Subclassification of nerve sheath tumors by gene expression profiling

Nerve sheath tumors are the most common tumors of Neurofibromatosis type 1 (NF1) patients. Dermal neurofibromas develop in nearly all NF1-patients, whereas plexiform neurofibromas are only observed in one-third of the patients. NF1-patients have about a 10% lifetime risk for developing malignant pheripheral nerve sheath tumors (MPNST). The origin of these tumors is thought to be the Schwann cell lacking functional neurofibromin. However, additional genetic alterations are likely to modulate tumor biology and to contribute to individual nerve sheath tumor entities. To gain insight into the molecular events and to determine whether these tumors can be classified according to gene expression profiles, we performed expression analysis applying cDNA array technology. Nine dermal neurofibromas, 7 plexiform neurofibromas, ten MPNST and two MPNST cell cultures were examined. All tumors but 6 sporadic MPNST were obtained from NF1-patients. We detected significant differences in gene expression patterns between neurofibromas and MPNST and between dermal neurofibromas and plexiform neurofibromas. Tumor class prediction agreed in all but one case with histological and clinical classification. NF1-associated and sporadic MPNST could not be distinguished by their gene expression patterns. We present a panel of discriminating genes that may assist subclassification of nerve sheath tumors.     

Harmonic imaging of the human brain. Visualization of brain perfusion with ultrasound

BACKGROUND AND PURPOSE: Through harmonic gray-scale imaging, it is possible to analyze brain tissue perfusion with different ultrasound methods. METHODS: In 12 healthy volunteers, 2 doses (0.5 and 1.5 mL) of Optison, a perfluoropropane-containing contrast agent, were injected intravenously and produced a strong increase of brightness in the brain parenchyma. We used harmonic imaging for quantification of ultrasound intensity in the thalamus, ipsilateral temporoparietal white matter (TPWM), and ipsilateral lateral fissure at both sides. Time-intensity curves were calculated, and peak increase (PI) of intensity and the area under the time-intensity curve (AUC) from baseline were compared. RESULTS: We found a significant dose dependence of the AUC in all regions at both sides. PI only showed a significant dose dependence in the TPWM but not in the ipsilateral thalamus and lateral fissure. No side differences for AUC and PI were detected in all regions and doses used. We found a significantly higher value of the PI insonating the thalamus from the ipsilateral side compared with the contralateral side. The same result was obtained for the AUC in the left thalamus for both doses and in the right thalamus for the high dose. Using 0.5 mL for insonation of the right thalamus AUC again showed a higher value for the insonation from the ipsilateral compared with the contralateral side but failed to show statistical significance (P=0.08, n=12). CONCLUSIONS: Harmonic gray-scale imaging with Optison showed a strong enhancement effect in the brain parenchyma. A quantitative analysis of perfusion seems difficult because of the depth dependence of the effect. The most robust parameter is the AUC.     

Phenotypic characterization of skin lesions in urticaria pigmentosa and mastocytomas

In order to identify possible cellular abnormalities in human mastocytosis, sections from 13 urticaria pigmentosa lesions and 5 mastocytomas were compared with 5 normal skin specimens using histochemical, enzyme histochemical and immunohistochemical techniques. All toluidine blue-positive mast cells also reacted with FcRI and c-kit antibodies, almost all stained for tryptase, many for chymase and the myeloid workshop mast cell antibodies, few for FcRII and none for the proliferation marker Ki-67. Urticaria pigmentosa lesions contained fewer epidermal Langerhans cells and a lower percentage of avidin-positive mast cells than mastocytomas and normal skin. Mastocytomas exhibited generally weaker staining for mast cell markers and mostly lacked FcRI-bound IgE on mast cells and Langerhans cells, although the receptor was able to bind IgE in tissue sections. Most of the mast cell antibodies also reacted with other cell types. Only toluidine blue, avidin, tryptase and chymase stains were mast cell specific. Mast cells in mastocytosis thus differed only to a minor degree from normal mast cells, although distinct pathomechanisms may play a role in urticaria pigmentosa and mastocytosis.     

