Michael H. A. Roehrl

Tissue Proteomics Reveals Differential and Compartment- Specific Expression of the Homologs Transgelin and Transgelin-2 in Lung Adenocarcinoma and Its Stroma

Discovery of tissue-specific biomarkers for human cancer is crucial for early diagnosis and molecular understanding of the disease. To overcome the limitations posed by the large dynamic concentration range and compositional complexity of tissue biomacromolecules, we applied heparin affinity fractionation for proteomic enrichment. Comparing the proteomes of five paired samples of normal lung and pulmonary adenocarcinoma tissue by 2-D difference gel electrophoresis, 14 spots were found to be differentially expressed. From these candidate spots, three proteins overexpressed in cancer were identified by mass spectrometry as transgelin (TAGLN, SM22-alpha, WS3-10), transgelin-2 (TAGLN2), and cyclophilin A (PPIA). Quantitative RT-PCR indicated that both TAGLN2 and PPIA were upregulated at the transcriptional level. Differential protein expression levels were validated by Western blot analysis using an independent set of 10 paired lung adenocarcinoma samples. Using immunohistochemistry on human tissue sections, we discovered that overexpression of TAGLN was strictly localized to the tumor-induced reactive myofibroblastic stromal tissue compartment, whereas overexpression of TAGLN2 was exclusively localized to the neoplastic glandular compartment. Thus, the highly homologous protein pair TAGLN and TAGLN2 displayed mutually exclusive, compartment-specific cell type expression regulation in tumor stroma vs neoplastic epithelial cells. Our data further suggest that TAGLN may be a marker of active stromal remodeling in the vicinity of invasive carcinomas. It may shed light on mechanisms of tumor-stroma interaction and could be useful for early diagnosis, treatment guidance, and treatment response monitoring.

Proteomic Expression Analysis of Surgical Human Colorectal Cancer Tissues: Up-Regulation of PSB7, PRDX1, and SRP9 and Hypoxic Adaptation in Cancer

Colorectal adenocarcinoma is one of the worldwide leading causes of cancer deaths. Discovery of specific biomarkers for early detection of cancer progression and the identification of underlying pathogenetic mechanisms are important tasks. Global proteomic approaches have thus far been limited by the large dynamic range of molecule concentrations in tissues and the lack of selective enrichment of the low-abundance proteome. We studied paired cancerous and normal clinical tissue specimens from patients with colorectal adenocarcinomas by heparin affinity fractionation enrichment (HAFE) followed by 2-D PAGE and tandem mass spectrometric (MS/MS) identification. Fifty-six proteins were found to be differentially expressed, of which 32 low-abundance proteins were only detectable after heparin affinity enrichment. MS/MS was used to identify 5 selected differentially expressed proteins as proteasome subunit beta type 7 (PSB7), hemoglobin alpha subunit (HBA), peroxiredoxin-1 (PRDX1), argininosuccinate synthase (ASSY), and signal recognition particle 9 kDa protein (SRP9). This is the first proteomic study detecting the differential expression of these proteins in human colorectal cancer tissue. Several of the proteins are functionally related to tissue hypoxia and hypoxic adaptation. The relative specificities of PSB7, PRDX1, and SRP9 overexpression in colon cancer were investigated by Western blot analysis of patients with colon adenocarcinomas and comparison with a control cohort of patients with lung adenocarcinomas. Furthermore, immunohistochemistry on tissue sections was used to define the specific locations of PSB7, PRDX1, and SRP9 up-regulation within heterogeneous primary human tumor tissue. Overexpression of the three proteins was restricted to the neoplastic cancer cell population within the tumors, demonstrating both cytoplasmic and nuclear localization of PSB7 and predominantly cytoplasmic localization of PRDX1 and SRP9. In summary, we describe heparin affinity fractionation enrichment (HAFE) as a prefractionation tool for the study of the human primary tissue proteome and the discovery of PSB7, PRDX1, and SRP9 up-regulation as candidate biomarkers of colon cancer.

Age-dependent reference ranges for automated assessment of immature granulocytes and clinical significance in an outpatient setting.

CONTEXT New generations of hematology analyzers have made the routine automated quantification of immature granulocytes (IGs) in peripheral blood samples accessible as a powerful clinical parameter. OBJECTIVE The use of IGs has previously been studied mostly in hospitalized patients with sepsis. We investigated the use of IGs in the outpatient setting. Establishment of precise normal outpatient IG reference ranges is a prerequisite for clinically meaningful interpretation of the parameter. DESIGN We analyzed a large outpatient population comprising more than 2400 samples to determine age-stratified normal reference ranges for IGs. RESULTS Using nonparametric statistical approaches, we show that 1-tailed 95th percentile estimates for relative and absolute IG concentrations up to the age of 10 years are 0.30% and 30.0 µL(-1), respectively. For individuals above the age of 10 years, the respective 95th percentile estimates are approximately twice as large at 0.74% and 60.0 µL(-1). No differences were seen between male and female reference ranges. Taking nonparametric 90% confidence intervals for each estimate into account, we recommend the following IG upper reference range limits for routine outpatient use: 0.30%/40.0 µL(-1) (≤10 years) and 0.90%/70.0 µL(-1) (>10 years). Up to the age of 10 years, the most common pathologies associated with elevated IG counts in outpatients were infections, in particular, otitis media, upper and lower respiratory infections, and gastroenteritis. By contrast, above the age of 10 years, the most common causes were hematologic malignancies, drug therapy (glucocorticoids, chemotherapy), severe infections, and pregnancy (young females). CONCLUSIONS The use of appropriate reference ranges makes IGs a powerful hematologic parameter for outpatient care that is associated with differential diagnoses that are distinctly characteristic of that setting.

