Biomedical subjects

Robert F Bonner

Publications and source records attributed to Robert F Bonner.

11 recordsLinked to original sources

PubMed2006

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA.

BACKGROUND: Gene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays. RESULTS: The MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases. CONCLUSION: RNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts.

Animals↗

PubMed2006

Tumor-associated endothelial cells display GSTP1 and RARbeta2 promoter methylation in human prostate cancer.

BACKGROUND: A functional blood supply is essential for tumor growth and proliferation. However, the mechanism of blood vessel recruitment to the tumor is still poorly understood. Ideally, a thorough molecular assessment of blood vessel cells would be critical in our comprehension of this process. Yet, to date, there is little known about the molecular makeup of the endothelial cells of tumor-associated blood vessels, due in part to the difficulty of isolating a pure population of endothelial cells from the heterogeneous tissue environment. METHODS: Here we describe the use of a recently developed technique, Expression Microdissection, to isolate endothelial cells from the tumor microenvironment. The methylation status of the dissected samples was evaluated for GSTP1 and RARbeta2 promoters via the QMS-PCR method. RESULTS: Comparing GSTP1 and RARbeta2 promoter methylation data, we show that 100% and 88% methylation is detected, respectively, in the tumor areas, both in epithelium and endothelium. Little to no methylation is observed in non-tumor tissue areas. CONCLUSION: We applied an accurate microdissection technique to isolate endothelial cells from tissues, enabling DNA analysis such as promoter methylation status. The observations suggest that epigenetic alterations may play a role in determining the phenotype of tumor-associated vasculature.

Journal Article↗

PubMed2005

Assessment of the correlation of platelet morphology with in vivo recovery and survival.

BACKGROUND: There is continuing interest in the development of in vitro tests evaluating the in vivo function, recovery, and survival of platelets stored for transfusion. A recent forum concluded that no completely reliable test exists, although discoid morphology indicates a platelet's good health. We evaluated a novel device, the NAPSAC (Noninvasive Assessment of Platelet Shape and Concentration), designed to determine noninvasively the proportion of discoid platelets in a stored concentrate, as well as platelet concentration. STUDY DESIGN AND METHODS: Twenty-eight plateletapheresis concentrates stored 24 hours in PL-146 were evaluated. Percent discoid platelet results were correlated with radiolabeled autologous recovery and survival performed using 111Indium oxyquinoline and calculated using linear (L) and multiple-hit (M) models. pH of 8 concentrates was raised at the end of storage with 6N NaOH. Platelet concentration measured by NAPSAC and Coulter Thrombocounter C was compared in 256 plateletapheresis products. RESULTS: Percent discoid platelets at 24 hours did not correlate significantly with platelet recovery or survival (recovery L = 0.29, M = 0.28; survival L = 0.16, M = 0.03). Raising the pH (mean 6.38 to 6.94) resulted in a significant increase in percent discoid platelets (21% to 41%). Platelet concentration values for both methods studied were linearly correlated with a slope of 1.01 +/- 0.03, r = 0.81. CONCLUSION: Percent discoid platelets was not predictive of posttransfusion platelet recovery or survival. The results suggest that non-discoid platelets may survive posttransfusion and even revert to discoid shape, since raising the pH approximately doubled the percent of discoid platelets. The NAPSAC was shown to be a reliable instrument for noninvasively determining platelet concentration in PL-146 concentrates.

Blood Platelets↗

PubMed2004

Selective gene expression in magnocellular neurons in rat supraoptic nucleus.

