Deficient CEBPA DNA binding function in normal karyotype AML patients is associated with favorable prognosis

CCAAT/enhancer binding protein-α (CEBPA) mutations in acute myeloid leukemia (AML) patients with a normal karyotype (NK) confer favorable prognosis, whereas NK-AML patients per se are of intermediate risk. This suggests that blocked CEBPA function characterizes NK-AML with favorable outcome. We determined the prognostic significance of CEBPA DNA binding function by enzyme-linked immunosorbent assay in 105 NK-AML patients. Suppressed CEBPA DNA binding was defined by 21 good-risk AML patients with inv(16) or t(8;21) (both abnormalities targeting CEBPA) and 8 NK-AML patients with dominant-negative CEBPA mutations. NK-AML patients with suppressed CEBPA function showed a better overall survival (P = .0231) and disease-free survival (P = .0069) than patients with conserved CEBPA function. Suppressed CEBPA DNA binding was an independent marker for better overall survival and disease-free survival in a multivariable analysis that included FLT3-ITD, NPM1 and CEBPA mutation status, white blood cell count, age and lactate dehydrogenase. These data indicate that suppressed CEBPA function is associated with favorable prognosis in NK-AML patients.

Nuclear cysteine cathepsin variants in thyroid carcinoma cells

The cysteine peptidase cathepsin B is important in thyroid physiology by being involved in thyroid prohormone processing initiated in the follicular lumen and completed in endo-lysosomal compartments. However, cathepsin B has also been localized to the extrafollicular space and is therefore suggested to promote invasiveness and metastasis in thyroid carcinomas through, e.g., ECM degradation. In this study, immunofluorescence and biochemical data from subcellular fractionation revealed that cathepsin B, in its single- and two-chain forms, is localized to endo-lysosomes in the papillary thyroid carcinoma cell line KTC-1 and in the anaplastic thyroid carcinoma cell lines HTh7 and HTh74. This distribution is not affected by thyroid stimulating hormone (TSH) incubation of HTh74, the only cell line that expresses a functional TSH-receptor. Immunofluorescence data disclosed an additional nuclear localization of cathepsin B immunoreactivity. This was supported by biochemical data showing a proteolytically active variant slightly smaller than the cathepsin B proform in nuclear fractions. We also demonstrate that immunoreactions specific for cathepsin V, but not cathepsin L, are localized to the nucleus in HTh74 in peri-nucleolar patterns. As deduced from co-localization studies and in vitro degradation assays, we suggest that nuclear variants of cathepsins are involved in the development of thyroid malignancies through modification of DNA-associated proteins.

Elevated level of metabotropic glutamate receptor 2/3 in the prefrontal cortex in major depression

Clinical, postmortem and preclinical research strongly implicates dysregulation of glutamatergic neurotransmission in major depressive disorder (MDD). Recently, metabotropic glutamate receptors (mGluRs) have been proposed as attractive targets for the discovery of novel therapeutic approaches against depression. The aim of this study was to examine mGluR2/3 protein levels in the prefrontal cortex (PFC) from depressed subjects. In addition, to test whether antidepressants influence mGluR2/3 expression we also studied levels of mGluR2/3 in fluoxetine-treated monkeys. Postmortem human prefrontal samples containing Brodmann's area 10 (BA10) were obtained from 11 depressed and 11 psychiatrically healthy controls. Male rhesus monkeys were treated chronically with fluoxetine (dose escalated to 3mg/kg, p.o.; n=7) or placebo (n=6) for 39 weeks. The mGluR2/3 immunoreactivity was investigated using Western blot method. There was a robust (+67%) increase in the expression of the mGlu2/3 protein in the PFC of depressed subjects relative to healthy controls. The expression of mGlu2/3 was unchanged in the PFC of monkeys treated with fluoxetine. Our findings provide the first evidence that mGluR2/3 is elevated in the PFC in MDD. This observation is consistent with reports showing that mGluR2/3 antagonists exhibit antidepressant-like activity in animal models and demonstrates that these receptors are promising targets for the discovery of novel antidepressants.

