Deliberate Microbial Infection Research Reveals Limitations to Current Safety Protections of Healthy Human Subjects.

Here we identify approximately 40,000 healthy human volunteers who were intentionally exposed to infectious pathogens in clinical research studies dating from late World War II to the early 2000s. Microbial challenge experiments continue today under contemporary human subject research requirements. In fact, we estimated 4,000 additional volunteers who were experimentally infected between 2010 and the present day.

Improving the Proteomic Analysis of Archival Tissue by Using Pressure-Assisted Protein Extraction: A Mechanistic Approach.

Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a valuable resource for the retrospective study of disease progression and response to therapy. However, the proteomic analysis of FFPE tissues has been hampered by formaldehyde-induced protein modifications, which reduce protein extraction efficiency and may lead to protein misidentification. Here, we demonstrate the use of heat augmented with high hydrostatic pressure (40,000 psi) as a novel method for the recovery of intact proteins from FFPE tissue.

An ultra-sensitive immunoassay for quantifying biomarkers in breast tumor tissue.

Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) have been validated at the highest level of evidence as clinical biomarkers of prognosis in breast cancer. The American Society of Clinical Oncology recommends using uPA and PAI-1 levels in breast tumors for deciding whether patients with newly diagnosed node-negative breast cancer can forgo adjuvant chemotherapy. The sole validated method for quantifying uPA and PAI-1 levels in breast tumor tissue is a colorimetric ELISA assay that takes 3 days to complete and requires 100-300 mg of fresh or frozen tissue.

Toward improving the proteomic analysis of formalin-fixed, paraffin-embedded tissue.

Archival formalin-fixed, paraffin-embedded (FFPE) tissue and their associated diagnostic records represent an invaluable source of retrospective proteomic information on diseases for which the clinical outcome and response to treatment are known. However, analysis of archival FFPE tissues by high-throughput proteomic methods has been hindered by the adverse effects of formaldehyde fixation and subsequent tissue histology. This review examines recent methodological advances for extracting proteins from FFPE tissue suitable for proteomic analysis.

Complete solubilization of formalin-fixed, paraffin-embedded tissue may improve proteomic studies.

Tissue-based proteomic approaches (tissue proteomics) are essential for discovering and evaluating biomarkers for personalized medicine. In any proteomics study, the most critical issue is sample extraction and preparation. This problem is especially difficult when recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissue sections.

Immunoliposome-PCR: a generic ultrasensitive quantitative antigen detection system.

Background: The accurate quantification of antigens at low concentrations over a wide dynamic range is needed for identifying biomarkers associated with disease and detecting protein interactions in high-throughput microarrays used in proteomics. Here we report the development of an ultrasensitive quantitative assay format called immunoliposome polymerase chain reaction (ILPCR) that fulfills these requirements. This method uses a liposome, with reporter DNA encapsulated inside and biotin-labeled polyethylene glycol (PEG) phospholipid conjugates incorporated into the outer surface of the liposome, as a detection reagent.

Pressure-assisted protein extraction: a novel method for recovering proteins from archival tissue for proteomic analysis.

Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a valuable resource for the retrospective study of disease progression and response to therapy. However, the proteomic analysis of FFPE tissues has been hampered by formaldehyde-induced protein modifications, which reduce protein extraction efficiency and may lead to protein misidentification. Here, we demonstrate the use of heat augmented with high hydrostatic pressure (40,000 psi) as a novel method for the recovery of intact proteins from FFPE mouse liver.

Novel mineral contrast agent for magnetic resonance studies of bone implants grown on a chick chorioallantoic membrane.

Magnetic resonance imaging (MRI) studies of tissue engineered constructs prior to implantation clearly demonstrate the utility of the MRI technique for studying the bone formation process. To test the utility of our MRI protocols for explant studies, we present a novel test platform in which osteoblast-seeded scaffolds were implanted on the chorioallantoic membrane of a chick embryo. Scaffolds from the following experimental groups were examined by high-resolution MRI: (a) cell-seeded implanted scaffolds (CIM), (b) unseeded implanted scaffolds (UCIM), (c) cell-seeded scaffolds in static culture (CIV) and (d) unseeded scaffolds in static culture (UCIV).

Paraffin embedding contributes to RNA aggregation, reduced RNA yield, and low RNA quality.

The RNA isolated from FFPE tissues is of poor quality and quantity. Other studies have indicated that formaldehyde fixation or the duration of storage of tissue blocks accounted for RNA damage. Herein we report a third source of harm to RNA: embedding in warm paraffin.

The effect of formaldehyde fixation on RNA: optimization of formaldehyde adduct removal.

Formalin-fixed, paraffin-embedded tissues generally provide low yields of extractable RNA that exhibit both covalent modification of nucleic acid bases and strand cleavage. This frustrates efforts to perform retrospective analyses of gene expression using archival tissue specimens. A variety of conditions have been reported to demodify formaldehyde-fixed RNA in different model systems.

Antigen retrieval causes protein unfolding: evidence for a linear epitope model of recovered immunoreactivity.

Antigen retrieval (AR), in which formalin-fixed paraffin-embedded tissue sections are briefly heated in buffers at high temperature, often greatly improves immunohistochemical staining. An important unresolved question regarding AR is how formalin treatment affects the conformation of protein epitopes and how heating unmasks these epitopes for subsequent antibody binding. The objective of the current study was to use model proteins to determine the effect of formalin treatment on protein conformation and thermal stability in relation to the mechanism of AR.

Protein mass spectrometry applications on FFPE tissue sections.

