Induction of p38, tumour necrosis factor-α and RANTES by mechanical stretching of keratinocytes expressing mutant keratin 10R156H.

BACKGROUND: Epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma), characterized by ichthyotic, rippled hyperkeratosis, erythroderma and skin blistering, is a rare autosomal dominant disease caused by mutations in keratin 1 or keratin 10 (K10) genes. A severe phenotype is caused by a missense mutation in a highly conserved arginine residue at position 156 (R156) in K10. OBJECTIVES: To analyse molecular pathomechanisms of hyperproliferation and hyperkeratosis, we investigated the defects in mechanosensation and mechanotransduction in keratinocytes carrying the K10(R156H) mutation. METHODS: Differentiated primary human keratinocytes infected with lentiviral vectors carrying wild-type K10 (K10(wt)) or mutated K10(R156H) were subjected to 20% isoaxial stretch. Cellular fragility and mechanosensation were studied by analysis of mitogen-activated protein kinase activation and cytokine release. RESULTS: Cultured keratinocytes expressing K10(R156H) showed keratin aggregate formation at the cell periphery, whereas the filament network in K10(wt) cells was normal. Under stretching conditions K10(R156H) keratinocytes exhibited about a twofold higher level of filament collapse compared with steady state. In stretched K10(R156H) cells, higher p38 activation, higher release of tumour necrosis factor-α and RANTES but reduced interleukin-1ß secretion compared with K10(wt) cells was observed. CONCLUSIONS: These results demonstrate that the R156H mutation in K10 destabilizes the keratin intermediate filament network and affects stress signalling and inflammatory responses to mechanical stretch in differentiated cultured keratinocytes.

Antifungal drug response in an in vitro model of dermatophyte nail infection.

Despite terbinafine being fungicidal against Trichophyton rubrum in standard NCCLS assays and rapidly accumulating in nails in vivo, onychomycosis patients require prolonged terbinafine treatment to be cured. To investigate this, we developed a more clinically relevant onychomycosis in vitro test model. Human nail powder inoculated with T. rubrum and incubated in liquid RPMI 1640 salt medium, which did not support growth alone, developed extensive and invasive mycelial growth. Antifungal drugs were added at different concentrations and cultures incubated for 1 to 4 weeks. Fungal survival was determined by spreading cultures on PDA plates without drug and measuring CFU after 1 to 4 weeks incubation. Drug activity was expressed as the nail minimum fungicidal concentration (Nail-MFC) required for 99.9% elimination of viable fungus. Terbinafine Nail-MFC was 4 microg/ml after 1 week exposure, decreasing to 1 microg/ml after 4 weeks exposure, much higher than MFCs < or = 0.03 microg/ml determined in standard NCCLS MIC assays. In contrast, other clinically used drugs were unable to kill T. rubrum after 4 weeks incubation in this model. Invasive mycelial growth on nail appears to protect T. rubrum from the cidal action of systemic drugs, thus providing a rationale for the long treatment periods in onychomycosis.

French multi-centric study of 2000 amniotic fluid interphase FISH analyses from high-risk pregnancies and review of the literature.

This prospective and multi-centric study confirms the accuracy and the limitations of interphase FISH and shows that any cytogenetics laboratory can perform this technique. With regard to the technical approach, we think that slides must be examined by two investigators, because the scoring may be subjective. The main problem with the AneuVysion kit concerns the alpha satellite probes, and especially the chromosome 18 probe, which is sometimes very difficult to interpret because of the high variability of the size of the spots, and this may lead to false negative and uninformative cases. The best solution would be to replace these probes by locus-specific probes. Concerning clinical management, we offer interphase FISH only in very high-risk pregnancies or/and at late gestational age because of the cost of the test. We think that an aberrant FISH result can be used for a clinical decision when it is associated with a corresponding abnormal ultrasound scan. In other cases, most of the time, we prefer to wait for the standard karyotype.