The transcriptional regulator megakaryoblastic leukemia-1 mediates serum response factor-independent activation of tenascin-C transcription by mechanical stress

The extracellular matrix protein tenascin-C (TNC) is up-regulated in processes influenced by mechanical stress, such as inflammation, tissue remodeling, wound healing, and tumorigenesis. Cyclic strain-induced TNC expression depends on RhoA-actin signaling, the pathway that regulates transcriptional activity of serum response factor (SRF) by its coactivator megakaryoblastic leukemia-1 (MKL1). Therefore, we tested whether MKL1 controls TNC transcription. We demonstrate that overexpression of MKL1 strongly induces TNC expression in mouse NIH3T3 fibroblasts and normal HC11 and transformed 4T1 mammary epithelial cells. Part of the induction was dependant on SRF and a newly identified atypical CArG box in the TNC promoter. Another part was independent of SRF but required the SAP domain of MKL1. An MKL1 mutant incapable of binding to SRF still strongly induced TNC, while induction of the SRF target c-fos was abolished. Cyclic strain failed to induce TNC in MKL1-deficient but not in SRF-deficient fibroblasts, and strain-induced TNC expression strongly depended on the SAP domain of MKL1. Promoter-reporter and chromatin immunoprecipitation experiments unraveled a SAP-dependent, SRF-independent interaction of MKL1 with the proximal promoter region of TNC, attributing for the first time a functional role to the SAP domain of MKL1 in regulating gene expression.

Expression and function of 5-hydroxytryptamine 4 receptors in smooth muscle preparations from the duodenum, ileum, and pelvic flexure of horses without gastrointestinal tract disease

OBJECTIVE: To evaluate the expression of the 5-hydroxytryptamine 4 (5-HT4) receptor subtype and investigate the modulating function of those receptors on contractility in intestinal tissues obtained from horses without gastrointestinal tract disease. SAMPLE POPULATION: Smooth muscle preparations from the duodenum, ileum, and pelvic flexure collected immediately after slaughter of 24 horses with no history or signs of gastrointestinal tract disease. PROCEDURES: In isometric organ baths, the contractile activities of smooth muscle preparations in response to 5-hydroxytryptamine and electric field stimulation were assessed; the effect of tegaserod alone or in combination with 5-hydroxytryptamine on contractility of intestinal specimens was also investigated. Presence and distribution of 5-HT4 receptors in intestinal tissues and localization on interstitial cells of Cajal were examined by use of an immunofluorescence technique. RESULTS: Widespread 5-HT4 receptor immunoreactivity was observed in all intestinal smooth muscle layers; 5-HT4 receptors were absent from the myenteric plexus and interstitial cells of Cajal. In electrical field-stimulated tissue preparations of duodenum and pelvic flexure, tegaserod increased the amplitude of smooth muscle contractions in a concentration-dependent manner. Preincubation with tegaserod significantly decreased the basal tone of the 5-HT-evoked contractility in small intestine specimens, compared with the effect of 5-HT alone, thereby confirming that tegaserod was acting as a partial agonist. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, 5-HT4 receptors on smooth muscle cells appear to be involved in the contractile response of the intestinal tract to 5-hydroxytryptamine. Results suggest that tegaserod may be useful for treatment of reduced gastrointestinal tract motility in horses.