Formalin-fixed, paraffin-embedded (FFPE) tissue archives and their associated diagnostic records represent an invaluable source of proteomic information on diseases where the patient outcomes are already known. Over the last few years, advances in methodology have made it possible to recover peptides from FFPE tissues that yield a reasonable representation of the proteins recovered from identical fresh or frozen specimens. These new methods, based largely upon heat-induced antigen retrieval techniques borrowed from immunohistochemistry, have developed sufficiently to allow at least a qualitative analysis of the proteome of FFPE archival tissues.

Regulatory and ethical issues on the utilization of FFPE tissues in research.

Formalin-fixed, paraffin-embedded (FFPE) archival tissues and their associated diagnostic records represent an invaluable source of information on diseases where the patient outcomes are already known. Older archives contain many unique FFPE tissue specimens that would be impossible to replicate today due to changes in medical practice and technology. Unfortunately, there is no single regulatory or bioethical standard that covers research with FFPE tissue specimens.

Hidden malignant cells within leukocyte aggregates: seeds for invasive and metastatic cancer?

Background: Our previous studies revealed that leukocyte infiltration into aged or injured myoepithelial cell layers is a key trigger for breast tumor invasion and metastasis. Our current study further assessed the possibility that leukocyte aggregates may harbor detached individual tumor cell or clusters of tumor cells.

Materials And Methods: Tissue sections from patients with pregnancy-associated breast cancer (PABC) and controls were subjected to morphological and immunohistochemical assessment with a panel of leukocyte and tumor cell related markers.

Elevated pressure improves the extraction and identification of proteins recovered from formalin-fixed, paraffin-embedded tissue surrogates.

Background: Proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing.

Elevated Pressure Improves the Rate of Formalin Penetration while Preserving Tissue Morphology.

Formaldehyde fixation and paraffin-embedding remains the most widely used technique for processing cancer tissue specimens for pathologic examination, the study of tissue morphology, and archival preservation. However, formaldehyde penetration and fixation is a slow process, requiring a minimum of 15 hr for routine processing of pathology samples. Routinely fixed samples often have a well-fixed outer rim, with a poorly-fixed inner core of tissue.

The liposome PCR assay is more sensitive than the Vibrio cholerae enterotoxin and Escherichia coli heat-labile enterotoxin reversed passive latex agglutination test at detecting cholera toxin in feces and water.

Practical detection of cholera toxin (CT) by a liposome PCR (LPCR) immunoassay was compared to that of an established V. cholerae enterotoxin and Escherichia coli heat-labile enterotoxin reversed passive latex agglutination (VET-RPLA) assay. LPCR detected CT in the range of 10 pg/ml to 100 ng/ml in simulated feces and environmental water.

Breast cancer heterogeneity: mechanisms, proofs, and implications.

Human breast cancer represents a group of highly heterogeneous lesions consisting of about 20 morphologically distinct subtypes with substantially different molecular and/or biochemical signatures, clinical courses, and prognoses. This study analyzed the possible correlation between the morphological presentations of breast cancer and two hypothesized models of carcinogenesis, in order to identify the intrinsic mechanism(s) and clinical implications of breast cancer heterogeneity.

Increased invasiveness and aggressiveness in breast epithelia with cytoplasmic p63 expression.

Our previous studies revealed that pregnancy associated breast cancer (PABC) had significantly reduced nuclear p63 expression in myoepithelia, while intense cytoplasmic p63 expression in associated epithelia. Our current study assessed these epithelia using immunohistochemistry with a panel of aggressiveness and invasiveness related markers and comparative genomic hybridization (array-CGH) with over 30,000 DNA probes. These epithelia showed several unique alterations, including (1) immunohistochemical and morphological resemblance to invasive cancer, (2) significant gain in copy numbers of DNA coding genes for morphogenesis, angiogenesis, and metastasis, and (3) significant loss in copy numbers of DNA coding genes for tumor suppressors, cell adhesion, and macromolecular complex assembly or intra-cellular trafficking.

Modeling formalin fixation and histological processing with ribonuclease A: effects of ethanol dehydration on reversal of formaldehyde cross-links.

Understanding the chemistry of protein modification by formaldehyde fixation and subsequent tissue processing is central to developing improved methods for antigen retrieval in immunohistochemistry and for recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissues for proteomic analysis. Our initial studies of single proteins, such as bovine pancreatic ribonuclease A (RNase A), in 10% buffered formalin solution revealed that upon removal of excess formaldehyde, monomeric RNase A exhibiting normal immunoreactivity could be recovered by heating at 60 degrees C for 30 min at pH 4. We next studied tissue surrogates, which are gelatin-like plugs of fixed proteins that have sufficient physical integrity to be processed using normal tissue histology.

Magnetic resonance microscopy of collagen mineralization.

A model mineralizing system was subjected to magnetic resonance microscopy to investigate how water proton transverse (T(2)) relaxation times and magnetization transfer ratios can be applied to monitor collagen mineralization. In our model system, a collagen sponge was mineralized with polymer-stabilized amorphous calcium carbonate. The lower hydration and water proton T(2) values of collagen sponges during the initial mineralization phase were attributed to the replacement of the water within the collagen fibrils by amorphous calcium carbonate.

Elevated hydrostatic pressure promotes protein recovery from formalin-fixed, paraffin-embedded tissue surrogates.

High-throughput proteomic studies on formalin-fixed, paraffin-embedded (FFPE) tissues have been hampered by inefficient methods to extract proteins from archival tissue and by an incomplete knowledge of formaldehyde-induced modifications to proteins. We previously reported a method for the formation of 'tissue surrogates' as a model to study formalin fixation, histochemical processing, and protein retrieval from FFPE tissues. In this study, we demonstrate the use of high hydrostatic pressure as a method for efficient protein recovery from FFPE tissue surrogates.