GLUT1 Expression in Cutaneous Sebaceous Lesions Determined by Immunohistochemical Staining Patterns

GLUT1 is a membrane associated carrier protein that functions in the physiologic transport of glucose across cell membranes. Multiple studies have shown an increased GLUT1 expression in various tumor types and a role in cancer prognosis. The aim of this study was to determine whether cutaneous sebaceous lesions have a differential expression of GLUT1 by immunohistochemistry (IHC). GLUT1 IHC was performed on excision specimens of ten cases of sebaceous carcinoma, nine of sebaceoma, ten of sebaceous adenoma, and ten of sebaceous hyperplasia. Intense, diffuse cytoplasmic staining was observed in sebaceous carcinoma. The pattern of GLUT1 staining in sebaceomas and sebaceous adenomas consisted of a gradient of intense cytoplasmic staining in the basaloid cells with a decreased intensity to membranous staining only and absent staining in mature sebaceous cells. In lesions of sebaceous hyperplasia, GLUT1 staining outlined the basal layer of each gland; cytoplasmic staining was minimal to absent. Increased cytoplasmic staining of GLUT1 may correlate with cellular metabolic and proliferative activity. GLUT1 has potential utility in differentiating sebaceous lesions.

Primary telencephalic lymphoma in a horse

A case of T small cell type lymphoma in the brain of a horse is described. A 20-year-old female Crioulo equine showed neurological signs characterized by ataxia, circling and partial loss of smell and sight. During necropsy, a whitish, firm, unencapsulated mass compressing the structures of the nervous tissue was observed, extending from the olfactory bulb to the internal capsule of the right telencephalon. Microscopic examination showed the proliferation of round cells with a small to moderate amount of eosinophilic cytoplasm. Nuclei were centrally located, irregularly round and occasionally cleaved and hyperchromatic. Immunohistochemistry for CD3 showed a moderate diffuse cytoplasmic staining. This is a rare primary central nervous system lymphoma in horses, with few reports in the veterinary literature. Nevertheless, it should be considered as a differential diagnosis in equines with neurological signs.

Detection of the BRAFV600E protein in human colon carcinomas by a mutation-specific antibody.

3576 Background: BRAF encodes a serine-threonine kinase that is a downstream effector of activated RAS. A point mutation (V600E) in BRAF occurs in a subset of colorectal cancers (CRCs) and is associated with adverse outcome and may predict non response to anti-EGFR antibodies. Detection of a BRAFV600E mutation in a CRC with microsatellite instability indicates a sporadic origin and excludes Lynch Syndrome. While BRAFV600E mutation status is determined using a DNA-based assay, antibodies against the BRAFV600E protein have recently been developed. We examined mutant BRAFV600E protein expression and its concordance with mutation status. Methods: Primary stage III colon carcinomas (50 BRAFV600E mutation carriers and 25 wild-type cases) were studied from a completed phase III adjuvant trial comparing FOLFOX +/- cetuximab (NCCTG N0147). In archival resection specimens, immunohistochemistry (IHC) was performed using a pan-BRAF antibody and a V600E mutation-specific antibody raised against an immunogenic synthetic peptide derived from the internal region of the BRAFV600E protein. BRAFV600E mutations in codon 15 were analyzed in extracted DNA using a multiplex, allele specific PCR–based assay. BRAF staining was scored independently by two pathologists blinded to mutation status. Results: In primary colon carcinomas stained with a pan-BRAF antibody, diffuse cytoplasmic staining for BRAF proteins was detected in 74 of 75 carcinomas with one case deemed non-evaluable. Using the mutation-specific BRAFV600E antibody, diffuse cytoplasmic staining was detected in 49 of 74 tumors without appreciable heterogeneity of expression. Among these 49 tumors expressing mutant BRAFV600E proteins, all (100%) were found to carry a BRAFV600E mutation according to a DNA-based assay. In contrast, absent BRAFV600E staining was observed in all 25 tumors that were found to have wild-type copies of BRAFV600E detected using a PCR-based assay. Conclusions: For the detection of mutant BRAFV600E, complete concordance was found between IHC and a DNA-based method in colon carcinomas. This finding supports the use of IHC as a simplified strategy to screen CRCs for mutant BRAFV600E proteins in routine clinical practice to inform clinical decision-making.

Immunohistochemical Study of Retinol-binding Protein in Livers of Polar Bears (Thalarctos maritimus)

Liver tumors of unknown cause have frequently been described in polar bears. Concurrent decrease of vitamin A levels and chronic liver disease are associated with hepatic carcinogenesis in humans. More than 90% of the body's vitamin A is stored in the liver, where it is bound to an intracellular retinol-binding protein (RBP). Therefore, in this retrospective study, RBP was assessed by immunohistochemistry in liver sections of 11 polar bears. Two of these polar bears had hepatocellular carcinoma, four showed other chronic liver changes, and five had normal livers. In normal livers, the cytoplasm stained diffusely positive with intensely staining cytoplasmic granules. RBP staining was evaluated and the abundance of diffuse cytoplasmic staining and intracytoplasmic large granules was determined. All cases with pathologic liver changes had markedly decreased staining intensities for RBP compared with normal livers. The findings of this study suggest that in polar bears, as in humans, vitamin A metabolism may play a role in hepatic carcinogenesis.

