Use of quantitative susceptibility mapping (QSM) in progressive multifocal leukoencephalopathy.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) is an opportunistic demyelinating encephalopathy related to JC virus. Its characteristics on conventional brain MRI are well known and are important for the diagnosis. OBJECTIVE: To analyze SWI hypointensities recently described in U-fibers and cortex adjacent to the white matter lesions of PML. METHODS: Prospective study including four patients with an history of definite diagnosis of PML. Clinical data were collected retrospectively. Brain MRI exams were done on a 3T magnet, including FLAIR, T2 GRE sequences and SWI. RESULTS: Four males were included (mean age: 47 years, mean PML duration: 24.2 months). Immunosuppression was related to AIDS (n=2), natalizumab for multiple sclerosis (n=1), B-cell lymphoma treated by chemotherapeutic agents and rituximab (n=1). All patients had SWI hypointensities in cortex and/or U-fibers adjacent to the white matter lesions. QSM always suggested a paramagnetic effect. CONCLUSION: SWI and T2 GRE hypointensities in cortex and U-fibers adjacent to the white matter lesions seem highly prevalent in PML, irrespective of the delay between PML onset and the MRI. QSM data suggest a paramagnetic effect.

CYLD regulates keratinocyte differentiation and skin cancer progression in humans.

CYLD is a gene mutated in familial cylindromatosis and related diseases, leading to the development of skin appendages tumors. Although the deubiquitinase CYLD is a skin tumor suppressor, its role in skin physiology is unknown. Using skin organotypic cultures as experimental model to mimic human skin, we have found that CYLD acts as a regulator of epidermal differentiation in humans through the JNK signaling pathway. We have determined the requirement of CYLD for the maintenance of epidermal polarity, keratinocyte differentiation and apoptosis. We show that CYLD overexpression increases keratinocyte differentiation while CYLD loss of function impairs epidermal differentiation. In addition, we describe the important role of CYLD in the control of human non-melanoma skin cancer progression. Our results show the reversion of the malignancy of human squamous cell carcinomas that express increased levels of CYLD, while its functional inhibition enhances the aggressiveness of these tumors which progress toward spindle cell carcinomas. We have found that the mechanisms through which CYLD regulates skin cancer progression include the control of tumor differentiation, angiogenesis and cell survival. These findings of the role of CYLD in human skin cancer prognosis make our results relevant from a therapeutic point of view, and open new avenues for exploring novel cancer therapies.

An inactivating CYLD mutation promotes skin tumor progression by conferring enhanced proliferative, survival and angiogenic properties to epidermal cancer cells.

In this study, we demonstrate that the expression in tumorigenic epidermal cells of a catalytically inactive form of CYLD (CYLD(C/S)) that mimics the identified mutations of cyld in human tumors and competes with the endogenous CYLD results in enhanced cell proliferation and inhibition of apoptosis; it also stimulates cell migration and induces the expression of angiogenic factors, including vascular endothelial growth factor-A. Altogether, these characteristics indicate an increased oncogenicity of the tumorigenic epidermal CYLD(C/S) mutant cells in vitro. Moreover, we show the increase in malignancy of epidermal squamous cell carcinomas that express the CYLD(C/S) transgene in an in vivo xenograft model. Tumors carrying the mutated CYLD(C/S) exhibit a fast growth, are poorly differentiated and present a robust angiogenesis. CYLD(C/S) tumors are also characterized by their elevated proliferation rate and decreased apoptosis. In contrast with previous studies showing the development of benign tumors by mutations in the CYLD gene, here we provide evidence that the occurrence of mutations in the CYLD gene in tumorigenic epidermal cells (carrying previous mutations) increases the aggressiveness of carcinomas, mainly through enhancement of the expression of angiogenic factors, having therefore a key role in epidermal cancer malignancy.

Effects of developer depletion on image quality of Kodak Insight and Ektaspeed Plus films.

