Image analysis algorithms for immunohistochemical assessment of cell death events and fibrosis in tissue sections.

Cell death is of broad physiological and pathological importance, making quantification of biochemical events associated with cell demise a high priority for experimental pathology. Fibrosis is a common consequence of tissue injury involving necrotic cell death. Using tissue specimens from experimental mouse models of traumatic brain injury, cardiac fibrosis, and cancer, as well as human tumor specimens assembled in tissue microarray (TMA) format, we undertook computer-assisted quantification of specific immunohistochemical and histological parameters that characterize processes associated with cell death. In this study, we demonstrated the utility of image analysis algorithms for color deconvolution, colocalization, and nuclear morphometry to characterize cell death events in tissue specimens: (a) subjected to immunostaining for detecting cleaved caspase-3, cleaved poly(ADP-ribose)-polymerase, cleaved lamin-A, phosphorylated histone H2AX, and Bcl-2; (b) analyzed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay to detect DNA fragmentation; and (c) evaluated with Masson's trichrome staining. We developed novel algorithm-based scoring methods and validated them using TMAs as a high-throughput format. The proposed computer-assisted scoring methods for digital images by brightfield microscopy permit linear quantification of immunohistochemical and histochemical stainings. Examples are provided of digital image analysis performed in automated or semiautomated fashion for successful quantification of molecular events associated with cell death in tissue sections.

Development of diabesity in mice with neuronal deletion of Shp2 tyrosine phosphatase.

Obesity and diabetes, termed "diabesity," are serious health problems that are increasing in frequency. However, the molecular mechanisms and neuronal regulation of these metabolic disorders are not fully understood. We show here that Shp2, a widely expressed Src homology 2-containing Tyr phosphatase, plays a critical role in the adult brain to control food intake, energy balance, and metabolism. Mice with a neuron-specific, conditional Shp2 deletion were generated by crossing a pan-neuronal Cre-line (CRE3) with Shp2(flox/flox) mice. These congenic mice, CRE3/Shp2-KO, developed obesity and diabetes and the associated pathophysiological complications that resemble those encountered in humans, including hyperglycemia, hyperinsulinemia, hyperleptinemia, insulin and leptin resistance, vasculitis, diabetic nephropathy, urinary bladder infections, prostatitis, gastric paresis, and impaired spermatogenesis. This mouse model may help to elucidate the molecular mechanisms that lead to the development of diabesity in humans and provide a tool to study the in vivo complications of uncontrolled diabetes.

Multiple antigen detection (MAD) western blotting.

A variation of ECL immunodetection method permits sequential detection of multiple antigens (MAD) on a single protein blot without stripping previously bound antibodies. Because antibody stripping is not involved, immobilized proteins are not lost from the membrane, which permits multiple sequential reprobings of the same membrane with different primary antibodies (> or = 12) and retention of strong signal intensities for all antibody probings. This procedure utilizes horseradish peroxidase (HRPase)-based detection with both chemiluminescent and colorimetric substrates. Initial incubation of the blot with secondary antibody followed by colorimetric development prior to probing the blot with primary antibodies markedly reduces background intensities in ECL-based detection procedures and permits sequential use of antibodies derived from a single species. By allowing large amounts of data to be obtained from a single blot, MAD immunoblotting has the potential to markedly streamline the work required to compare the expression levels of several proteins within biological samples. This technique could be particularly valuable for analyzing cellular populations that are difficult to isolate in large numbers or clinical specimens where the amount of protein samples is limited or available on a one-time basis.

Bcl-B expression in human epithelial and nonepithelial malignancies.