Baculovirus p33 Binds Human p53 and Enhances p53-Mediated Apoptosis

ABSTRACT In vertebrates, p53 participates in numerous biological processes including cell cycle regulation, apoptosis, differentiation, and oncogenic transformation. When insect SF-21 cells were infected with a recombinant of the baculovirus Autographa californicanuclear polyhedrosis virus (AcMNPV) overexpressing human p53, p53 formed a stable complex with the product of the AcMNPV orf92, a novel protein p33. The interaction between p53 and p33 was further confirmed by immunoprecipitation studies. When individually expressed in SF-21 cells, human p53 localized mainly in the nucleus whereas baculovirus p33 displayed diffuse cytoplasmic staining and punctuate nuclear staining. However, coexpression of p33 with p53 resulted in exclusive nuclear localization of p33. In both SF-21 and TN-368 cells, p53 expression induced typical features of apoptosis including nuclear condensation and fragmentation, oligonucleosomal ladder formation, cell surface blebbing, and apoptotic body formation. Coexpression of p53 with a baculovirus inhibitor of apoptosis, p35, OpIAP, or CpIAP, blocked apoptosis, whereas coexpression with p33 enhanced p53-mediated apoptosis approximately twofold. Expression of p53 in SF-21 cells stably expressing OpIAP inhibited cell growth in the presence or absence of p33. Thus, human p53 can influence both insect cell growth and death and baculovirus p33 can modulate the death-inducing effects of p53.

Applications of Wide Field Fluorescence and Confocal Reflectance Microscopy to Bone and Cartilage Studies

Bisphosphonate Uptake by Living CellsBisphosphonates are drugs which inhibit bone resorption and are being pursued as therapies for osteoporosis. The molecular mechanisms by which bisphosphonates exert their biological effects are still unclear. It is generally assumed that uptake of these highly polar molecules by cells is very limited. In order to determine whether bisphosphonates are internalized and therefore may act intracellularly, we have examined the extent of their cellular uptake using a fluorescent naphthalene-dicarboxaldehyde (NDA) derivative of alendronate (ALN).The distribution of NDA-ALN was examined in Ros 17/2.8 osteoblast-like osteosarcoma cells and freshly isolated neonatal rat osteoclasts by fluorescence microscopy following 0.5-24 hours of treatment in vitro. Data were collected using a Nikon Diaphot 300 equipped with a 60x planapochromat objective and high N.A. DIC optics. Images captured using a Photometries PXL camera were processed using UIC Metamorph software.Uptake of the bisphosphonate into the cells was rapid and could be detected after 30 minutes of treatment in cells exposed to an external concentration of 10–5M NDA-ALN. Bisphosphonate appeared to accumulate over time within vacuolar, often perinuclear structures similar in appearance to secondary lysosomes (Figure 1). NDA-ALN was retained within cells after washing. These observations are consistent with the uptake of NDA-ALN proceeding via fluid-phase endocytosis. Uptake was greatly reduced when cells were incubated with the bisphosphonate at 20°C rather than 37°C. Freshly isolated osteoclasts also exhibited a noticeable amount of diffuse cytoplasmic staining (Figure 1), possibly indicating the presence of a second path of entry into the cell.

Developmental expression of NOS isoforms in fetal rat lung: implications for transitional circulation and pulmonary angiogenesis

To better understand the role of nitric oxide (NO) in fetal lung development, specifically in the transition of the fetal circulation at birth, we studied the timing of cell-specific expression of NO synthase (NOS) isoforms from formation of lung buds (13th day of gestation) to 7 days postnatal. Expression of NOS was studied using immunohistochemical labeling with antibodies against the three known NOS isoforms and the NADPH diaphorase technique (NADPH-d). Endothelial NOS (eNOS) immunoreactivity was found in the cells of the 14-day fetal lung. As gestation proceeded, the quantity of these immunopositive cells increased, and they coalesced to form an inner (endothelial) layer of pulmonary vessels. This process of angiogenesis marked by eNOS-positive cells was seen from 15 days of gestation to at least 7 days postnatal. A majority of the eNOS immunoreactivity appeared densely in one focal spot in the cytoplasm, indicating that during development the eNOS may be primarily located in a cytoplasmic organelle. Epithelial cells of the rat airway from the same developmental period were positively stained with both brain NOS antibody (bNOS) and NADPH-d at the beginning of 13 days of gestation. Then the intensity of stainings began to decrease and reached the lowest level in the 16-day fetal lung. However, the NOS stainings of the epithelium, especially in small canalicular structures of the airways, began to increase at 18 days of gestation and was dramatically elevated at 20 days of gestation (term is 22 days). Postnatally, NOS in epithelium was decreased in distal airways in conjunction with the formation of alveolar structure. Inducible NOS (iNOS) immunoreactivity was also found in the epithelium of rat lung airways after 16 days of gestation. Unlike the bNOS staining, iNOS immunoreactivity exhibited a pattern of a small dot-like staining within epithelial cytoplasm during gestation and the first day postnatal, then changed to a pattern of diffuse cytoplasmic staining by the 7th postnatal day. This study concludes that 1) expression of three isoforms of NOS is present and regulated during lung development; 2) markedly increased NOS in epithelium near term supports a role for NO in mediating the pulmonary transition from fetal to neonatal life; and 3) eNOS immunohistochemistry serves as an effective marker to follow the process of pulmonary angiogenesis and suggests the concept of in situ formation of endothelial vesicles in developing mesenchyme.