TNFα inhibits the development of osteoclasts through osteoblast-derived GM-CSF

Inflammatory cytokines such as tumor necrosis factor-alpha (TNFα) are potent stimulators of osteoclast formation and bone resorption and are frequently associated with pathologic bone metabolism. The cytokine exerts specific effects on its target cells and constitutes a part of the cellular microenvironment. Previously, TNFα was demonstrated to inhibit the development of osteoclasts in vitro via an osteoblast-mediated pathway. In the present study, the molecular mechanisms of the inhibition of osteoclastogenesis were investigated in co-cultures of osteoblasts and bone marrow cells (BMC) and in cultures of macrophage-colony stimulating factor (M-CSF) dependent, non-adherent osteoclast progenitor cells (OPC) grown with M-CSF and receptor activator of NF-κB ligand (RANKL). Granulocyte-macrophage colony stimulating factor (GM-CSF), a known inhibitor of osteoclastogenesis was found to be induced in osteoblasts treated with TNFα and the secreted protein accumulated in the supernatant. Dexamethasone (Dex), an anti-inflammatory steroid, caused a decrease in GM-CSF expression, leading to partial recovery of osteoclast formation. Flow cytometry analysis revealed that in cultures of OPC, supplemented with 10% conditioned medium (CM) from osteoblasts treated with TNFα/1,25(OH)(2)D(3), expression of RANK and CD11c was suppressed. The decrease in RANK expression may be explained by the finding, that GM-CSF and the CM from wt osteoblasts were found to suppress the expression of c-Fos, Fra-1, and Nfatc-1. The failure of OPC to develop into CD11c(+) dendritic cells suggests that cell development is not deviated to an alternative differentiation pathway, but rather, that the monocytes are maintained in an undifferentiated, F4/80(+), state. The data further implies possible interactions among inflammatory cytokines. GM-CSF induced by TNFα acts on early hematopoietic precursors, inhibiting osteoclastogenesis while acting as the growth factor for M-CSF independent inflammatory macrophages. These in turn may condition a microenvironment enhancing osteoclast differentiation and bone resorption upon migration of the OPC from circulation to the bone/bone marrow compartment.

Glomerulonephropathy in Aged Captive Key Largo Woodrats (Neotoma floridana smalli)

A retrospective review of mortality records of Key Largo woodrats (Neotoma floridana smalli) in a captive breeding program revealed chronic renal disease in 5 of 6 woodrats older than 4 years of age. Two of the 5 woodrats with chronic renal disease also had clinical evidence of diabetes mellitus. Kidneys from all 5 woodrats were examined via light microscopy, histochemical staining, immunohistochemical staining, and transmission electron microscopy. The dietary histories of the affected animals were examined as well. The most striking histopathologic abnormality in the affected kidneys was the presence of large protein casts within cortical and medullary tubules in combination with lesions of membranous glomerulopathy and glomerulosclerosis. Transmission electron microscopy revealed thickening and undulation of the tubular and glomerular mesangial basement membranes with the variable presence of electron-dense deposits within the capillary endothelial basement membrane. Patchy glomerular immunoreactivity for IgG was noted in 2 cases, but IgA and IgM immunoreactivity were not present. The pathologic changes in the kidneys of the Key Largo woodrats mirrored many of the features of chronic progressive nephropathy commonly diagnosed in laboratory rats. Woodrats in the captive population were fed an ad libitum high-protein diet similar to diets that have been shown in laboratory rats to exacerbate the development and progression of chronic progressive nephropathy. It is concluded that Key Largo woodrats develop glomerulonephropathy with features similar to chronic progressive nephropathy described in laboratory rats. Age, concomitant disease, and dietary factors may contribute to the development and severity of this potentially age-limiting disease in Key Largo woodrats.

Sarcomatous evolution of oligodendroglioma ("oligosarcoma"): confirmatory report of an uncommon pattern of malignant progression in oligodendroglial tumors

By analogy to gliosarcoma, the neologism "oligosarcoma" is to describe an uncommon form of biphasic central nervous system tumor composed of contiguous neuroepithelial and mesenchymal elements, each of which individually meet the criteria of oligodendroglioma and sarcoma, respectively. By virtue of its distinctive genotype (codeletion 1p/19q), oligodendroglioma is a particularly inviting paradigm to test the assumption that such mixed tumors are clonally derived from a glial primary. We observed this constellation in a 41-year-old male who underwent two resection procedures for a recurring right frontal tumor at five years' interval. On imaging, both lesions were contrast-enhancing, and measured 7 cm × 7 cm × 6.8 cm and 7 cm × 6.5 cm × 4cm, respectively. Following the first operation, temozolomide monotherapy was administered. Whereas initial histology showed conventional anaplastic oligodendroglioma, the recurrence consisted mostly of a fibrosarcoma-like, fascicular neoplasm that was immunoreactive for vimentin, smooth muscle actin, S100 protein, and focally epithelial membrane antigen. In between, a subset of otherwise indistinguishable spindle cells expressed GFAP, and focally merged with residues of oligodendroglioma. Molecular testing for loss of heterozygosity confirmed codeletion of 1p/19q in both the primary tumor and the sarcomatous recurrence. Similarly, generalized immunoreactivity for the mutant R132H form of isocitrate dehydrogenase in both lesions indicated an identical mutation of the IDH1 gene. By the above standards, biologically consistent "oligosarcomas" are felt to be exceedingly rare, and possibly participate of a nosologically heterogeneous group of combined glial/mesenchymal lesions that may also include iatrogenically induced second malignancies as well as true collision tumors.

