Guanosine promotes B16F10 melanoma cell differentiation through PKC-ERK 1/2 pathway

Malignant melanoma is one of the most lethal cancers. Nowadays, several anti-melanoma therapies have been employed. However, the poor prognosis and/or the increased toxicity of those treatments clearly demonstrate the requirement of searching for new drugs or novel combined chemotherapeutic protocols, contemplating both effectiveness and low toxicity. Guanosine (Guo) has been used in combination with acriflavina to potentiate the latter`s antitumor activity, through still unknown mechanisms. Here, we show that Guo induces B16F10 melanoma cell differentiation, attested by growth arrest, dendrite-like outgrowth and increased melanogenesis, and also reduced motility. A sustained ERK 1/2 phosphorylation was observed after Guo treatment and ERK inhibition led to blockage of dendritogenesis. Intracellular cyclic AMP was not involved in ERK activation, since its levels remained unchanged. Protein kinase C (PKC), in contrast to phospholipase C (PLC), inhibition completely prevented ERK activation. While the classical melanoma differentiation agent forskolin activates cAMP-PKA-Raf-MEK-ERK pathway in B16F10 cells, here we suggest that a cAMP-independent, PKC-ERK axis is involved in Guo-induced B16F10 differentiation. Altogether, our results show that Guo acts as a differentiating agent, with cytostatic rather than cytotoxic properties, leading to a decreased melanoma malignancy. Thus, we propose that Guo may be envisaged in combination with lower doses of conventional anti-melanoma drugs, in an attempt to prevent or diminish their adverse effects. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

PTCH1 gene mutations in exon 17 and loss of heterozygosity on D9S180 microsatellite in sporadic and inherited human basal cell carcinomas

Background Basal cell carcinomas (BCCs) are the most frequent human cancer that results from malignant transformation of basal cells in the epidermis. Gorlin syndrome is a rare inherited autosomal dominant disease that predisposes with multiple BCCs and other birth defects. Both sporadic and inherited BCCs are associated with mutations in the tumor suppressor gene PTCH1, but there is still uncertainty on the role of its homolog PTCH2. Objectives To search for mutations and genomic instability in sporadic and inherited BCCs. Methods DNA obtained from leukocytes and tumor cells was amplified by polymerase chain reaction regarding five exons of PTCH1 and PTCH2 and neighboring microsatellites. Exons were sequenced and compared with the GenBank database. Results Only D9S180, of six microsatellites, showed loss of heterozygosity in three BCCs (two sporadic and one inherited). One sporadic BCC presented the mutation g. 2885G>C in exon 17 of PTCH1, which predicts the substitution p.R962T in an external domain of the protein. In addition, the leukocytes and tumor cells of one patient with Gorlin syndrome showed the mutation g. 2839T>G in the same exon and gene, which predicts a p.E947stop and truncated protein. All control and tumor samples presented IVS9 + 217T in intron 9 of PTCH1. Conclusion Mutations found in the PTCH1 gene and neighboring repetitive sequences may have contributed to the development of the studied BCCs.

Comparative analysis of the expression of cytokeratins (1,10,14,16,4), involucrin, filaggrin and e-cadherin in plane warts and epidermodysplasia verruciformis plane wart-type lesions

Background: Epidermodysplasia verruciformis (EV) is a rare genodermatosis with susceptibility to human papillomavirus (HPV) infection, and high risk of skin cancer considered a model of viral oncogenesis. Methods: Fifteen cases of EV plane wart (PW)-type lesions (EV) and 14 cases of PW in healthy individuals were subjected to immunohistochemical technique for cytokeratins (K) 1, 10, 14, 16, 4, involucrin, filaggrin and e-cadherin. Results: K1/10 showed retarded or negative expression in EV, being substituted by K14. Expression of K14 occurred in the basal and suprabasal layers in both groups, but in EV, its expression was observed up to the more superficial layers. Both groups showed positivity for K16 and K4, involucrin expression in lower levels of the spinous layer and unaltered filaggrin expression. E-cadherin expression was diminished at the koilocytotic foci of both lesions, more superficially in EV. Conclusion: Infection by HPV may alter the differentiation status of the epidermis, leading to a major expression of K14, delayed or absent expression of K1/10 and earlier involucrin expression, especially in EV. It also stimulates the expression of K16 and K4. Filaggrin expression is not altered, and e-cadherin is diminished in superficial koilocytotic cells` foci in EV.