Phenotypic evaluation of cultured human mast and basophilic cells and of normal human skin mast cells

In order to evaluate various markers for human mast cells, two human mast/basophilic cell lines (HMC-1/KU812), cultured mast cells from the peripheral blood monocytic fraction and peripheral blood monocytes were compared with mast cells in tissue sections from normal skin, using histochemistry, enzyme histochemistry and immunohistochemistry. All reagents stained normal skin mast cells, with toluidine blue, tryptase reactivity and antibodies against the FcRI and the stem cell factor receptor (c-kit) being most active. The cell lines and mast cells cultured from peripheral blood were negative for avidin, safranin and chymase, strongly positive for c-kit and variably reactive with all other reagents. All antibodies except AA1 against tryptase also stained one or several epidermal and dermal cell types or blood monocytes. Histochemical stains (toluidine blue, avidin) and reagents for the enzymes tryptase and chymase are thus specific markers for mast cells. The frequent reactivity of antibodies against mast cells with other cell types indicates interesting functional and ontogenetic relationships between these cells.     

Distemper virus encephalitis exerts detrimental effects on hippocampal neurogenesis

E.‐L. von Rüden, J. Avemary, C. Zellinger, D. Algermissen, P. Bock, A. Beineke, W. Baumgärtner, V. M. Stein, A. Tipold and H. Potschka (2012) Neuropathology and Applied Neurobiology 38, 426–442 Distemper virus encephalitis exerts detrimental effects on hippocampal neurogenesis Aims: Despite knowledge about the impact of brain inflammation on hippocampal neurogenesis, data on the influence of virus encephalitis on dentate granule cell neurogenesis are so far limited. Canine distemper is considered an interesting model of virus encephalitis, which can be associated with a chronic progressing disease course and can cause symptomatic seizures. Methods: To determine the impact of canine distemper virus (CDV) infection on hippocampal neurogenesis, we compared post‐mortem tissue from dogs with infection with and without seizures, from epileptic dogs with non‐viral aetiology and from dogs without central nervous system diseases. Results: The majority of animals with infection and with epilepsy of non‐viral aetiology exhibited neuronal progenitor numbers below the age average in controls. Virus infection with and without seizures significantly decreased the mean number of neuronal progenitor cells by 43% and 76% as compared to age‐matched controls. Ki‐67 labelling demonstrated that hippocampal cell proliferation was neither affected by infection nor by epilepsy of non‐viral aetiology. Analysis of CDV infection in cells expressing caspase‐3, doublecortin or Ki‐67 indicated that infection of neuronal progenitor cells is extremely rare and suggests that infection might damage non‐differentiated progenitor cells, hamper neuronal differentiation and promote glial differentiation. A high inter‐individual variance in the number of lectin‐reactive microglial cells was evident in dogs with distemper infection. Statistical analyses did not reveal a correlation between the number of lectin‐reactive microglia cells and neuronal progenitor cells. Conclusions: Our data demonstrate that virus encephalitis with and without seizures can exert detrimental effects on hippocampal neurogenesis, which might contribute to long‐term consequences of the disease. The lack of a significant impact of distemper virus on Ki‐67‐labelled cells indicates that the infection affected neuronal differentiation and survival of newborn cells rather than hippocampal cell proliferation.     

Mast cell chymase and tryptase during tissue turnover: analysis on in vitro mitogenesis of fibroblasts and keratinocytes and alterations in cutaneous scars

In order to shed further light on the potential role of mast cells during tissue turnover, we have investigated the number of mast cells containing only tryptase and those storing both tryptase and chymase by enzyme histochemistry in normal versus healing skin. Furthermore, we have studied the in vitro effect of these enzymes on the mitogenesis of subconfluent quiescent fibroblast and HaCaT keratinocyte cultures, using flowcytometric DNA analysis. Chymase-containing mast cell numbers were markedly decreased in scars (P<0.001), whereas the overall number of tryptase-containing mast cells was not decreased, although these cells were smaller and stained more faintly in scars. Chymase (5 to 300 mU/ml) induced a marked, dose-dependent in vitro mitogenic response in 3T3 fibroblasts, whereas the effects of tryptase, at up to 60 nM, were only moderate, compared to the known fibroblast mitogens EGF, TGF-alpha, alpha-thrombin and trypsin at optimal concentrations. Coincubation of either protease with EGF or alpha-thrombin had additive effects. In contrast to fibroblasts, keratinocytes showed only minor mitogenic responses to tryptase and chymase, also in comparison to other known mitogenic stimuli, and responses to EGF and alpha-thrombin were inhibited on costimulation of cells with the proteases. These findings document for the first time a potential role of mast cell chymase in connective tissue repair, with tryptase being less active on fibroblasts, and with inhibitory effects of both mast cell proteases on keratinocytes.     