Characteristics of aleveolar bone associated with physiological movement of molar in mice: a histological and histochemical study

Mouse molars undergo distal movement, during which new bone is formed at the mesial side of the tooth root whereas the preexisting bone is resorbed at the distal side of the root. However, there is little detailed information available regarding which of the bones that surround the tooth root are involved in physiological tooth movement. In the present study, we therefore aimed to investigate the precise morphological differences of the alveolar bone between the bone formation side of the tooth root, using routine histological procedures including silver impregnation, as well as by immunohistochemical analysis of alkaline phosphatase and tartrate-resistant acid phosphatase activity, and immunohistochemical analysis of the expression of the osteocyte markers dentin matrix protein 1, sclerostin, and fibroblast growth factor 23. Histochemical analysis indicated that bone formation by osteoblasts and bone resorption by osteoclasts occurred at the bone formation side and the bone resorption side, respectively. Osteocyte marker immunoreactivity of osteocytes at the surface of the bone close to the periodontal ligament differed at the bone formation and bone resorption sides. We also showed different specific features of osteocytic lacunar canalicular systems at the bone formation and bone resorption sides by using silver staining. This study suggests that the alveolar bone is different in the osteocyte nature between the bone formation side and the bone resorption side due to physiological distal movement of the mouse molar.

Assessment of Alcohol Reward in Prairie Voles (Microtus ochrogaster)

The purpose of our study was to assess whether prairie voles find alcohol rewarding. Prairie voles have recently become a species of interest for alcohol studies, which have traditionally used other rodent model species including several different strains of mice and rats. The prairie vole is one of only two known rodent species that readily administers high levels of unsweetened alcohol, implicating it as a potentially effective animal model for studying alcohol abuse. However, voluntary consumption does not necessarily imply that prairie voles find it rewarding. Therefore the purpose of our study was to investigate if alcohol has rewarding properties for prairie voles using three different approaches: place conditioning, flavor conditioning, and immunohistochemistry. Furthermore, we sought to characterize their reward profile and compare it to other commonly used rodent models ¿ C57BL/6 mice, DBA/2J mice, and Sprague-Dawley rats. Place and flavor conditioning are behavioral methods that rely on the learned association between a stimulus and the effects of a drug; the drug of interest in these studies is alcohol. To assess whether prairie voles will demonstrate a conditioned preference for alcohol-paired stimuli, seven place conditioning studies were run that investigated a range of different doses, individual conditioning session durations, and trial durations. Video analysis revealed no difference in the amount of time spent on the alcohol-paired floor, suggesting no conditioned place preference for alcohol. Two flavor conditioning tests were conducted to assess whether voles would demonstrate a preference for an alcohol-paired flavored saccharin solution. Voles demonstrated reduced consumption of the alcohol-paired flavored saccharin solution, regardless of dose or flavor, when alcohol administration occurred after conditioning sessions (p=<0.001). When alcohol was administered before conditioning sessions, no difference in consumption of the alcohol-paired and saline-paired flavored saccharin solutions was seen (p=0.545). Previous studies that have documented similar behavior have hypothesized that this is an example of an anticipatory contrast effect. This theory proposes that prairie voles reduce their intake of a hedonic solution (flavored saccharin solution) in anticipation of later drug administration (alcohol). However, conditioning-based behavioral methods of studying alcohol reward are highly sensitive to the parameters of the conditioned stimulus, thus it is possible that voles will not show preference for alcohol-related stimuli, even if they do find alcohol rewarding. Immunohistochemical analysis supplemented this behavioral data by allowing us to identify specific neural regions that were directly activated in response to the acute administration of alcohol. No difference in the number of activated c-Fos neurons in the Nucleus Accumbens (NAc) core or shell was seen (p=0.3364; p=0.6698) in animals that received an acute injection of alcohol or saline. There was a significant increase in the number of activated c-Fos neurons in the Paraventricular Nucleus of the Hypothalamus (PVN) in alcohol-treated animals compared to saline-treated animals (p=0.0034). There was no difference in the pixel count of activated c-Fos neurons or in the % area activated in the Arcuate Nucleus between alcohol and saline-treated animals (p=0.4523; p=0.3304). In conclusion, the place conditioning studies that were conducted in this thesis suggest that prairie voles do not demonstrate preference or aversion towards alcohol-paired stimuli. The flavor conditioning studies suggest that prairie voles do not demonstrate aversion but rather avoidance of the alcohol-paired flavor in anticipation of future alcohol administration. The preliminary immunohistochemical data collected is inconclusive but cannot rule out the possibility of neuronal activation patterns indicative of reward. Taken together, our data indicate that prairie voles hav