The Nasolabial Cyst: A Nonodontogenic Oral Cyst Related to Nasolacrimal Duct Epithelium

Nasolabial cysts are interesting, relatively uncommon benign extraosseous maxillary lesions. We review current knowledge about epidemiology, symptoms, imaging modalities, pathogenesis, histopathologic and ultrastructural features, treatment options, and prognosis. Nasolabial cyst lining epithelium is characteristically composed of a basal layer of cuboidal cells and a luminal layer of columnar secretory cells with interspersed mucous goblet cells. In addition, areas of multilayered epithelium and squamous metaplasia may be seen. The cyst stroma is characterized by collagen-rich fibrovascular tissue with variably admixed chronic inflammatory cells. Furthermore, to our knowledge, we report the first example of immunohistochemical protein expression profiling of nasolabial cyst lining epithelium, discovering that basal layer cells express p63 and cytokeratin 5/6, while goblet cells express MUC-2 and MUC-5AC mucins, supporting the notion that nasolabial cysts can be understood as hamartomatous, locally expansile remnants of distal nasolacrimal duct development.

Eosinophilic myocarditis in hypereosinophilic syndrome

A 49-year-old woman presented with cough, fatigue, stomach pain, and fevers to 38.0 8C. An echocardiogram showed a mildly reduced left ventricular ejection fraction and global hypokinesis. A CBC was remarkable for a leukocyte count of 21.4 3 10 lL with a relative and absolute eosinophil count of 67% and 14.4 3 10 lL, respectively. The serum IgE concentration was 290 IU/mL (normal, 0– 150 IU/mL). An endomyocardial biopsy of the right ventricle revealed abundant, focally degranulating eosinophils infiltrating the myocardium in both perivascular and interstitial patterns (Image 1A, H&E stain, 6003). There was associated multifocal myocyte damage and drop-out, compensatory myocyte hypertrophy, and significant interstitial and replacement fibrosis, as highlighted by expanded blue areas on a trichrome stain (Image 1B, 2003). The myocardium also showed scattered CD4 T cells but virtually no CD8 T cells or CD20 B cells. A bone marrow biopsy revealed a hypercellular marrow with trilineage maturation and a prominent component of mature-appearing eosinophils comprising 60–70% of total cellularity (Image 1C, H&E stain, 6003). Cytogenetic analysis revealed a normal 46,XX karyotype. FISH testing was negative for BCRABL1, PDGFRA, and PDGFRB rearrangements. Because of documented cardiac tissue damage, a diagnosis of idiopathic hypereosinophilic syndrome (HES) was deemed most appropriate [1]. Our patient was treated with 60 mg of oral prednisone per day starting on hospital day 8 (Image 1D, arrow), achieving a near-normal eosinophil count on day 18 (dashed line, upper limit of normal). Response to steroids is thought to indicate an overall better prognosis. Approximately one third of patients, however, will not respond to steroids. Hydroxyurea and interferon alpha are currently second-line drugs of choice and can be complemented by attempts of high dose imatinib, monoclonal antibodies against IL-5 (mepolizumab) or CD52 (alemtuzumab), or hematopoietic stem cell transplantation [2]. Myocardial involvement is a common and serious complication of HES, potentially resulting in death due to cardiac fibrosis, congestive heart failure, and arrhythmia Image 1.

Human Proteins with Affinity for Dermatan Sulfate Have the Propensity to Become Autoantigens

The mystery of why and how a small, seemingly disparate subset of all self molecules become functional autoantigens holds a key to understanding autoimmune diseases. Here and in a companion article in this issue, we show that affinity of self molecules to the glycosaminoglycan dermatan sulfate (DS) is a common property of autoantigens and leads to a specific autoreactive B-1a cell response. Autoimmune ANA/ENA reference sera react preferentially with DS affinity-fractionated cellular proteins. Studying patients with autoimmune diseases, we discovered patient-specific complex autoantigen patterns that are far richer and more diverse than previously thought, indicating significant pathological heterogeneity even within traditionally defined clinical entities, such as systemic lupus erythematosus. By shotgun sequencing of DS affinity-enriched proteomes extracted from cell lines, we identified approximately 200 autoantigens, both novel and previously linked to autoimmunity, including several well-known families of autoantigens related to the nucleosome, ribonucleoproteins, the cytoskeleton, and heat shock proteins. Using electron microscopy, we recognized direct interaction with dead cells as an origin of autoantigenic association of DS with self molecules. DS affinity may be a unifying property of the human autoantigen-ome (ie, totality of self molecules that can serve as functional autoantingens) and thus provides a promising tool for discovery of autoantigens, molecular diagnosis of autoimmune diseases, and development of cause-specific therapies.

