Characterization of serum and tissue oxytocinase and tissue oxytocin in the pregnant and non-pregnant mare.

Oxytocin is a hormone with functions in: reproduction, maternal bonding, milk ejection, and feeding/social behavior, and is reported to be present in a variety of tissues. Our goal is to characterize oxytocin and leucyl and cystinyl aminopeptidase (LNPEP/oxytocinase), a key regulator of oxytocin in mares. We measured serum and tissue LNPEP by ELISA from ovulation (D0) until D21-22 in non-pregnant (n = 5) and pregnant mares (n = 6); and in periparturient and postpartum mares (n = 18). Placenta (n = 7) and homogenized tissue of diestrus mares (n = 6) were evaluated using protein determinations and LNPEP ELISAs. Identification of LNPEP and OXT protein in tissues was also performed via western blot, immunohistochemistry and liquid chromatography-mass spectrometry (LC-MS/MS). Furthermore, in situ hybridization was performed for LNPEP and OXT on endometrium, myometrium, pituitary and corpus luteum (CL). Serum LNPEP concentration were similar. Placental LNPEP U/mg protein was highest in the body and pregnant horn. The highest to lowest LNPEP U/mg protein by tissue were: myometrium > follicle wall > endometrium > kidney > CL > liver. Oxytocin was identified in the equine pituitary, CL and placenta and is likely to act in autocrine or paracrine manner, while LNPEP may act systemically and locally to regulate the availability of OXT.

Expression of Oxytocin/Neurophysin I and Oxytocinase in the Equine Conceptus from Day 8 to Day 21 Post-Ovulation.

Leucyl and cystinyl aminopeptidase (LNPEP/oxytocinase) is an enzyme that metabolizes oxytocin in serum and tissues. The presence of oxytocin/neurophysin I (OXT), oxytocin and LNPEP and their relationship to other genes is unknown in the equine conceptus. Our objective was to characterize gene expression of LNPEP and OXT on D8, 10, 12, 14, 15, 16 and 21 conceptuses in relationship to other genes. Immunohistochemistry, western blot and liquid chromatography with tandem mass spectrometry (LC-MS/MS) were used for identification of oxytocin and LNPEP in D15, 16 and 18 conceptuses. LNPEP was increased at D15 compared to D10, was immunolocalized in the equine trophectoderm and endoderm, and protein was confirmed by LC-MS/MS. Maximal abundance of OXT was at D21, and lowest on D12 and D14, but no protein was identified. OXTR abundance was highest on D14 and D21. LNPEP was correlated with PTGFR and PTGES on D12 and D14-D15, and high expression of PTGES, PTGS2 was found on D14, D15 and D21; PTGFR was found on D8 and D12-21. LNPEP may have a role in prostaglandin regulation and conceptus fixation by decreasing the availability of oxytocin. Further investigation on the role embryonic LNPEP during pregnancy is warranted.

Detection of Epithelial Cell Adhesion Molecule in Feline Normal and Tumor Cell Lines and Tissues With Selected Commercial Anti-human EpCAM Antibodies.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane protein expressed at intercellular junctions in epithelial cells. As an epithelial biomarker, it used for immunologic-based capture of epithelial-derived circulating tumor cells (CTCs) in human patients with different carcinomas. EpCAM expression has not been described in normal or neoplastic epithelial tissues in cats. Our goal was to find a commercial antibody that recognizes surface EpCAM expression for CTC detection. We tested two anti-human EpCAM antibodies, designated for use with flow cytometry, for detection of surface EpCAM expression on feline cell lines derived from normal mammary and renal epithelia and mammary and oropharyngeal squamous cell carcinomas in cats. Only one of the antibodies, a goat polyclonal antibody, labeled normal and neoplastic feline mammary epithelial cells and oropharyngeal squamous cell carcinoma cells; no labeling was observed for normal feline kidney epithelial cells. At low dilution, this antibody immunohistochemically stained the intercellular junctions of normal pancreatic, intestinal and mammary epithelium, as well as neoplastic mammary epithelium in feline tissues; however, oral mucosa, skin, and an oropharyngeal squamous cell carcinoma showed no positive immunostaining. The antibody only weakly bound feline squamous cell carcinoma cell lines under static adhesion. Our results indicate that EpCAM is expressed in specific epithelia in cats but is variably expressed in feline mammary tumors and oropharyngeal squamous cell carcinoma. A higher avidity cross-reactive or feline-specific antibody will be required to further investigate EpCAM expression in normal and neoplastic feline tissue or for detecting CTCs in the blood of tumor-bearing cats.