The LPS receptor, CD14, in experimental autoimmune encephalomyelitis and multiple sclerosis

Innate immune receptors are crucial for defense against microorganisms. Recently, a cross-talk between innate and adaptive immunity has been considered. Here, we provide first evidence for a role of the key innate immune receptor, LPS receptor (CD14) in pathophysiology of experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. Indicating a functional importance in vivo, we show that CD14 deficiency increased clinical symptoms in active experimental autoimmune encephalomyelitis. Consistent with these observations, CD14 deficient mice exhibited a markedly enhanced infiltration of monocytes and neutrophils in brain and spinal cord. Moreover, we observed an increased immunoreactivity of CD14 in biopsy and post mortem brain tissues of multiple sclerosis patients compared to age-matched controls. Thus, the key innate immune receptor, CD14, may be of pathophysiological relevance in experimental autoimmune encephalomyelitis and multiple sclerosis.

Papillary glioneuronal tumor

The descriptive term papillary glioneuronal tumor (PGNT) has been repeatedly applied to a morphologic subset of low-grade mixed glial-neuronal neoplasia of juvenile and young adult patients. We report on a 13-year-old boy with PGNT of the left temporal lobe, who presented with headaches and a single generalized seizure. On magnetic resonance imaging, tumor was seen as a large, moderately enhancing paraventricular mass with cyst-mural nodule configuration and slight midline shift. Perifocal edema was virtually absent. Gross total resection could be performed, followed by an uneventful recovery. Histologically, the tumor exhibited similar, if not identical, features as reported previously. These comprised a patterned biphasic mixture of sheets of synaptophysin-expressing small round cells and pseudorosettes of GFAP-positive rudimentary astrocytes along vascular cores. Focally, the latter imprinted a pseudopapillary aspect on this otherwise solid lesion. Both cellular components expressed non-polysialylated neural cell adhesion molecule (NCAM)-L species, and several overlapping areas of synaptophysin and GFAP immunoreactivity were present. The mean MIB-1 labeling index remained below 1%. Signs of anaplasia, in particular mitotic figures, endothelial proliferation, or necrosis were consistently lacking. We perceive PGNT as a clinically and morphologically well-delineated subgroup of extraventricular neurocytic neoplasia, whose paradigmatic presentation may allow for consideration as an entity.

Leiomyomatoid angiomatous neuroendocrine tumor (LANT) of the pituitary: a distinctive biphasic neoplasm with primitive secretory phenotype and smooth muscle-rich stroma