Primary Cutaneous Blastoid Mantle Cell Lymphoma-Case Report

Mantle cell lymphoma (MCL) commonly involves extranodal sites, usually as a manifestation of disseminated disease. In rare cases, MCLs may arise as a primary tumor in the skin. Blastoid mantle cell lymphoma (BV-MCL) is a rare variant and has a more aggressive clinical course. The phenotype of BV-MCL is characterized as CD20(+), CD5(+), cyclin D1(+), CD23(-), and CD10(-). Interphase fluorescence in situ hybridization shows a characteristic t(11; 14) fusion pattern. We report a case of a BV-MCL arising in skin as primary cutaneous MCL with the characteristic immunophenotype and translocation.

Pathogenesis of Endemic Pemphigus Foliaceus

Pemphigus refers to a group of human autoimmune blistering diseases involving skin and/or mucous membranes. Endemic pemphigus foliaceus (EPF), or fogo selvagem is an organ-specific autoimmune blistering disease, first reported in the beginning of the 20th century in rural areas of Brazil. The disease follows the course of streams and creeks, and vanishes after urbanization of the endemic areas. The auto-antigen related to EPF is desmoglein 1, a 160 kDa glycoprotein of the desmossomal core, targeted by in situ and circulating IgG autoantibodies, mainly of the IgG4 subclass.

Autopathogenic T helper cell type 1 (Th1) and protective Th2 clones differ in their recognition of the autoantigenic peptide of myelin proteolipid protein

We previously generated a panel of T helper cell 1 (Th1) clones specific for an encephalitogenic peptide of myelin proteolipid protein (PLP) peptide 139-151 (HSLGKWLGHPDKF) that induces experimental autoimmune encephalomyelitis (EAE) upon adoptive transfer. In spite of the differences in their T cell receptor (TCR) gene usage, all these Th1 clones required W144 as the primary and most critical TCR contact residue for the activation. In this study, we determined the TCR contact residues of a panel of Th2/Th0 clones specific for the PLP peptide 139-151 generated either by immunization with the PLP 139-151 peptide with anti-B7-1 antibody or by immunization with an altered peptide Q144. Using alanine-substituted peptide analogues of the native PLP peptide, we show that the Th2 clones have shifted their primary contact residue to the NH2-terminal end of the peptide. These Th2 cells do not show any dependence on the W144, but show a critical requirement for L141/G142 as their major TCR contact residue. Thus, in contrast with the Th1 clones that did not proliferate to A144-substituted peptide, the Th2 clones tolerated a substitution at position 144 and proliferated to A144 peptide. This alternative A144 reactive repertoire appears to have a critical role in the regulation of autoimmune response to PLP 139-151 because preimmunization with A144 to expand the L141/G142-reactive repertoire protects mice from developing EAE induced with the native PLP 139-151 peptide. These data suggest that a balance between two different T cell repertoires specific for same autoantigenic epitope can determine disease phenotype, i.e., resistance or susceptibility to an autoimmune disease.

Nuclear localization of RelB is associated with effective antigen-presenting cell function

Dendritic cells (DC) are potent APCs that enter resting tissues as precursors and, after Ag exposure, differentiate and migrate to draining lymph nodes. The phenotype of RelB knockout mice implicates this member of the NF kappa B/Rel family in DC differentiation. To further elucidate the role of RelB in DC differentiation, mRNA, intracellular protein expression, and DNA binding activity of RelB were examined in immature and differentiated human DC, as well as other PB mononuclear cell populations. RelB protein and mRNA were detected constitutively in lymphocytes and in activated monocytes, differentiated DC, and monocyte-derived DC. Immunohistochemical staining demonstrated RelB within the differentiated lymph node interdigitating DC and follicular DC, but not undifferentiated DC in normal skin. Active nuclear RelB was detected by supershift assay only in differentiated DC derived from either PB precursors or monocytes and in activated B cells. These RelB(+) APC were potent stimulators of the MLR. The data indicate that RelB expression is regulated both transcriptionally and post-translationally in myeloid cells. Within the nucleus, RelB may specifically transactivate genes that are critical for APC function.

Colorectal cancer in familial adenomatous polyposis: Are there clinical predictive factors?