Ileal Lymphoma in Swine

Eleven cases of alimentary lymphoma affecting the ileum were observed among 26 cases of swine lymphoma detected by meat inspection in Kochi, Japan. The ileal lymphomas were located in the Peyer's patches, along with early involvement of regional lymph nodes, and showed a characteristic pattern of follicular invasion leading to diffuse growth. Following the National Cancer Institute Working Formulation, 10 neoplasms were classified as diffuse, large, noncleaved cell lymphomas and one neoplasm was a diffuse, mixed, small to large cell lymphoma. Both types of lymphoma featured numerous intermingled “starry sky” histiocytes. The lymphoma cells tended to infiltrate into the muscular layer of the ileum in an “Indian file” pattern. Two cases also showed transserosal metastasis into the abdomen and leukemic change. The lymphoma cells showed membrane positivity for alkaline phosphatase and diffuse cytoplasmic staining for acid phosphatase and nonspecific esterase. Monoclonal intracytoplasmic immunoglobulins were demonstrated in nine neoplasms (IgM-λ in seven, IgG-λ in one, and IgG-λ in one). In the areas of follicular invasion, an attenuated network of follicular dendritic cells was visualised via an antiserum against the β subunit of S-100 protein. Ultrastructurally, strands of dilatated rough endoplasmic reticulum and scattered or clustered dense bodies were noted. When compared with feline and human alimentary lymphoma, including Burkitt's lymphoma, the present neoplasms possessed distinctive features, such as originating in Peyer's patches, transserosal metastasis, and predominantly large B cell type with IgM-λ type immunoglobulin expression, although some features were similar.

Selective staining of neurons for thick-section intermediate and High-Voltage EM studies

Methods are needed that enable the visualization of selectively stained neurons, neuronal processes and intracellular structures which provide higher resolution than can be obtained with the light microscope. Procedures have been developed using thick sections that allow direct visualization of the three-dimensional structure of the neuron and the visualization of intracellular organelles. Using these methods the geometry of neurons may be accurately measured and this information may then be used to test theoretical predictions about the way in which electrical signals of synaptic origin are processed by the cells. Intermediate and high voltage electron microscopes (HVEM) are well suited to this application because of their high resolution and ability to form images of thick sections. Use of these instruments requires development of selective stains that can produce diffuse cytoplasmic staining of specific cells, cell populations or organelle systems on the basis of their functional properties, or organelle systems.

Keratinocyte transglutaminase in human skin and oral mucosa: cytoplasmic localization and uncoupling of differentiation markers

Expression of keratinocyte transglutaminase, a specific differentiation marker, has been examined by immunogold-silver cytochemistry in human epidermis and oral epithelium, and in oral mucosal hyperplasia and neoplasia. Two major findings have been obtained. First, considerable immunoreactivity was evident not only at the plasma membrane (the site of cross-linked envelope formation) but also in the cytoplasm of spinous cells, suggesting a cytoplasmic function for this transglutaminase. Staining at the cell border was seen principally in the granular layer of orthokeratinized epithelium (epidermis, hard palate), the outer spinous cells of ortho- and parakeratinized epithelium and in the suprabasal cells showing squamous differentiation in benign and malignant neoplasms. By contrast, diffuse cytoplasmic staining was observed in the upper spinous layer of the normal epithelium and benign lesions. The cytoplasmic immunoreactivity, which extended nearly to the basal layer in hyperkeratosis of the oral mucosa, was evident in two of three verrucous carcinomas examined. In keeping with their undifferentiated character, invasive nests of squamous cell carcinoma and basaloid epithelium in benign and neoplastic lesions were immunonegative for transglutaminase. The second major finding was that lesions of severe oral epithelial dysplasia, immunonegative for transglutaminase, were capable of expressing involucrin immunoreactivity, indicating an uncoupling of keratinocyte programming. These results suggest that immunogold-silver staining for transglutaminase may be useful in evaluating the degree of differentiation in benign and malignant oral epithelial proliferation.