Is the distribution of dermal neurofibromas in neurofibromatosis type 1 (NF1) related to the pattern of the skin surface temperature?

The formation of dermal neurofibromas is a hallmark of the neurofibromatosis type 1 (NF1). A total loss of the NF1 gene product by stochastic events inactivating the wild type allele in Schwann cells should precede the development of neurofibromas. Dermal neurofibromas tend to be located mainly on the surface of the trunk and not in the body periphery. This distribution partly resembles the density of sensitive nerve endings in the epidermis. Our hypothesis is that a better correlation concerns the pattern of normal body surface temperature. According to our clinical observations we assume that in skin areas with higher temperatures the number of visible dermal neurofibromas is higher than in colder areas such as the arms/legs or nose. It is known that differences in temperature are able to determine differentiation. We suggest that the regulation of skin temperature is also involved in the formation of NF1 dermal neurofibromas and is related to the intrafamilial variability in NF1.     

Differential expression of CRABP II,psoriasin and cytokeratin 1 mRNA in human skin diseases

In order to confirm and further explore the significance of the overexpression of the CRABP II (cellular retinoic acid binding protein type II) and psoriasin genes in psoriatic versus normal skin, we examined the mRNA expression levels of these two genes by in situ hybridization in skin samples from psoriatic plaques and in one case from the border between a psoriatic plaque and uninvolved skin. Both genes were markedly upregulated in lesional skin, with a shift from low to high expression in the transitional zone of the plaque. Expression of the cytokeratin 1 (K1) gene was, in contrast, high in normal skin and decreased in the transition from uninvolved skin to psoriatic plaque, Examination of mRNA levels of CRABP II and psoriasin in other hyperproliferative and inflammatory skin diseases showed high expression of psoriasin, and in some cases also of CRABP II, in atopic dermatitis, mycosis fungoides, Darier’s disease and inflammatory lichen sclerosus et atrophicus. In atrophic lesions of lichen sclerosus et atrophicus that lacked an inflammatory infiltrate, these changes were only weakly expressed. These findings demonstrate that altered epidermal gene expression of K1, psoriasin and CRABP II is not disease-specific and may reflect instead an altered state of epidermal differentiation and/or may be linked to the inflammation and cellular infiltration common to all the conditions studied.     

Altered expression of mast cell chymase and tryptase and of c-Kit in human cutaneous scar tissue

In order to explore a possible involvement of mast cells during human wound healing, we studied sections from scars (4-369-d-old) (N = 20) and normal skin (N = 10) for mast-cell-specific tryptase and chymase by enzyme histochemistry, for the stem cell factor receptor c-Kit and the melanosomal marker TA99 by immunohistochemistry, and for simultaneous c-Kit expression and avidin fluorescence by double staining. Enzyme activities and mRNA expression were also studied in tissue extracts. Chymase-reactive mast cell numbers as well as chymase activity and mRNA expression were reduced in all scars, whereas overall numbers of tryptase-reactive cells did not differ from normal skin, although tryptase activity and mRNA expression were increased in scar extracts. In contrast, numbers of c-Kit positive cells were significantly increased in old scars, and in the mid and lower dermis of all scars. A marked reduction of c-Kit reactivity was noted, however, in avidin-positive dermal mast cells and in epidermal basal cells, despite unchanged numbers of melanosome-positive cells, with an associated overall decrease of c-Kit mRNA in scar extracts. These data thus show that numbers of resident mast cells are very low in human cutaneous scars, suggesting massive mediator release from these cells into fresh wounds. Downregulation of stem cell factor receptors may also prevent these cells from increasing in number even in old scars. Instead, scar tissue is populated by a mast cell subpopulation that is chymase-, avidin-, tryptase +, c-Kit +, reflecting most probably an increased immigration and/or proliferation of immature mast cells and their precursors.