Background: Familial Adenomatous Polyposis (FAP) is a hereditary disorder with multiple colorectal polyps that exhibit an almost inevitable risk of colorectal cancer (CRC) in untreated patients. Goals: To evaluate clinical features related to CRC risk at diagnosis. Material and methods: Charts from 88 patients were reviewed to collect information regarding age, family history, symptoms, polyposis severity and association with CRC. Results: 41 men (46.6%) and 47 women (53.4%) were assisted. CRC was detected in 53 patients (60.2%), with a frequency of 9.1% under 20 years, 58% between 21-40 and 85% over 41 years of age. Average age of patients without CRC was lower at treatment (29.5 vs. 40.0 years; p=0.001). Family history was reported by 58 patients (65.9%), whose average age did not differ from those who didn`t report it (33.4 vs. 34.4; p=0.17). Asymptomatic patients comprised 10.2% of the total; in this group, CRC incidence was much lower when compared to those presenting symptoms (1.1% vs. 65.8%; p=0.001). Patients without CRC presented a shorter length of symptoms (15.2 vs. 26.4 months; p=0.03) and less frequent weight loss (11.4% vs. 33.9%; p=0.01). At colonoscopy, polyposis was classified as attenuated in 12 patients (14.3%), who presented greater average age (48.2 vs. 33.3 years; p=0.02) and equal CRC incidence (58.3% vs. 58.3%; p=0.6) when compared to those with classic polyposis. Conclusions: The risk of CRC in FAP patients 1) increases significantly after the second decade; 2) is associated with higher age, weight loss, presence and duration of simptomatology; 3) is similar in patients with attenuated or classic phenotype. (C) 2010 AEC. Published by Elsevier Espana, S.L. All rights reserved.

Expression of constitutively activated human c-Kit in myb transformed early myeloid cells leads to factor independence, histiocytic differentiation, and tumorigenicity

The cDNAs encoding wild type (WT) human receptor tyrosine kinase c-Kit and a constitutively activated mutant, V816Kit, were introduced into granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent early murine hemopoietic cells, which had been transformed with activated Myb, WTKit cells were able to grow in the presence of the human ligand for Kit, stem cell factor (SCF), but displayed reduced growth and clonogenic potential in either SCF or GM-CSF compared with the parental cells in GM-CSF. In contrast, V816Kit cells grew without factor at a higher rate than the parental cells in GM-CSF and displayed increased clonogenicity. Dissection of the growth characteristics in liquid culture showed that in the presence of appropriate factors, the different populations had similar proliferation rates, but that V816Kit profoundly increased cell survival compared with WTKit or parental cells, This suggests that the signals transduced by WTKit activated with SCF, and by V816Kit, were not identical. Also, WTKit and V816Kit-expressing cells both varied from the early myeloid progenitor phenotype of the parental cells and gave rise to a small number of large to giant adherent cells that expressed macrophage (alpha-naphthyl acetate) esterase and neutrophil (naphtol-AS-D-chloroacetate) esterase, were highly phagocytic and phenotypically resembled histiocytes. Thus, WTKit activated by SCF and V816Kit were able to induce differentiation in a proportion of Myb-transformed myeloid cells. The factor independent V816Kit cells, unlike the parental and WTKit expressing cells, were shown to produce tumors of highly mitotic, invasive cells at various stages of differentiation in syngeneic mice. These results imply that constitutively activated Kit can promote the development of differentiated myeloid tumors and that its oncogenic effects are not restricted to lineages (mast cell and B-cell acute lymphoblastic leukemia), which have been reported previously. Furthermore, the mixed populations of cells in culture and in the tumors phenotypically resembled the leukemic cells from patients with monocytic leukemia with histiocytic differentiation (acute myeloid leukemia-M5c), a newly proposed subtype of myeloid leukemia. (C) 1997 by The American Society of Hematology.