Minor dysplastic changes are frequently observed in the bone marrow aspirate in elderly patients without haematological disease.

Bone marrow aspirates were obtained by sternal puncture prior to sternotomy in 54 volunteers (40 males and 14 females) aged 60 years or more. All underwent surgery for cardiological diseases and had normal blood counts, without any haematological abnormalities. Quantitative examination of these bone marrow aspirates yielded reference ranges for each cell type similar to those obtained in younger adults. However, qualitative analysis revealed certain discrepancies: dysplastic changes were observed frequently, mainly in megakaryocytes and erythroblasts, with a normal growth pattern of haematopoietic progenitor cells. A low proportion of macrophages and mast cells were noted in 30% of the bone marrow aspirates. Lymphoid aggregates, seen in 13% of these samples, were generally of moderate size, contained few mast cells and were non-clonal on immunophenotypic analysis.

The hemidesmosomal protein bullous pemphigoid antigen 1 and the integrin beta 4 subunit bind to ERBIN. Molecular cloning of multiple alternative splice variants of ERBIN and analysis of their tissue expression.

The bullous pemphigoid antigen 1 (eBPAG1) is a constituent of hemidesmosomes (HDs), cell-substrate adhesion complexes in stratified epithelia. Although its COOH terminus interacts with intermediate filaments, its NH(2) terminus is important for its recruitment into HDs. To identify proteins that interact with the NH(2) terminus of human eBPAG1, we performed a yeast two-hybrid screen, which uncovered a protein belonging to the LAP/LERP (for LRR and PDZ domain) protein family with 16 NH(2)-terminal leucine-rich repeats and a COOH-terminal PDZ domain. The gene for this LAP/LERP protein comprises at least 26 exons located on the long arm of chromosome 5. In most human tissues, several transcripts were detected differing in the coding region situated upstream of or within the PDZ domain. One of the encoded variants was found to correspond to the recently described protein ERBIN. In yeast and in vitro binding experiments, ERBIN was shown to interact not only with eBPAG1 but also with the COOH-terminal region of the cytoplasmic domain of the integrin beta4 subunit, another component of HDs. Antibodies raised against the COOH terminus showed that ERBIN is expressed in keratinocytes. In transfected epithelial cells the protein, however, was not localized in HDs but was either diffusely distributed over the cytoplasm or concentrated at the basolateral plasma membrane. Because ERBIN had been shown previously to interact with the transmembrane tyrosine kinase receptor Erb-B2, which in turn associates with the integrin beta4 subunit, we suggest that ERBIN provides a link between HD assembly and Erb-B2 receptor signaling.

Autoantibodies to the extracellular and intracellular domain of bullous pemphigoid 180, the putative key autoantigen in bullous pemphigoid, belong predominantly to the IgG1 and IgG4 subclasses.

BACKGROUND: Autoantibodies to the extracellular domain (ECD) of bullous pemphigoid (BP) antigen 180 (BP180) are thought to play a crucial part in the pathophysiology of BP. OBJECTIVES: As the various IgG subclasses have different biological properties, we have sought to assess the relative isotype distribution of IgG to BP180 and their reactivity against the ECD and intracellular domain (ICD) of BP180. METHODS: The reactivity of 27 sera from patients with BP was assayed by immunoblotting against recombinant proteins covering the ECD and ICD of BP180. RESULTS: Twenty-seven (100%) and 21 (77%) of 27 BP sera, respectively, contained IgG1 and IgG4 autoantibodies binding to the ECD of BP180. Fourteen (82%) and six (35%) of the 17 BP sera that were reactive with the ICD of BP180 had autoantibodies of the IgG1 and IgG4 subclass, respectively. The profile of the isotype restriction appeared to be similar when the response to the ECD vs. that to the ICD was compared. IgG2 and IgG3 reactivity with BP180 was found less frequently. Patients with BP of longer duration showed a tendency to have, in addition to IgG1, an IgG4 response. CONCLUSIONS: Consistent with prior evidence indicating that subepidermal blister formation in BP is dependent upon complement activation, the frequent finding of complement-fixing IgG1 autoantibodies to both the ECD and ICD of BP180 might have pathogenic relevance in BP. These findings provide new insights relevant for our understanding of the immune response to BP180, the putative key autoantigen in BP.