PURPOSE: Apoptosis plays an important role in neoplastic processes. Bcl-B is an antiapoptotic Bcl-2 family member, which is known to change its phenotype upon binding to Nur77/TR3. The expression pattern of this protein in human malignancies has not been reported. EXPERIMENTAL DESIGN: We investigated Bcl-B expression in normal human tissues and several types of human epithelial and nonepithelial malignancy by immunohistochemistry, correlating results with tumor stage, histologic grade, and patient survival. RESULTS: Bcl-B protein was strongly expressed in all normal plasma cells but found in only 18% of multiple myelomas (n = 133). Bcl-B immunostaining was also present in normal germinal center centroblasts and centrocytes and in approximately half of diffuse large B-cell lymphoma (n = 48) specimens, whereas follicular lymphomas (n = 57) did not contain Bcl-B. In breast (n = 119), prostate (n = 66), gastric (n = 180), and colorectal (n = 106) adenocarcinomas, as well as in non-small cell lung cancers (n = 82), tumor-specific overexpression of Bcl-B was observed. Bcl-B expression was associated with variables of poor prognosis, such as high tumor grade in breast cancer (P = 0.009), microsatellite stability (P = 0.0002), and left-sided anatomic location (P = 0.02) of colorectal cancers, as well as with greater incidence of death from prostate cancer (P = 0.005) and shorter survival of patients with small cell lung cancer (P = 0.009). Conversely, although overexpressed in many gastric cancers, Bcl-B tended to correlate with better outcome (P = 0.01) and more differentiated tumor histology (P < 0.0001). CONCLUSIONS: Tumor-specific alterations in Bcl-B expression may define subsets of nonepithelial and epithelial neoplasms with distinct clinical behaviors.

Tumor-associated alterations in caspase-14 expression in epithelial malignancies.

PURPOSE: Caspase-14 is unique among caspase family proteases in that its proteolytic processing has been principally associated with epithelial cell differentiation rather than apoptosis or inflammation. We investigated caspase-14 expression in several types of human epithelial malignancy by immunohistochemistry, correlating results with stage, histologic grade, and patient survival. EXPERIMENTAL DESIGN: Tumor-associated alterations in caspase-14 expression were observed for cervical, ovarian, breast, gastric, and colon cancers. RESULTS: In cervical (n = 445), ovarian (n = 91), and colon (n = 106) specimens, expression of caspase-14 was significantly reduced in cancers compared with normal epithelium. Decreases in caspase-14 immunopositivity correlated with the histologic progression of cervical cancer (P < 0.0001, ANOVA). In localized gastric cancers, caspase-14 immunostaining was significantly lower in poorly differentiated tumors compared with well-differentiated tumors (P = 0.02, Pearson's chi(2) analysis). Lower caspase-14 expression was associated with advanced clinical stage in ovarian cancer (P = 0.04, ANOVA) and with shorter overall survival among ovarian cancer patients with serous tumors (n = 62) in both univariate (P = 0.005) and multivariate (P = 0.03) analysis. Lower caspase-14 expression correlated with shorter overall survival among patients with T(3)N(0)M(0) stage gastric cancers (n = 94; P = 0.006, log-rank test). In contrast to cervical, ovarian, and colon cancers, caspase-14 expression was increased in ductal carcinoma in situ and invasive cancers compared with normal mammary epithelium (P = 0.001, t test). CONCLUSIONS: The findings reveal tumor-specific alterations in caspase-14 expression and suggest that differences in its expression may define subsets of epithelial cancers with distinct clinical behaviors.

Elevated expression of inhibitor of apoptosis proteins in prostate cancer.

PURPOSE: Inhibitor of apoptosis (IAP) family proteins are suppressors of apoptosis that have been implicated in apoptosis resistance in some cancers. Their expression and relevance to the prognosis of prostate cancer were investigated. EXPERIMENTAL DESIGN: The expression of four members of the IAP family (cellular inhibitor of apoptosis protein 1, cellular inhibitor of apoptosis protein 2, X chromosome-linked IAP, and survivin) was examined by immunohistochemistry and immunoblotting in human prostate cancers and in prostate tissues from transgenic mice expressing SV40 large T antigen under control of a probasin promoter. RESULTS: Tumor-associated elevations in the levels of all four IAP family members were common in prostate cancers of both humans and mice, suggesting concomitant up-regulation of multiple IAP family proteins. Compared with normal prostatic epithelium, increased IAP expression was often evident even in prostatic intraepithelial neoplasia lesions (carcinoma in situ), suggesting that deregulation of IAP expression occurs early in the pathogenesis of prostate cancer. IAP expression did not correlate with Gleason grade or prostate-specific antigen levels. CONCLUSIONS: The findings demonstrate that tumor- associated elevations in the expression of several IAP family proteins occur as a frequent and early event in the etiology of prostate cancer.