Inactivation of a c-Myb/estrogen receptor fusion protein in transformed primary cells leads to granulocyte/macrophage differentiation and down regulation of c-kit but not c-myc or cdc2

Primary murine fetal hemopoietic cells were transformed with a fusion protein consisting of the ligand-binding domain of the estrogen receptor and a carboxyl-terminally truncated c-Myb protein (ERMYB), The ERMYB-transformed hemopoietic cells exhibit an immature myeloid phenotype when grown in the presence of beta-estradiol. Upon removal of beta-estradiol, the ERMYB cells display increased adherence, decreased clonogenicity and differentiate to cells exhibiting granulocyte or macrophage morphology, The expression of the c-myc, c-kit, cdc2 and bcl-2 genes, which are putatively regulated by Myb, was investigated in ERMYB cells grown in the presence or absence of beta-estradiol. Neither c-myc nor cdc2 expression was down-regulated after removal of beta-estradiol demonstrating that differentiation is not a consequence of decreased transactivation of these genes by ERMYB. While bcl-2 expression was reduced by 50% in ERMYB cells grown in the absence of beta-estradiol, there was no increase in DNA laddering, suggesting that Myb was not protecting ERMYB cells from apoptosis, In contrast, a substantial (200-fold) decrease in c-kit mRNA level was observed following differentiation of ERMYB cells, and c-kit mRNA could be partially re-induced by the re-addition of beta-estradiol. Furthermore, a reporter construct containing the c-kit promoter was activated when cotransfected with a Myb expression vector, providing further evidence of a role for Myb in the regulation of c-kit.

Limited Ca(2+) and PKA-pathway dependent neurogenic differentiation of human adult mesenchymal stem cells as compared to fetal neuronal stem cells

The ability of mesenchymal stem cells to generate functional neurons in culture is still a matter of controversy. In order to assess this issue, we performed a functional comparison between neuronal differentiation of human MSCs and fetal-derived neural stem cells (NSCs) based on morphological, immunocytochemical, and electrophysiological criteria. Furthermore, possible biochemical mechanisms involved in this process were presented. NF200 immunostaining was used to quantify the yield of differentiated cells after exposure to CAMP. The addition of a PKA inhibitor and Ca(2+) blockers to the differentiation medium significantly reduced the yield of differentiated cells. Activation of CREB was also observed on MSCs during maturation. Na(+)-, K(+)-, and Ca(2+)-voltage-dependent currents were recorded from MSCs-derived cells. In contrast, significantly larger Na(+) currents, firing activity, and spontaneous synaptic currents were recorded from NSCs. Our results indicate that the initial neuronal differentiation of MSCs is induced by CAMP and seems to be dependent upon Ca(2+) and the PKA pathway. However, compared to fetal neural stem cells, adult mesenchymal counterparts are limited in their neurogenic potential. Despite the similar yield of neuronal cells, NSCs achieved a more mature functional state. Description of the underlying mechanisms that govern MSCs` differentiation toward a stable neuronal phenotype and their limitations provides a unique opportunity to enhance our understanding of stem cell plasticity. (C) 2009 Elsevier Inc. All rights reserved.

Stem cells in endometrium and their role in the pathogenesis of endometriosis

The human endometrium is a dynamic tissue that undergoes cycles of growth and regression with each menstrual cycle. Adult progenitor stem cells are likely responsible for this remarkable regenerative capacity; these same progenitor stem cells may also have an enhanced capacity to generate endometriosis if shed in a retrograde fashion. The progenitor stem cells reside in the uterus; however, less-committed mesenchymal stem cells may also travel from other tissues such as bone marrow to repopulate the progenitor population. Mesenchymal stem cells are also involved in the pathogenesis of endometriosis and may be the principle source of endometriosis outside of the peritoneal cavity when they differentiate into endometriosis in ectopic locations. Finally, besides progenitor stem cells, recent publications have identified multipotent stem cells in the endometrium. These multipotent stem cells are a readily available source of cells that are useful in tissue engineering and regenerative medicine. Endometrial stem cells have been used to generate chondrocytes, myocytes, neurons, and adiposites in vitro as well as to replace dopaminergic neurons in a murine model of Parkinson`s disease.