Approach for semi-automated measurement of fiber diameter in murine and canine skeletal muscle.

Currently available software tools for automated segmentation and analysis of muscle cross-section images often perform poorly in cases of weak or non-uniform staining conditions. To address these issues, our group has developed the MyoSAT (Myofiber Segmentation and Analysis Tool) image-processing pipeline. MyoSAT combines several unconventional approaches including advanced background leveling, Perona-Malik anisotropic diffusion filtering, and Steger's line detection algorithm to aid in pre-processing and enhancement of the muscle image. Final segmentation is based upon marker-based watershed segmentation. Validation tests using collagen V labeled murine and canine muscle tissue demonstrate that MyoSAT can determine mean muscle fiber diameter with an average accuracy of ~92.4%. The software has been tested to work on full muscle cross-sections and works well even under non-optimal staining conditions. The MyoSAT software tool has been implemented as a macro for the freely available ImageJ software platform. This new segmentation tool allows scientists to efficiently analyze large muscle cross-sections for use in research studies and diagnostics.

Evaluation of two methods to isolate Schwann cells from murine sciatic nerve.

BACKGROUND: Schwann cells (SC) and macrophages play key roles in the response to peripheral nerve injury (PNI). Accurate isolation of such cells is essential for further analyses that can lead to better understanding of the repair process after PNI. Separation of live SC from the injury site without culture enrichment is necessary for targeted gene expression analysis. NEW METHODS: Two flow cytometric techniques are presented for rapid enrichment of live SC and macrophages from injured murine peripheral nerve without the need for culture. RESULTS: SC were isolated by fluorescent activated cell sorting (FACS) using transgenic expression of eGFP in SC, or by exclusion of other cell types collected from the injury site. COMPARISON WITH EXISTING METHOD(S): Gene expression analyses of peripheral nerve repair have commonly used whole nerve lysates. Isolating SC allows more accurate understanding of their specific role in repair. SC are commonly enriched from nerve by culture, however this changes gene expression patterns and limits the utility for transcriptomic analysis. The surface marker p75-NTR has variable expression in different SC phenotypes and during the course of injury and repair. Using p75-NTR for SC isolation might enrich only a subset of SC. More stably expressed lineage markers for SC are intracellular and not suitable for sorting for gene expression. The methods used here avoid the requirement for surface marker labeling of SC. CONCLUSION: Gene expression analysis of sorted cells from both methods showed successful enrichment of SC. Lineage markers such as Map1b, p75-NTR and S100b were enriched in the sorted SC population. SC sorting by eGFP expression showed improved enrichment, particularly of mature myelinating genes, although this could represent sampling of a subset of SC.

Differential Gene Expression Profiles and Selected Cytokine Protein Analysis of Mediastinal Lymph Nodes of Horses with Chronic Recurrent Airway Obstruction (RAO) Support an Interleukin-17 Immune Response.

Recurrent airway obstruction (RAO) is a pulmonary inflammatory condition that afflicts certain mature horses exposed to organic dust particulates in hay. Its clinical and pathological features, manifested by reversible bronchoconstriction, excessive mucus production and airway neutrophilia, resemble the pulmonary alterations that occur in agricultural workers with occupational asthma. The immunological basis of RAO remains uncertain although its chronicity, its localization to a mucosal surface and its domination by a neutrophilic, non-septic inflammatory response, suggest involvement of Interleukin-17 (IL-17). We examined global gene expression profiles in mediastinal (pulmonary-draining) lymph nodes isolated from RAO-affected and control horses. Differential expression of > 200 genes, coupled with network analysis, supports an IL-17 response centered about NF-κB. Immunohistochemical analysis of mediastinal lymph node sections demonstrated increased IL-17 staining intensity in diseased horses. This result, along with the finding of increased IL-17 concentrations in lymph node homogenates from RAO-affected horses (P = 0.1) and a down-regulation of IL-4 gene and protein expression, provides additional evidence of the involvement of IL-17 in the chronic stages of RAO. Additional investigations are needed to ascertain the cellular source of IL-17 in this equine model of occupational asthma. Understanding the immunopathogenesis of this disorder likely will enhance the development of therapeutic interventions beneficial to human and animal pulmonary health.