Plectin, an unusual target antigen in bullous pemphigoid.

BACKGROUND: Bullous pemphigoid (BP) is a blistering disease associated with autoantibodies directed against two components of hemidesmosomes, BP180 and BP230. OBJECTIVES: To assess whether BP patients have autoantibodies targeting plectin, another hemidesmosomal component showing extensive homology to BP230. METHODS: Examination of sera from 16 patients with BP, using immunoprecipitation studies followed by immunoblotting. RESULTS: Serum of one of the 16 (6%) patients with BP contain autoantibodies binding to plectin, while no reactivity was found with sera from three control subjects. Sera from all 16 BP patients immunoprecipitated BP230 from extracts of biosynthetically radiolabelled human keratinocytes. CONCLUSIONS: Our results indicate that sera from BP patients might contain autoantibodies binding to plectin. Although this protein and BP230 are closely sequence-related, the occurrence of autoantibodies binding to plectin is a rare phenomenon in BP.

The 8p12 myeloproliferative disorder. t(8;19)(p12;q13.3): a novel translocation involving the FGFR1 gene.

Translocations affecting the chromosomal locus 8p12 are hallmarks of an atypical stem cell myeloproliferative disorder. These events disrupt the fibroblast growth factor receptor 1 (FGFR1) gene and fuse the FGFR1 C-terminus catalytic domain with unrelated proteins. Here, we report on the characterization of the 19q13.3 locus as the fifth FGFR1 chromosomal partner.

Acute myeloid leukemia with hypergranular cytoplasm: a differential diagnosis of acute promyelocytic leukemia.

We report here the case of a woman with acute myeloid leukemia with some blast cells exhibiting acute promyelocytic leukemia (APL)-like hypergranular cytoplasm. The cytologic and cytochemical aspects as well as the mature myeloid phenotype and hemostasis disorders were consistent with the diagnosis of APL. However, no t(15;17), or RARalpha gene, MLL gene or PML gene rearrangement was observed, or any other cytogenetic clonal abnormality. Coexpression on blast cells of CD33 and CD56 without CD34, CD16 or HLA-DR, suggested a myeloid/natural killer cell acute leukemia.

Immunophenotype of a transient myeloproliferative disorder in a newborn with trisomy 21.

Cytologic, immunologic, and cytogenetic studies were performed on the blast cells of a newborn with Down syndrome and transient myeloproliferative disease. This hematologic disorder is uncommon, and occurs primarily in infants with Down syndrome. This boy presented with a high white blood cell count and a high percentage of blast cells, without anemia or thrombocytopenia. Chromosome analysis showed a constitutional trisomy 21 without any other clonal abnormality. A three-color flow cytometric analysis was performed and revealed two different CD45 dim, CD34(+), CD117(+), CD56(+) immature subpopulations: the normal immature myeloid precursor and an immature blast cell population that expressed CD41, CD42, CD61, CD36, CD13, CD1a, and CD2. We postulate that this population could be the leukemic precursor involved in the acute megakaryoblastic leukemia frequently observed in children with Down syndrome.

IgG autoantibodies from bullous pemphigoid patients recognize multiple antigenic reactive sites located predominantly within the B and C subdomains of the COOH-terminus of BP230.