We describe a hitherto undocumented variant of dimorphic pituitary neoplasm composed of an admixture of neurosecretory cells and profuse leiomyomatous stroma around intratumoral vessels. Radiologically perceived as a macroadenoma of 3.8 cm in diameter, this pituitary mass developed in an otherwise healthy 43-year-old female. At the term of a yearlong history of amenorrhea and progressive bitemporal visual loss, subtotal resection was performed via transsphenoidal microsurgery. Discounting mild hyperprolactinemia, there was no evidence of excess hormone production. Histologically, solid sheets, nests and cords of epithelial-looking, yet cytokeratin-negative cells were seen growing in a richly vascularized stroma of spindle cells. While strong immunoreactivity for NCAM, Synaptophysin and Chromogranin-A was detected in the former, the latter showed both morphological and immunophenotypic hallmarks of smooth muscle, being positive for vimentin, muscle actin and smooth muscle actin. Architectural patterns varied from monomorphous stroma-dominant zones through biphasic neuroendocrine-leiomyomatous areas, to pseudopapillary fronds along vascular cores. Only endothelia were labeled with CD34. Staining for S100 protein and GFAP, characteristics of sustentacular cells, as well as bcl-2 and c-kit was absent. Except for alpha-subunit, anterior pituitary hormones tested negative in tumor cells, as did a panel of peripheral endocrine markers, including serotonin, somatostatin, calcitonin, parathormone and vasoactive intestinal polypeptide. Mitotic activity was absent and the MIB-1 labeling index low (1-2%). While assignment of this lesion to any established neoplastic entity is not forthcoming, we propose it is being considered as a low-grade neuroendocrine tumor possibly related to null cell adenoma.

Clinicopathologic correlations of silent corticotroph adenomas of the pituitary: report of four cases and literature review

Silent corticotroph adenomas (SCA) are rare pituitary tumors with histologic hallmarks of corticotroph differentiation, including ACTH immunoreactivity, but lacking clinical evidence of Cushing's syndrome. We report on four female patients, aged 19-66 years, each presenting with a nonfunctional macroadenoma. Leading symptoms were headache in two cases and visual field deficits in one. One patient was incidentally diagnosed while undergoing cranial MRI for an unrelated condition. Three patients had marked obesity; none of them presented constitutional signs of Cushing's syndrome. Serum cortisol levels were moderately elevated in the two patients systematically tested in this respect. Marginal to moderate hyperprolactinemia was present in two cases. Two patients also were shown to be deficient in either gonadotroph or thyrotroph axis, while a third had a combined insufficiency of both gonadotroph and thyrotroph axis. MRI scans revealed intratumoral hemorrhage and/or cystic change in three cases, as well as tumor-related occlusive hydrocephalus in one. The latter patient was biopsied only, while the remaining underwent gross total resection. Histologically, all four lesions were diagnosed as SCA subtype I displaying intense immunoreactivity for ACTH. In three tumors, scattered cells coexpressed PRL as well. In addition, Crooke's hyaline change was noted in a significant number of tumor cells and in residual non-neoplastic corticotrophs in one case each. With MIB-1 labeling indices of 1-3%, none of the tumors qualified as atypical adenoma. We conclude that SCAs are more likely to be discovered as expansile tumors, whose advanced local space-occupying character at surgery rather than an inherently aggressive growth potential may negatively influence the clinical outcome. Subtle morphologic evidence of corticotroph suppression in residual pituitary adjacent to tumor lends further support to literature data indicating minimal or intermittent functional activity in this tumor type.

ATRA resolves the differentiation block in t(15;17) acute myeloid leukemia by restoring PU.1 expression

Tightly regulated expression of the transcription factor PU.1 is crucial for normal hematopoiesis. PU.1 knockdown mice develop acute myeloid leukemia (AML), and PU.1 mutations have been observed in some populations of patients with AML. Here we found that conditional expression of promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA), the protein encoded by the t(15;17) translocation found in acute promyelocytic leukemia (APL), suppressed PU.1 expression, while treatment of APL cell lines and primary cells with all-trans retinoic acid (ATRA) restored PU.1 expression and induced neutrophil differentiation. ATRA-induced activation was mediated by a region in the PU.1 promoter to which CEBPB and OCT-1 binding were induced. Finally, conditional expression of PU.1 in human APL cells was sufficient to trigger neutrophil differentiation, whereas reduction of PU.1 by small interfering RNA (siRNA) blocked ATRA-induced neutrophil differentiation. This is the first report to show that PU.1 is suppressed in acute promyelocytic leukemia, and that ATRA restores PU.1 expression in cells harboring t(15;17).