A new FOXL2 gene mutation in a woman with premature ovarian failure and sporadic blepharophimosis-ptosis-epicanthus inversus syndrome

Objective: To describe a new FOXL2 gene mutation in a woman with sporadic blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) and hypergonadotropic hypogonadism. Design: Case report. Setting: University medical center. Patient(s): A 28-year-old woman. Intervention(s): Clinical evaluation, hormone assays, gene mutation research. Main Outcome Measure(s): FOXL2 gene mutation. Result(s): The patient with hypergonadotropic hypogonadism was diagnosed with BPES due to a new FOXL2 gene mutation. Conclusion(s): Blepharophimosis-ptosis-epicanthus inversus syndrome is a rare disorder associated with premature ovarian failure (POF). The syndrome is an autosomal dominant trait that causes eyelid malformations and POF in affected women. Mutations in FOXL2 gene, located in chromosome 3, are related to the development of BPES with POF (BPES type I) or without POF (BPES type II). This report demonstrates a previously undescribed de novo mutation in the FOXL2 gene-a thymidine deletion, c. 627delT (g. 864delT)-in a woman with a sporadic case of BPES and POF. This mutation leads to truncated protein production that is related to a BPES type I phenotype. This report shows the importance of family history and genetic analysis in the evaluation of patients with POF and corroborates the relationship between mutations on the FOXL2 gene and ovarian insufficiency. (Fertil Steril (R) 2010; 93: 1006.e3-e6. (C) 2010 by American Society for Reproductive Medicine.)

TNF receptor-associated periodic syndrome (TRAPS): Description of a novel TNFRSF1A mutation and response to etanercept

TRAPS is the most common of the autosomal dominant periodic fever syndromes. It is caused by mutations in the TNFRSF1A gene, which encodes for the type 1 TNF-receptor (TNFR1). We describe here a Brazilian patient with TRAPS associated to a novel TNFRSF1A de novo mutation and the response to anti-TNF therapy. The patient is a 9-year-old girl with recurrent fevers since the age of 3 years, usually lasting 3 to 7 days, and recurring every other week. These episodes are associated with mild abdominal pain, nausea, vomiting and generalized myalgia. Recurrent conjunctivitis and erysipela-like skin lesions in the lower limbs also occur. Laboratory studies show persistent normocytic normochromic anemia, thrombocytosis, elevated erythrocyte sedimentation rate and C-reactive protein. IgD levels are normal. Mutational screening of TNFRSF1A revealed the association of a novel C30F mutation with the common R92Q low-penetrance mutation. The R92Q mutation is seen in 5% of the general population and is associated with an atypical inflammatory phenotype. The patient had a very good response to etanercept, with cessation of fever and normalization of inflammatory markers. Our report expands the spectrum of TNFRSF1A mutations associated with TRAPS, adding further evidence for possible additive effects of a low-penetration R92Q and cysteine residue mutations, and confirms etanercept as an efficacious treatment alternative.

Decreased AIRE Expression and Global Thymic Hypofunction in Down Syndrome

The Down syndrome (DS) immune phenotype is characterized by thymus hypotrophy, higher propensity to organ-specific autoimmune disorders, and higher susceptibility to infections, among other features. Considering that AIRE (autoimmune regulator) is located on 21q22.3, we analyzed protein and gene expression in surgically removed thymuses from 14 DS patients with congenital heart defects, who were compared with 42 age-matched controls with heart anomaly as an isolated malformation. Immunohistochemistry revealed 70.48 +/- 49.59 AIRE-positive cells/mm(2) in DS versus 154.70 +/- 61.16 AIRE-positive cells/mm(2) in controls (p < 0.0001), and quantitative PCR as well as DNA microarray data confirmed those results. The number of FOXP3-positive cells/mm(2) was equivalent in both groups. Thymus transcriptome analysis showed 407 genes significantly hypoexpressed in DS, most of which were related, according to network transcriptional analysis (FunNet), to cell division and to immunity. Immune response-related genes included those involved in 1) Ag processing and presentation (HLA-DQB1, HLA-DRB3, CD1A, CD1B, CD1C, ERAP) and 2) thymic T cell differentiation (IL2RG, RAG2, CD3D, CD3E, PRDX2, CDK6) and selection (SH2D1A, CD74). It is noteworthy that relevant AIRE-partner genes, such as TOP2A, LAMNB1, and NUP93, were found hypoexpressed in DNA microarrays and quantitative real-time PCR analyses. These findings on global thymic hypofunction in DS revealed molecular mechanisms underlying DS immune phenotype and strongly suggest that DS immune abnormalities are present since early development, rather than being a consequence of precocious aging, as widely hypothesized. Thus, DS should be considered as a non-monogenic primary immunodeficiency. The Journal of Immunology, 2011, 187: 3422-3430.