Bullous pemphigoid is a subepidermal bullous disorder characterized by an autoantibody response against the bullous pemphigoid antigen 230 (BP230) and the bullous pemphigoid antigen 180 (BP180), a cytoplasmic component and a transmembrane component, respectively, of hemidesmosomes. Although immunodominant sequences within the extracellular domain of BP180 have been identified, characterization of the antigenic sites on BP230 is still incomplete. To identify autoantibody-reactive sites on BP230 and to examine whether the targeted regions are contained within functionally important domains, recombinant fragments encompassing almost the entire BP230 were used to assess the reactivity of 25 bullous pemphigoid sera by immunoblotting. Our results demonstrate that (i) the region bearing the B and C subdomains of the COOH-terminus of BP230 contains immunodominant sequences recognized by the majority of bullous pemphigoid sera; (ii) additional autoantibody- reactive sites are present over extended regions of the NH2-terminal half of BP230 without evidence for antigenic cross-reactivity between the NH2- and COOH-termini of BP230; and, finally, (iii) autoantibodies reacting with the BP230 tail predominantly belong to the IgG4 and IgG1 subclasses, suggesting that both autoreactive TH2 and autoreactive TH1 cells regulate the autoantibody response to immunodominant sequences of BP230. As the COOH- terminus of BP230 mediates the attachment of keratin intermediate filaments to the hemidesmosomal plaque, whereas its NH2-terminus contains sequences important for its interaction with other constituents of hemidesmosomes, autoantibodies to BP230 might precipitate subepidermal blister formation and perpetuate the disease not only by eliciting an inflammatory reaction but also by interfering with the function of BP230 and thus the stability of hemidesmosomes.

Multiple amino acid substitutions in lanosterol 14alpha-demethylase contribute to azole resistance in Candida albicans.

Lanosterol 14alpha-demethylase (14DM) is the target of the azole antifungals, and alteration of the 14DM sequence leading to a decreased affinity of the enzyme for azoles is one of several potential mechanisms for resistance to these drugs in Candida albicans. In order to identify such alterations the authors investigated a collection of 19 C. albicans clinical isolates demonstrating either frank resistance (MICs > or = 32 microg ml(-1)) or dose-dependent resistance (MICs 8-16 microg ml(-1)) to fluconazole. In cell-free extracts from four isolates, including the Darlington strain ATCC 64124, sensitivity of sterol biosynthesis to inhibition by fluconazole was greatly reduced, suggesting that alterations in the activity or affinity of the 14DM could contribute to resistance. Cloning and sequencing of the 14DM gene from these isolates revealed 12 different alterations (two to four per isolate) leading to changes in the deduced amino acid sequence. Five of these mutations have not previously been reported. To demonstrate that these alterations could affect fungal susceptibility to azoles, the 14DM genes from one sensitive and three resistant C. albicans strains were tagged at the carboxyl terminus with a c-myc epitope and expressed in Saccharomyces cerevisiae under control of the endogenous promoter. Transformants receiving 14DM genes from resistant strains had fluconazole MICs up to 32-fold higher than those of transformants receiving 14DM from a sensitive strain, although Western blot analysis indicated that the level of expressed 14DM was similar in all transformants. Amino acid substitutions in the 14DM gene from the Darlington strain also conferred a strong cross-resistance to ketoconazole. In conclusion, multiple genetic alterations in C. albicans 14DM, including several not previously reported, can affect the affinity of the enzyme for azoles and contribute to resistance of clinical isolates.

Two cases of terminal deletion of chromosome 13: clinical features, conventional and molecular cytogenetic analysis.

We report the cases of two unrelated patients with psychomotor retardation and craniofacial abnormalities, in whom cytogenetic studies have revealed a terminal deletion of chromosome 13 confirmed by fluorescence in situ hybridization (FISH). This del(13)(q33.2) is the smallest terminal deletion of the 13q reported so far. Interestingly enough, the serum level of coagulation factors VII and X, whose genes are located in 13q34, were reduced in both patients. These cases illustrate the difficulties in identifying precisely chromosome deletions and demonstrate that FISH techniques allow to obtain a more precise correlation between clinical phenotype and cytogenetic abnormalities.

IgG autoantibodies from bullous pemphigoid (BP) patients bind antigenic sites on both the extracellular and the intracellular domains of the BP antigen 180.