Oxytocin- and vasopressin-producing magnocellular neurons (MCNs) of the hypothalamo-neurohypophysial system are the only neuronal phenotypes present in the rat supraoptic nucleus (SON). Laser microdissection of the SON, extraction and T7-based amplification of its RNAs, and analysis of the resulting cDNAs by hybridization on a 35, 319 element DNA microarray have provided a detailed composite view of the gene expression profile of the MCNs. The genes expressed in the SON were compared with those expressed in a reference tissue consisting of total hypothalamus, and this "expression ratio" indicated which genes were preferentially expressed in the SON. Of the 26,000 unique genes on the array, 1385 were found to be expressed in the SON at levels more than two times greater than in the hypothalamus as a whole. Of these, 123 were expressed > or =3.4-fold higher in the SON versus hypothalamus. Most of these preferentially expressed genes were not previously known to be expressed in the MCNs. Quantitative and double-label in situ hybridization histochemistry was used selectively to confirm a number of these microarray observations and to evaluate the osmotic regulation and cell-specific expression of these genes, respectively.

Animals↗

PubMed2004

Assessment of gene expression in head and neck carcinoma using laser capture microdissection and real-time reverse transcription polymerase chain reaction.

OBJECTIVES: To quantify gene expression in tumor cells from human head and neck squamous cell carcinomas (HNSCC) using laser capture microdissection (LCM). STUDY DESIGN: Histopathologically identified HNSCC cells were microdissected from frozen sections, RNA was isolated, and vascular endothelial growth factor (VEGF) gene expression was measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR). MATERIALS AND METHODS: Two human HNSCC tumor samples and matched normal mucosal biopsies and five human xenograft tumor specimens were harvested, embedded, and frozen in OCT. The frozen tumors were sectioned to 8 to 10 mum in thickness, and hematoxylin-eosin (H&E) staining was performed before LCM. An estimated 2,000 to 3,000 tumor cells were microdissected from frozen sections and processed for RNA isolation. mRNA for VEGF was analyzed by real time RT-PCR (TaqMan) with commercially available primers and probes. RESULTS: Two thousand to 3000 cells were necessary to obtain a suitable quantity of RNA for subsequent gene expression study by real-time RT-PCR. The gene expression of VEGF, a major tumor angiogenic factor, was tested in microdissected HNSCC and compared with uninvolved normal mucosal controls. A greater than seven-fold increase of VEGF expression in tumor specimens versus mucosal controls was observed. CONCLUSIONS: LCM is a novel sample conserving technique that allows the precise selection of tumor cells from a heterogeneous architecture. The combination of LCM and real-time RT-PCR appears particularly efficacious for studying HNSCC molecular pathogenesis and identifying tissue-specific biomarkers.

Biopsy, Needle↗

PubMed2004

Expression microdissection: operator-independent retrieval of cells for molecular profiling.

Tissue microdissection is an important method for the study of disease states. However, it is difficult to perform high-throughput molecular analysis with current techniques. We describe here a prototype version of a novel technique (expression microdissection) that allows for the procurement of desired cells via molecular targeting. Expression microdissection (xMD) offers significant advantages over available methods, including an increase in dissection speed of several orders of magnitude. xMD may become a valuable tool for investigators studying cancer or other disease states in patient specimens and animal models.

Animals↗

PubMed2004

A model of spectral filtering to reduce photochemical damage in age-related macular degeneration.

BACKGROUND/PURPOSE: Cumulative sunlight exposure and cataract surgery are reported risk factors for advanced age-related macular degeneration (AMD). Laboratory studies suggest that accumulation and photochemical reactions of A2E (N-retinylidene-N-retinylethanolamine) and its epoxides, components of lipofuscin, are important in AMD. To relate this data to the clinical setting, we modeled the effects of macular irradiance and spectral filtering on production of A2E and reactive oxygen intermediates (ROIs) in pseudophakic eyes with a clear or "yellow" intraocular lens (IOL) and in phakic eyes. METHODS: We calculated relative changes of macular irradiance as a function of light (390 to 700 nm) intensity, pupil size, age, and lens status, and modeled resulting all-trans-retinal concentration and rates of production of A2E-related photochemicals and photon-induced ROIs in rods and retinal pigment epithelium (RPE). We compared these photoproducts following cataract surgery and IOL implantation with and without spectral sunglasses to normal age-related nuclear sclerotic lens changes. RESULTS: Following cataract and IOL surgery, all-trans-retinal and lipofuscin photochemistry would theoretically increase average generation of 1) A2E-related photochemicals, 2) ROI in rods and 3) ROI in RPE, respectively, 2.6-, 15- and 6.6-fold with a clear IOL, and 2.1-, 4.1- and 2.6 fold with a yellow IOL, but decrease approximately 30-, approximately 20- and 4-fold with a vermillion filter sunglass and clear IOL compared to an average 70 year old phakic eye. CONCLUSION: Sunglasses that strongly decrease both deep blue light and rod photobleaching, while preserving photopic sensitivity and color perception, would provide upstream protection from potential photochemical damage in subjects at risk for AMD progression after cataract surgery.