OBJECTIVE: To evaluate the effect of processing solution depletion on the image quality of F-speed dental X-ray film (Insight), compared with Ektaspeed Plus. METHODS: The films were exposed with a phantom and developed in manual and automatic conditions, in fresh and progressively depleted solutions. The comparison was based on densitometric analysis and subjective appraisal. RESULTS: The processing solution depletion presented a different behaviour depending on whether manual or automatic technique was used. The films were distinctly affected by depleted processing solutions. CONCLUSIONS: The developer depletion was faster in automatic than manual conditions. Insight film was more resistant than Ektaspeed Plus to the effects of processing solution depletion. In the present study there was agreement between the objective and subjective appraisals.

Opposite functions for E2F1 and E2F4 in human epidermal keratinocyte differentiation.

Proteins of the retinoblastoma family (pRb, p107, and p130) modulate cell proliferation, a function related to their capacity to control the activity of the E2F transcription factor family. The Rb proteins also control cell differentiation in different tissues. We have recently described their involvement in human epidermal keratinocyte differentiation (Paramio, J. M., Lain, S., Segrelles, C., Lane, E. B. , and Jorcano, J. L. (1998) Oncogene 17, 949-957). Here we show that E2F proteins are also involved in this process. We found that E2F1 and E2F4 are expressed differentially during the in vitro differentiation of human epidermal keratinocytes, with the former uniformly present throughout the process, whereas the second is predominantly expressed at the onset of differentiation. This pattern is also observed in human skin by confocal microscopy. Electrophoretic mobility shift assays and immunoprecipitation experiments demonstrated that the complexes formed by E2F1 and E2F4 and Rb family proteins vary throughout in vitro keratinocyte differentiation. In agreement with this observation, several E2F-responsive genes are differentially regulated during this process. To test the functional implications of these observations, we transfected HaCaT keratinocytes with plasmids coding for E2F1 and E2F4. Transfected cells display opposite in vitro differentiation properties. Although E2F1-transfected cells are unable to differentiate, E2F4-transfected cells show an increased differentiation rate compared with Neo-transfected control cells. Our data demonstrate that the differential and coordinated expression and interaction of E2F and Rb proteins modulate the process of epidermal differentiation and provide clear evidence that members of the E2F family of transcription factors play specific and opposite roles during cell differentiation.

Exocrine pancreatic disorders in transsgenic mice expressing human keratin 8.

Keratins K8 and K18 are the major components of the intermediate-filament cytoskeleton of simple epithelia. Increased levels of these keratins have been correlated with various tumor cell characteristics, including progression to malignancy, invasive behavior, and drug sensitivity, although a role for K8/K18 in tumorigenesis has not yet been demonstrated. To examine the function of these keratins, we generated mice expressing the human K8 (hk8) gene, which leads to a moderate keratin-content increase in their simple epithelia. These mice displayed progressive exocrine pancreas alterations, including dysplasia and loss of acinar architecture, redifferentiation of acinar to ductal cells, inflammation, fibrosis, and substitution of exocrine by adipose tissue, as well as increased cell proliferation and apoptosis. Histological changes were not observed in other simple epithelia, such as the liver. Electron microscopy showed that transgenic acinar cells have keratins organized in abundant filament bundles dispersed throughout the cytoplasm, in contrast to control acinar cells, which have scarce and apically concentrated filaments. The phenotype found was very similar to that reported for transgenic mice expressing a dominant-negative mutant TGF-beta type II receptor (TGFbetaRII mice). We show that these TGFbetaRII mutant mice also have elevated K8/K18 levels. These results indicate that simple epithelial keratins play a relevant role in the regulation of exocrine pancreas homeostasis and support the idea that disruption of mechanisms that normally regulate keratin expression in vivo could be related to inflammatory and neoplastic pancreatic disorders.

Modulation of cell proliferation by cytokeratins K10 and K16.

