma cell lines were characterized by molecular phenotyping and by flow cytometry for HLA class I antigen expression. Twelve of the 20 cell lines, as well as analyses of freshly resected glioma tissues, were further characterized for protein and/or mRNA expression of 16 tumor antigen precursor proteins or TAA. Results: These 20 human glioma cell lines potentially cover 77%, 85%, and 78% of the U.S. Caucasian population at HLA-A, HLA-B, and HLA-C alleles, respectively. All cells exhibited multiple TAA expressions. Most glioma cells expressed antigen isolated from immunoselected melanoma-2 (Aim-2), B-cyclin, EphA2, GP100, β1,6-N-acetylglucosaminyltransferase V (GnT-V), IL13Rα2, Her2/neu, hTert, Mage, Mart-1, Sart-1, and survivin. Real-time PCR technology showed that glioblastoma specimens expressed most of the TAA as well. Tumor-infiltrating lymphocytes and CD8+ CTL killed T2 cells when loaded with specific HLA-A2+ restricted TAA, or gliomas that were both HLA-A2+ and also positive for specific TAA (Mart-1, GP100, Her2/neu, and tyrosinase) but not those cells negative for HLA-A2 and/or lacking the specific epitope. Conclusions: These data provide proof-in-principle for the use of allogeneic, partially HLA patient–matched glioma cells for vaccine generation or for peptide pulsing with allogeneic glioma cell extracts of autologous patient dendritic cells to induce endogenous CTL in brain tumor patients.

Mechanism of extracellular calcium regulation of intestinal epithelial tight junction permeability: Role of cytoskeletal involvement

Recent studies suggest that an abnormal increase in intestinal tight junction (TJ) permeability may be an important etiologic factor in number of diseases including Crohns disease, NSAID‐associated enteritis, and various infectious diarrheal syndromes. The intracellular processes involved in regulation of intestinal epithelial TJ permeability, however, remain poorly understood. In this study, we used cultured Caco‐2 intestinal epithelial cells to examine the intracellular processes involved in extracellular Ca++ modulation of intestinal epithelial monolayer TJ barrier. Incubation of the filter‐grown Caco‐2 intestinal monolayers in Ca++‐free solution (CFS), consisting of modified Krebs‐buffer solution containing 0 mM Ca++ and 1 mM EGTA, resulted in a rapid drop in Caco‐2 epithelial resistance and increase in epithelial permeability to paracellular markers mannitol and inulin, indicating an increase in TJ permeability. The increase in Caco‐2 TJ permeability was rapidly reversed by the re‐introduction of Ca++ (1.8 mM) into the incubation medium. The CFS‐induced increase in Caco‐2 TJ permeability was associated with separation of the cytoplasmic and transmembrane TJ proteins, ZO‐1 and occludin, and formation of large intercellular openings between the adjoining cells. The CFS‐induced modulation of TJ barrier was associated with activation of myosin light chain kinase (MLCK) activity and centripetal retraction of peri‐junctional actin and myosin filaments. The inhibition of CFS‐induced activation of Caco‐2 MLCK with MLCK inhibitor (ML‐7) prevented the CFS‐induced retraction of actin and myosin filaments and the subsequent alteration of TJ barrier function and structure. Our results suggested that the CFS‐induced alteration of TJ proteins and functional increase in TJ permeability was mediated by Caco‐2 MLCK activation and the resultant contraction of the peri‐junctionally located actin‐myosin filaments. Consistent with the role of MLCK in this process, selected inhibitors of Mg++‐myosin ATPase and metabolic energy, but not protein synthesis inhibitors, also prevented the CFS‐induced retraction of actin and myosin filaments and the subsequent increase in TJ permeability. In conclusion, our results indicate that extracellular Ca++ is crucial for the maintenance of intestinal epithelial TJ barrier function. The removal of extracellular Ca++ from the incubation medium causes activation of Caco‐2 MLCK, which in turn leads to an increase in intestinal monolayer TJ permeability. Microsc. Res. Tech. 51:156–168, 2000.