Bullous pemphigoid (BP) and gestational pemphigoid (PG) are subepidermal blistering disorders associated with autoantibodies directed against two components of hemidesmosomes: the BP antigen 180 (BP180) and the BP antigen 230 (BP230). Autoantibodies against the extracellular domain (ECD) of BP180 are thought to play an initiatory role in subepidermal blister formation. To characterize the targeted antigenic sites on BP180, we have assessed the reactivity of sera from BP and PG patients against eukaryotic recombinant proteins encompassing various portions of the ECD and the intracellular domain (ICD) of BP180. Twenty-two of 22 (100%) BP sera that immunoblotted BP180 in keratinocyte extracts, bound a mutant form consisting of the entire ECD of BP180, whereas only three of these 22 sera (14%) reacted against the ECD of BP180 lacking the NC16A membrane proximal region. Thirteen out of the 22 (59%) BP sera recognized the ICD of BP180. Circulating IgG from a representative BP patient that was affinity purified against the ECD of BP180 did not bind the ICD when reblotted, indicating that there was no antigenic cross-reactivity between the ECD and the ICD of BP180. Reactivity against the ICD of BP180 was further ascertained by immunofluorescence microscopy studies showing that nine of the 22 (41%) BP sera stained COS-7 cells expressing the ICD of BP180. Using deletion mutants of the ICD of BP180, the majority of the sera was found to recognize the central region of the ICD of BP180. Specifically, an immunodominant region was localized to an 87-amino acid segment located towards the NH2-terminus of BP180. In contrast to BP sera, five of six (83%) PG sera contained IgG that recognized exclusively the NC16A region, whereas none bound to the ICD of BP180. Together, the results indicate that in BP, autoantibody reactivity to BP180 is not exclusively restricted to the NC16A region, but that additional antigenic determinants exist on the ICD of BP180. The observed heterogeneous immune response against BP180 might reflect intramolecular epitope spreading. Because the ICD ofBP180 harbors functionally important regions, it is possible that autoantibodies against the ICD of BP180 have pathogenic significance for the progression of the disease.

Translocation 1;19 in two brain tumors.

We report two-cases of brain tumors, one childhood medulloblastoma and one adult glioblastoma with an unusual chromosomal abnormality: a t(1;19)(q23;q13). We analyzed these karyotypes using fluorescence in situ hybridization (FISH) and wonder if this chromosomal aberration could represent a particular entity in these brain tumors like t(1;19) in ALL.

Modulation of the enzymatic properties of protein phosphatase 2A catalytic subunit by the recombinant 65-kDa regulatory subunit PR65alpha.

All protein phosphatase 2A (PP2A) holoenzymes contain a 36-kDa catalytic subunit (PP2Ac) and a regulatory subunit of 65 kDa (PR65). We have studied the interaction between PP2Ac and PR65 in an in vitro system, using PP2Ac isolated from rabbit skeletal muscle and recombinant PR65alpha expressed in bacteria or insect cells. Bacterially expressed PR65alpha exhibited identical biochemical properties to the protein expressed and isolated from the baculoviral expression system. The association of recombinant PR65 with PP2Ac was very tight (K(D)app = 85 pM) and led to a suppression of PP2A activity, which was maximal (70-80%) when phosphoproteins were used as substrates. When less-structured or smaller substrates (such as phosphopeptides) were used, this inhibition was only 30%. PR65 stimulated PP2Ac activity when the assays were performed in the presence of polycations. This indicates that the PR65 not only serves the previously predicted structural role as a molecular scaffold, but also allosterically modulates the enzymatic properties of PP2Ac. Furthermore, we identified a site of interaction between PP2Ac and PR65alpha by disruption of a stretch of basic amino acids by introduction of a glutamate at position 416. This produced an almost 100-fold reduced affinity for PP2Ac and indicated that this basic motif is an important determinant for the interaction of PR65 and PP2Ac.