Nonstripping "Rainbow" and Multiple Antigen Detection (MAD) Western Blotting.

A variation of immunoblotting method (the "Rainbow Western"), permits sequential detection of multiple antigens (MAD) on a single protein blot without stripping off prior antibodies. Because no stripping is involved, immobilized proteins are not lost from the membrane, thus allowing for multiple reprobings of the same membrane with different primary antibodies (≥12), retaining strong signal intensities for all sequential antibody probings. The procedure utilizes horseradish peroxidase (HRPase)-based detection with both a chemiluminescent and colorimetric substrate. Initial incubation of the blot with secondary antibody followed by colorimetric development prior to probing the blot with primary antibodies markedly reduces background in ECL-based detection procedures and permits sequential use of antibodies derived from a single species. In the "Rainbow Western," four different HRPase-colorimetric substrates that produce black, brown, red, and green colors are employed sequentially for detection and simultaneous display of four different antigens on the same membrane. By allowing large amounts of data to be obtained from a single blot, the MAD-immunoblotting and Rainbow Western methods have the potential for researchers to compare the expression of several proteins within a single biological sample. Both techniques could be particularly valuable for analysis of cellular populations that are difficult to isolate in large numbers or of clinical specimens where the amounts of protein samples is minute or only available on a one-time basis.

Neuronal deletion of caspase 8 protects against brain injury in mouse models of controlled cortical impact and kainic acid-induced excitotoxicity.

BACKGROUND: Acute brain injury is an important health problem. Given the critical position of caspase 8 at the crossroads of cell death pathways, we generated a new viable mouse line (Ncasp8(-/-)), in which the gene encoding caspase 8 was selectively deleted in neurons by cre-lox system. METHODOLOGY/PRINCIPAL FINDINGS: Caspase 8 deletion reduced rates of neuronal cell death in primary neuronal cultures and in whole brain organotypic coronal slice cultures prepared from 4 and 8 month old mice and cultivated up to 14 days in vitro. Treatments of cultures with recombinant murine TNFα (100 ng/ml) or TRAIL (250 ng/mL) plus cyclohexamide significantly protected neurons against cell death induced by these apoptosis-inducing ligands. A protective role of caspase 8 deletion in vivo was also demonstrated using a controlled cortical impact (CCI) model of traumatic brain injury (TBI) and seizure-induced brain injury caused by kainic acid (KA). Morphometric analyses were performed using digital imaging in conjunction with image analysis algorithms. By employing virtual images of hundreds of brain sections, we were able to perform quantitative morphometry of histological and immunohistochemical staining data in an unbiased manner. In the TBI model, homozygous deletion of caspase 8 resulted in reduced lesion volumes, improved post-injury motor performance, superior learning and memory retention, decreased apoptosis, diminished proteolytic processing of caspases and caspase substrates, and less neuronal degeneration, compared to wild type, homozygous cre, and caspase 8-floxed control mice. In the KA model, Ncasp8(-/-) mice demonstrated superior survival, reduced seizure severity, less apoptosis, and reduced caspase 3 processing. Uninjured aged knockout mice showed improved learning and memory, implicating a possible role for caspase 8 in cognitive decline with aging. CONCLUSIONS: Neuron-specific deletion of caspase 8 reduces brain damage and improves post-traumatic functional outcomes, suggesting an important role for this caspase in pathophysiology of acute brain trauma.