Tumor antigen precursor protein profiles of adult and pediatric brain tumors identify potential targets for immunotherapy

Objectives We evaluated and compared tumor antigen precursor protein (TAPP) profiles in adult and pediatric brain tumors of 31 genes related to tumor associated antigens (TAA) for possible use in immunotherapy. Antigens were selected based on their potential to stimulate T cell responses against tumors of neuroectodermal origin. Methods Thirty-seven brain tumor specimens from 11 adult and 26 pediatric patients were analyzed by quantitative real-time PCR for the relative expression of 31 TAPP mRNAs. The age range of adults (4F:7M) was 27–77xa0years (median 51.5xa0±xa014.5xa0years) and for pediatrics (12F:14M) was 0.9–19xa0years (median 8.3xa0±xa05.5xa0years). Histological diagnoses consisted of 16 glioblastomas, 4 low grade astrocytomas, 10 juvenile pilocytic astrocytomas, and 7 ependymomas. Results The adult gliomas expressed 94% (29 of 31) of the TAPP mRNAs evaluated compared with pediatric brain tumors that expressed 55–74% of the TAPP mRNAs, dependent on tumor histological subtype. Four types of TAPP expression patterns were observed: (1) equal expression among adult and pediatric cases, (2) greater expression in adult than pediatric cases, (3) expression restricted to adult GBM and (4) a random distribution. The pediatric brain tumors lacked expression of some genes associated with engendering tumor survival, such as hTert and Survivin. Conclusions The potential TAA targets identified from the TAPP profiles of 31 genes associated with adult and pediatric brain tumors may help investigators select specific target antigens for developing dendritic cell- or peptide-based vaccines or T cell-based immunotherapeutic approaches against brain tumors.

Human monocytes kill M-CSF-expressing glioma cells by BK channel activation.

In this study, human monocytes/macrophages were observed to kill human U251 glioma cells expressing membrane macrophage colony-stimulating factor (mM-CSF) via a swelling and vacuolization process called paraptosis. Human monocytes responded to the mM-CSF-transduced U251 glioma cells, but not to viral vector control U251 glioma cells (U251-VV), by producing a respiratory burst within 20u2009min. Using patch clamp techniques, functional big potassium (BK) channels were observed on the membrane of the U251 glioma cell. It has been previously reported that oxygen indirectly regulates BK channel function. In this study, it was demonstrated that prolonged BK channel activation in response to the respiratory burst induced by monocytes initiates paraptosis in selected glioma cells. Forced BK channel opening within the glioma cells by BK channel activators (phloretin or pimaric acid) induced U251 glioma cell swelling and vacuolization occurred within 30u2009min. U251 glioma cell cytotoxicity, induced by using BK channel activators, required between 8 and 12u2009h. Swelling and vacuolization induced by phloretin and pimaric acid was prevented by iberiotoxin, a specific BK channel inhibitor. Confocal fluorescence microscopy demonstrated BK channels co-localized with the endoplasmic reticulum and mitochondria, the two targeted organelles affected in paraptosis. Iberiotoxin prevented monocytes from producing death in mM-CSF-expressing U251glioma cells in a 24u2009h assay. This study demonstrates a novel mechanism whereby monocytes can induce paraptosis via the disruption of internal potassium ion homeostasis.

Antiangiogenic Drugs Synergize with a Membrane Macrophage Colony-Stimulating Factor-Based Tumor Vaccine to Therapeutically Treat Rats with an Established Malignant Intracranial Glioma