Dermatan Sulfate Interacts with Dead Cells and Regulates CD5+ B-Cell Fate: Implications for a Key Role in Autoimmunity

CD5(+) (B-1a) B cells play pivotal roles in autoimmunity through expression of autoreactive B-cell receptors and production of autoantibodies. The mechanism underlying their positive selection and expansion is currently unknown. This study demonstrates that dermatan sulfate (DS) expands the B-1a cell population and augments the specific antibody response to an antigen when it is in complex with DS. DS displays preferential affinity for apoptotic and dead cells, and DS-stimulated cell cultures produce antibodies to various known autoantigens. The companion article further illustrates that autoantigens can be identified by affinity to DS, suggesting that molecules with affinity to DS have a high propensity to become autoantigens. We thus propose that the association of antigens from dead cells with DS is a possible origin of autoantigens and that autoreactive B-1a cells are positively selected and expanded by DS∙autoantigen complexes. This mechanism may also explain the clonal expansion of B-1a cells in certain B-cell malignancies.

Computed Tomography of Granulomatous Pneumonia with Oxalosis in an American Alligator (Alligator mississippiensis) Associated with Metarhizium anisopliae var anisopliae

Abstract:  An 18-yr-old, male, albino, American alligator (Alligator mississippiensis) was evaluated for decreased appetite and abnormal buoyancy. Computed tomography (CT) of the coelomic cavity showed multifocal mineral and soft tissue attenuating pulmonary masses consistent with pulmonary fungal granulomas. Additionally, multifocal areas of generalized, severe emphysema and pulmonary and pleural thickening were identified. The alligator was euthanized and necropsy revealed severe fungal pneumonia associated with oxalosis. Metarhizium anisopliae var. anisopliae was cultured from lung tissue and exhibited oxalate crystal formation in vitro. Crystals were identified as calcium oxalate monohydrate by X-ray powder defractometry. Fungal identification was based on morphology, including tissue sporulation, and DNA sequence analysis. This organism is typically thought of as an entomopathogen. Clinical signs of fungal pneumonia in nonavian reptiles are often inapparent until the disease is at an advanced stage, making antemortem diagnosis challenging. This case demonstrates the value of CT for pulmonary assessment and diagnosis of fungal pneumonia in the American alligator. Fungal infection with associated oxalosis should not be presumed to be aspergillosis.

Immature granulocytes in pregnancy: A story of Virchow, anxious fathers, and expectant mothers†

A 20-year-old woman (G2P1) was followed for an uncomplicated second pregnancy. She was not taking any medications besides a daily oral multivitamin and iron supplement. At 23 weeks and 5 days, she was feeling well, and vital signs, physical exam, and urinalysis were normal. A complete peripheral venous blood count using a Sysmex XT-1800i instrument showed a white cell count of 15.6 3 10 ll, red cell count of 3.6 3 10 ll, hemoglobin concentration of 10.5 g/dl, hematocrit of 30.8%, mean corpuscular volume of 86.0 fl, mean corpuscular hemoglobin of 29.3 pg, and a platelet count of 351 3 10 ll. An automated differential count (Image 1A, left panel; SSC, side scatter; SFL, side fluorescence) revealed 59.3% neutrophils (population colored in cyan/teal), 15.3% lymphocytes (magenta), 9.0% monocytes (green), 2.9% eosinophils (red), 1.2% basophils (cyan/teal; overlapping with neutrophils in this projection), and 12.3% (1,920 ll) immature granulocytes (i.e., promyelocytes, myelocytes, and/or metamyelocytes [1,2]; dark blue, yellow arrows). Manual review of a blood smear confirmed these results and showed that the immature granulocytes were composed of 5.0% myelocytes and 7.3% metamyelocytes (Image 1A, middle and right panels). No promyelocytes, myeloblasts, or dysplastic features were seen. Not surprisingly, the marked increase of immature myeloid cells caused significant anxiety in this young patient and her family. At 39 weeks, the woman delivered a healthy daughter by Cesarean section without complications. 2 weeks post partum, a complete blood count showed a white cell count of 7.3 3 10 ll, red cell count of 5.0 3 10 ll, hemoglobin concentration of 14.3 g/dl, hematocrit of 42.7%, mean corpuscular volume of 85.1 fl, mean corpuscular hemoglobin of 28.5 pg, and a platelet count of 304 3 10 ll. An Image 1