Aged↗

PubMed2003

Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks.

Histone H2AX is rapidly phosphorylated in the chromatin micro-environment surrounding a DNA double-strand break (DSB). Although H2AX deficiency is not detrimental to life, H2AX is required for the accumulation of numerous essential proteins into irradiation induced foci (IRIF). However, the relationship between IRIF formation, H2AX phosphorylation (gamma-H2AX) and the detection of DNA damage is unclear. Here, we show that the migration of repair and signalling proteins to DSBs is not abrogated in H2AX(-/-) cells, or in H2AX-deficient cells that have been reconstituted with H2AX mutants that eliminate phosphorylation. Despite their initial recruitment to DSBs, numerous factors, including Nbs1, 53BP1 and Brca1, subsequently fail to form IRIF. We propose that gamma-H2AX does not constitute the primary signal required for the redistribution of repair complexes to damaged chromatin, but may function to concentrate proteins in the vicinity of DNA lesions. The differential requirements for factor recruitment to DSBs and sequestration into IRIF may explain why essential regulatory pathways controlling the ability of cells to respond to DNA damage are not abolished in the absence of H2AX.

Animals↗

PubMed2002

A comparison of hydraulic and laser capture microdissection methods for collection of single B cells, PCR, and sequencing of antibody VDJ.

During the development of B lymphocytes, a series of gene rearrangements assemble the sequences that encode immunoglobulin heavy and light chains (VDJ). Earlier studies of VDJ sequence diversification during expansion of cells in splenic or appendix germinal centers used hydraulic micromanipulation (HM) to collect single B cells for PCR amplification of rearranged antibody heavy and light chain genes. PCR products were directly sequenced without a cloning step. Hydraulic micromanipulation is a very tedious method. Once capability to collect single cells by laser capture microdissection (LCM) was developed, we modified previous tissue staining and fixation methods so that we could collect cells from a given stained tissue section by HM and LCM and directly compare our success rates using these two methods. Cells were alkaline lysed and after two rounds of nested PCR products were recovered and directly sequenced. Because each rearrangement of genomic DNA that occurs to form the immunoglobulin heavy-chain-encoding sequence in developing B cells is unique, this system allowed us to verify our success rate in recovering single lymphocytes from tissue sections and amplifying a single allele. The methods developed have now made LCM an efficient alternative to HM for the collection of single B cells.

Animals↗

PubMed2002

A preservation method that allows recovery of intact RNA from tissues dissected by laser capture microdissection.

We report a novel method for preparing samples for laser capture microdissection. The procedure described here permits extraction of intact RNA while preserving morphology, thus being suitable both for identification of specific cells and for analysis of their gene expression. The method is applicable to both mouse embryos and human tumors and may improve the preparation of cDNA libraries from specific cell types without interfering with histological diagnosis.

Animals↗

PubMed2002

Post-analysis follow-up and validation of microarray experiments.

Measurement of gene-expression profiles using microarray technology is becoming increasingly popular among the biomedical research community. Although there has been great progress in this field, investigators are still confronted with a difficult question after completing their experiments: how to validate the large data sets that are generated? This review summarizes current approaches to verifying global expression results, discusses the caveats that must be considered, and describes some methods that are being developed to address outstanding problems.

Animals↗