Combining a T9/9L glioma vaccine, expressing the membrane form of M-CSF, with a systemic antiangiogenic drug-based therapy theoretically targeted toward growth factor receptors within the tumor’s vasculature successfully treated >90% of the rats bearing 7-day-old intracranial T9/9L gliomas. The antiangiogenic drugs included (Z)-3-[4-(dimethylamino)benzylidenyl]indolin-2-one (a platelet-derived growth factor receptor β and a fibroblast growth factor receptor 1 kinase inhibitor) and oxindole (a vascular endothelial growth factor receptor 2 kinase inhibitor). A total of 20–40% of the animals treated with the antiangiogenic drugs alone survived, while all nontreated controls and tumor vaccine-treated rats died within 40 days. In vitro, these drugs inhibited endothelial cells from proliferating in response to the angiogenic factors produced by T9/9L glioma cells and prevented endothelial cell tubulogenesis. FITC-labeled tomato lectin staining demonstrated fewer and constricted blood vessels within the intracranial tumor after drug therapy. Magnetic resonance imaging demonstrated that the intracranial T9 glioma grew much slower in the presence of these antiangiogenic drugs. These drugs did not affect in vitro glioma cell growth nor T cell mitogenesis. Histological analysis revealed that the tumor destruction occurred at the margins of the tumor, where there was a heavy lymphocytic infiltrate. Real-time PCR showed more IL-2-specific mRNA was present within the gliomas in the vaccinated rats treated with the drugs. Animals that rejected the established T9/9L glioma by the combination therapy proved immune against an intracranial rechallenge by T9/9L glioma, but showed no resistance to an unrelated MADB106 breast cancer.

New mechanistic explanation for the localization of ulcers in the rat duodenum: Role of iron and selective uptake of cysteamine

Cysteamine, a coenzyme A metabolite, induces duodenal ulcers in rodents. Our recent studies showed that ulcer formation was aggravated by iron overload and diminished in iron deficiency. We hypothesized that cysteamine is selectively taken up in the duodenal mucosa, where iron absorption primarily occurs, and is transported by a carrier-mediated process. Here we report that cysteamine administration in rats leads to cysteamine accumulation in the proximal duodenum, where the highest concentration of iron in the gastrointestinal tract is found. In vitro, iron loading of intestinal epithelial cells (IEC-6) accelerated reactive oxygen species (ROS) production and increased [(14)C]cysteamine uptake. [(14)C]Cysteamine uptake by isolated gastrointestinal mucosal cells and by IEC-6 was pH-dependent and inhibited by unlabeled cysteamine. The uptake of [(14)C]cysteamine by IEC-6 was Na(+)-independent, saturable, inhibited by structural analogs, H(2)-histamine receptor antagonists, and organic cation transporter (OCT) inhibitors. OCT1 mRNA was markedly expressed in the rat duodenum and in IEC-6, and transfection of IEC-6 with OCT1 siRNA decreased OCT1 mRNA expression and inhibited [(14)C]cysteamine uptake. Cysteamine-induced duodenal ulcers were decreased in OCT1/2 knockout mice. These studies provide new insights into the mechanism of cysteamine absorption and demonstrate that intracellular iron plays a critical role in cysteamine uptake and in experimental duodenal ulcerogenesis.

Basic fibroblast growth factor stimulates repair of wounded hepatocyte monolayer: modulatory role of protein kinase A and extracellular matrix.

The two important repair mechanisms after hepatocyte injury are proliferation and migration of the nearby healthy hepatocytes. Although previous studies have shown that basic fibroblast growth factor (bFGF) levels are markedly elevated after liver injury, the role of bFGF in the repair of the wounded hepatocytes is not well understood. The aim of this study was to delineate the role of bFGF in the repair of the wounded hepatocyte monolayers. Specifically, we examined the role of bFGF in cellular proliferation and migration of hepatocytes with an in vitro wound model. Standardized excisional wounds were created in clone 9 rat hepatocyte monolayers by a razor blade, and the extent of epithelial proliferation and migration was measured. After wound formation, bFGF (30 ng/mL) significantly stimulated proliferation of hepatocytes at the wound margin. bFGF also stimulated the migration of hepatocytes at the wound front. bFGF stimulation of hepatocyte migration correlated with increased formation of actin stress fibers and bFGF-receptor protein level. The bFGF stimulation of hepatocyte migration was abolished by various protein kinase A activating agents including 3-isobutyl-1-methylxanthine, 8-bromoadenosine-3, 5-cyclic monophosphate, forskolin, and cholera toxin. In addition, protein kinase A activating agents almost completely prevented bFGF-induced actin stress fiber formation in the cells at the wound front. Varying the basement membrane composition of the extracellular matrix had a selective enhancing effect on the basal rates of hepatocyte migration (collagen IV > or = laminin > collagen I > fibronectin > control (plastic)). bFGF treatment resulted in a similar additive increase in hepatocyte migration across all coated surfaces studied. We conclude that bFGF promotes hepatocyte wound repair by stimulating both proliferation and migration of the hepatocyte at the margin of the wound.