The members of the large keratin family of cytoskeletal proteins are expressed in a carefully regulated tissue- and differentiation-specific manner. Although these proteins are thought to be involved in imparting mechanical integrity to epithelial cells, the functional significance of their complex differential expression is still unclear. Here we provide new data suggesting that the expression of particular keratins may influence cell proliferation. Specifically, we demonstrate that the ectopic expression of K10 inhibits the proliferation of human keratinocytes in culture, while K16 expression appears to promote the proliferation of these cells. Other keratins, such as K13 or K14, do not significantly alter this parameter. K10-induced inhibition is reversed by the coexpression of K16 but not that of K14. These results are coherent with the observed expression pattern of these proteins in the epidermis: basal, proliferative keratinocytes express K14; when they terminally differentiate, keratinocytes switch off K14 and start K10 expression, whereas in response to hyperproliferative stimuli, K16 replaces K10. The characteristics of this process indicate that K10 and K16 act on the retinoblastoma (Rb) pathway, as (i) K10-induced inhibition is hampered by cotransfection with viral oncoproteins which interfere with pRb but not with p53; (ii) K10-mediated cell growth arrest is rescued by the coexpression of specific cyclins, cyclin-dependent kinases (CDKs), or cyclin-CDK complexes; (iii) K10-induced inhibition does not take place in Rb-deficient cells but is restored in these cells by cotransfection with pRb or p107 but not p130; (iv) K16 efficiently rescues the cell growth arrest induced by pRb in HaCaT cells but not that induced by p107 or p130; and (v) pRb phosphorylation and cyclin D1 expression are reduced in K10-transfected cells and increased in K16-transfected cells. Finally, using K10 deletion mutants, we map this inhibitory function to the nonhelical terminal domains of K10, hypervariable regions in which keratin-specific functions are thought to reside, and demonstrate that the presence of one of these domains is sufficient to promote cell growth arrest.

Keratin intermediate filament dynamics in cell heterokaryons reveals diverse behaviour of different keratins.

To study the dynamics of keratin intermediate filaments, we fused two different types of epithelial cells (PtK2 and BMGE+H) and studied how the keratins from the parental cells recombine and copolymerize to form the heterokaryon cytoskeleton. The behaviour of the keratins during this process was followed by immunofluorescence using specific antibodies. After fusion, the parental cytoskeletons undergo a depolymerization process most apparent in the region adjacent to the fusion area. The depolymerized subunits spread throughout the heterokaryon and copolymerize into a new hybrid cytoskeleton. The complete process is very rapid, occurring in 3-4 hours, thus demonstrating the highly dynamic nature of the keratin cytoskeleton. Although newly synthesised subunits contribute to the formation of the hybrid cytoskeleton, the process takes place with similar kinetics in the absence of protein synthesis, showing the dynamic nature of the keratins from pre-existing cytoskeletons. During this process, specific keratins behave differently. Keratins K8, K18, K5 and K10 are mobilised from the parental cytoskeletons and reassemble rapidly into the hybrid cytoskeleton (3-6 hours), whereas K14 requires a substantially longer period (9-24 hours). Thus, different keratins, even when they form part of the same heterodimeric/tetrameric complexes, as is the case for K5 and K14, exhibit different dynamics. This suggests that individual polypeptides or homopolymeric complexes rather than exclusively heterodimeric/ tetrameric subunits, as is currently thought, can also take part in keratin intermediate filament assembly and dynamics. Biochemical analysis performed in the absence of protein synthesis revealed greater amounts of K5 than of K14 in the soluble pool of BMGE+H cells. Crosslinking and immunoprecipitation experiments indicated an excess of monomeric K5, as well as of K5/K14 heterodimers and K5 homodimers in the soluble pool. These results are in agreement with the different dynamic behaviour of these keratins observed in immunofluorescence. On the contrary, the phosphorylation levels of K5 and K14 are similar in both the soluble pool and the polymerized fraction, suggesting that phosphorylation does not play an important role in the different dynamics displayed by these two proteins. In summary, our results demonstrate that, following fusion, the keratin intermediate filament network reshapes rather rapidly and that keratins are highly dynamic proteins, although this mobility depends on each particular polypeptide.