Generation and characterization of a monoclonal antibody against canine tissue factor.

Tissue factor (TF, coagulation factor III) has recently identified roles in innate immunity and cancer. We generated a murine mAb against canine TF (cTF) cloned from Madin-Darby canine kidney cells and expressed in Chinese Hamster Ovarian (CHO) cells, with an equine IL-4 tag. One clone was selected for purification based on initial screening of CHO cell supernatants. The mAb was further characterized with flow cytometry, immunofluorescent microscopy, immunoblotting and immunohistochemical staining of normal and neoplastic canine tissue. The mAb labeled high, but not low, TF-expressing canine breast cancer (CMT25) and osteosarcoma (HMPOS) cells with flow cytometry and immunofluorescent microscopy. Immunoblotting revealed a 42kDa protein with homogenized canine brain and CMT25, but not HMPOS, lysates. The mAb labeled renal tubules and glomeruli, intestinal and dermal epithelium, and arteriolar adventitial cells in frozen tissues. Using immunofluorescent microscopy, increased numbers of labeled PBMCs were observed after LPS stimulation. Our results indicate that the anti-cTF mAb detects a protein with the expected tissue distribution and molecular weight of TF in normal, LPS-stimulated and neoplastic canine cells. This mAb may prove useful for exploring the role of TF in neoplastic and infectious disorders in dogs.

Alkaline phosphatase is a useful cytochemical marker for the diagnosis of acute myelomonocytic and monocytic leukemia in the dog.

BACKGROUND: Immunophenotyping has replaced cytochemical staining as the preferred technique for classifying acute leukemia. However, some acute myeloid leukemias (AML) lack lineage-associated markers. In our experience, alkaline phosphatase (ALP) is expressed in immature canine monocytes. We hypothesized that ALP is a useful marker for monocytic AML. OBJECTIVES: The objective was to compare ALP expression in neoplastic cells from dogs with lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoid leukemia (ALL), and AML. METHODS: Alkaline phosphatase results were retrieved from medical records of dogs with acute leukemia. Smears from dogs with lymphoma or leukemia were also prospectively stained for ALP activity. CLL was based on persistent lymphocytosis (10 × 10(9) /L) and acute leukemia on ≥ 20% blasts in blood or bone marrow. ALL was classified based on positive phenotyping for T- or B-lymphocyte antigens, and AML on positive phenotyping for CD11b, CD11c or CD14, or cytochemical staining for chloroacetate esterase, Sudan Black B, or myeloperoxidase. RESULTS: There was no ALP activity in all 49 lymphomas and 7 CLLs. Weak ALP activity was seen in 31% of 14 ALL (all T-ALL). ALP activity was seen in all 20 AML (P < .001 vs ALL) with strong activity in 64% (vs 25% ALL) in most neoplastic cells (median 75% vs 9% ALL, P = .020). Of AML, 80% were CD34+ (vs 39% ALL, P = .027) and 100% were MHCII- (vs 43% ALL, P = .002). CONCLUSIONS: ALP activity may be useful for AML confirmation in dogs, particularly if neoplastic cells only express CD34+ on immunophenotyping.

Sirtuin-targeting drugs: Mechanisms of action and potential therapeutic applications.

The sirtuins are NAD+-dependent histone/protein deacetylases that are similar to Saccharomyces cerevisiae silent information regulator 2 (Sir2). Sirtuins regulate various normal and abnormal cellular and metabolic processes, including tumorigenesis, neurodegeneration, and processes associated with type 2 diabetes and obesity. Several age-related diseases, such as Alzheimer's disease, and longevity have also been linked to the functions of sirtuins. A thorough understanding of the mechanisms of action of the sirtuins may therefore yield novel therapeutic strategies targeting these processes; several small-molecule and naturally occurring inhibitors and activators of these enzymes have been identified. This review describes the mechanisms regulating sirtuin activity, as well as how these modes of regulation may be exploited to manipulate activity in the context of various pathological states (ie, metabolic diseases, cancer and neurodegenerative diseases). The possible metabolic outcomes of the pharmacological manipulation of sirtuins are also discussed.