Interaction of Hepatocyte Growth Factor and Non-Steroidal Anti-Inflammatory Drugs during Gastric Epithelial Wound Healing

Background/Aims: Expression of the hepatocyte growth factor (HGF) and cyclooxygenase-2 (COX-2) is upregulated at the margins of healing gastric ulcers. We investigated in vitrothe interference of HGF, the selective COX-2 inhibitor NS-398 and the nonselective COX inhibitor indomethacin with gastric epithelial wound healing and actin microfilament (actin-MF) formation. Methods: Standardized gastric epithelial wounds, created in confluent RGM1 rat cell monolayers were treated with: HGF (10 ng/ml), NS-398 (1–100 µM) or indomethacin (0.01– 0.5 mM). The areas of re-epithelialization and cell proliferation were measured 24 h after wounding. Actin-MFs were labeled with fluorescein-conjugated phalloidin and their distribution was examined using a Nikon epifluorescence microscope. Results: HGF caused a significant increase in gastric monolayer wound re-epithelialization and this was not affected by mitomycin C. Both indomethacin and NS-398 inhibited HGF-stimulated re-epithelialization, but the basal wound re-epithelialization rate and cell proliferation was only significantly inhibited by indomethacin. HGF triggered actin stress fiber formation which was inhibited by both indomethacin and NS-398, but only indomethacin interfered with actin-MF formation at the baseline condition. Conclusions: HGF significantly increased gastric wound re-epithelialization by activating cell migration which may be mediated by the COX-2 pathway.

Using REMBRANDT to Paint in the Details of Glioma Biology: Applications for Future Immunotherapy

Microarray technology developed in the late 1990’s allows one to simultaneously analyze the entire transcriptome of a given population of cells at a single time. It is essentially a microchip with each spot containing about a picogram of DNA immobilized at defined locations. This DNA captures mRNA or cDNA from defined cell populations (tissue, cell line, etc) in a quantitative manner. This technique allows complex patterns of global gene expressions to be quickly and easily detected between two specimens. Microarrays were initially used to describe cell cycle patterns of Saccharomyces cerevisiae [1]. Its powerful potential was quickly recognized and applied towards cancer. The first successful application of this technology was to show that diffuse large cell B lymphomas had two different subtypes. One subset of this cancer was easily treatable with chemotherapy; whereas, the second phenotype required a more aggressive therapy [2].

Cytotoxic T cell immunity against the non-immunogenic, murine, hepatocellular carcinoma Hepa1-6 is directed towards the novel alternative form of macrophage colony stimulating factor

Mouse Hepa1-6 hepatocellular carcinoma (HCC) cells were transduced with the membrane form of macrophage colony stimulating factor (mM-CSF). When mM-CSF transduced Hepa1-6 cells were injected subcutaneously into mice, these cells did not form tumors. The spleens of these immunized mice contained cytotoxic CD8+ T lymphocytes (CTL) that killed the unmodified Hepa1-6 cells. We show that the alternative form of macrophage colony stimulating factor (altM-CSF) induced CTL-mediated immunity against Hepa1-6 cells. AltM-CSF is restricted to the H-2D(b) allele. CTLs killed RMA-S cells loaded with exogenous altM-CSF peptide. Vaccination of mice with dendritic cells pulsed with the altM-CSF peptide stimulated anti-Hepa1-6 CTLs. Hyper-immunization of mice with mM-CSF Hepa1-6 cells showed inflammation of the liver and kidneys. Although altM-CSF was expressed within liver and kidney cells, its intensity was lower than Hepa1-6 cells. AltM-CSF was detected within the human HepG2 cell line. These studies suggest that altM-CSF may be a tumor antigen for HCC.