CCAAT/enhancer-binding protein alpha (CEBPA) gene haploinsufficiency does not alter hematopoiesis or induce leukemia in Lck-CALM/AF10 transgenic mice.

Although rare, CALM/AF10 is a chromosomal rearrangement found in immature T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia, and mixed phenotype acute leukemia of T/myeloid lineages with poor prognosis. Moreover, this translocation is detected in 50% of T-ALL patients with gamma/delta T cell receptor rearrangement, frequently associated with low expression of transcription factor CCAAT/enhancer-binding protein alpha (CEBPA). However, the relevance of CEBPA low expression for CALM/AF10 leukemogenesis has not yet been evaluated. We generated double mutant mice, which express the Lck-CALM/AF10 fusion gene and are haploinsufficient for the Cebpa gene. To characterize the hematopoiesis, we quantified hematopoietic stem cells, myeloid progenitor cells, megakaryocyte-erythrocyte progenitor cells, common myeloid progenitor cells, and granulocyte-macrophage progenitor cells. No significant difference was detected in any of the progenitor subsets. Finally, we tested if Cebpa haploinsufficiency would lead to the expansion of Mac-1+/B220+/c-Kit+ cells proposed as the CALM/AF10 leukemic progenitor. Less than 1% of bone marrow cells expressed Mac-1, B220, and c-Kit with no significant difference between groups. Our results showed that the reduction of Cebpa gene expression in Lck-CALM/AF10 mice did not affect their hematopoiesis or induce leukemia. Our data corroborated previous studies suggesting that the CALM/AF10 leukemia-initiating cells are early progenitors with lymphoid/myeloid differentiating potential.

CCAAT/enhancer-binding protein alpha (CEBPA) gene haploinsufficiency does not alter hematopoiesis or induce leukemia in Lck-CALM/AF10 transgenic mice

Although rare, CALM/AF10 is a chromosomal rearrangement found in immature T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia, and mixed phenotype acute leukemia of T/myeloid lineages with poor prognosis. Moreover, this translocation is detected in 50% of T-ALL patients with gamma/delta T cell receptor rearrangement, frequently associated with low expression of transcription factor CCAAT/enhancer-binding protein alpha (CEBPA). However, the relevance of CEBPA low expression for CALM/AF10 leukemogenesis has not yet been evaluated. We generated double mutant mice, which express the Lck-CALM/AF10 fusion gene and are haploinsufficient for the Cebpa gene. To characterize the hematopoiesis, we quantified hematopoietic stem cells, myeloid progenitor cells, megakaryocyte-erythrocyte progenitor cells, common myeloid progenitor cells, and granulocyte-macrophage progenitor cells. No significant difference was detected in any of the progenitor subsets. Finally, we tested if Cebpa haploinsufficiency would lead to the expansion of Mac-1+/B220+/c-Kit+ cells proposed as the CALM/AF10 leukemic progenitor. Less than 1% of bone marrow cells expressed Mac-1, B220, and c-Kit with no significant difference between groups. Our results showed that the reduction of Cebpa gene expression in Lck-CALM/AF10 mice did not affect their hematopoiesis or induce leukemia. Our data corroborated previous studies suggesting that the CALM/AF10 leukemia-initiating cells are early progenitors with lymphoid/myeloid differentiating potential.

Qualitative analysis of striated skeletal muscles in thedystrophynopathy of mdx mice submitted to physical activity

Duchenne’s Muscular Dystrophy (DMD) is a recessive hereditary myopathy linked to the chromosome X,caused by a mutation in the dystrophin gene, which strengthens and stabilizes the sarcolemma during the stressof muscular contraction and, when absent, the sarcollema ruptures and allows calcium to enter, which causesthe muscle fiber to necrotize. The object of the present study was to perform the morphologic analysis of theanterior tibial and the gastrocnemius muscles with (w/pa) or without (n/pa) physical activity for five weeks.We used 72 mice, divided in 12 experimental groups – 6 of them mdx and 6 control groups (C57/10J) aged4, 7, and 10 weeks. The samples were collected, processed and stained with hematoxylin-eosin. They wereanalyzed by light microscopy, selected and photomicrographed. On the cross-sections of control animals aged4, 7 and 10 weeks w/pa and n/pa, polygonal muscle fibers of many different sizes, ellipse-shaped and withseveral peripheral nucleuses were observed. In the mdx mice w/pa aged 4, 7 and 10 weeks, the muscle fibersshowed different shapes, sizes and stain affinities, rounded edges, anuclear or centered nucleuses; hyalines andmyofibrillas were highly contracted. Muscular regeneration and nectrotic areas with inflammatory infiltrateswere identified in the mdx animals aged 4, 7 and 10 weeks w/pa, as well as in animals aged 10 weeks n/pa.With the progression of the disease in the animals submitted to physical activity, there was evidence of failurein the regeneration and muscular degeneration, intensified and characterized by the gradual replacement ofthe striated skeletal muscle tissue by fibroadipose connective tissue.

Sclerocorneal limbal stem cell autograft transplantation in dogs

The effects of sclerocorneal limbal stem cell autograft transplantation in dogs with corneal wounds were studied. Eighteen dogs were divided in two groups (GI and GII). The animals of GI (n=12) underwent limbal transplantation 30 days after the destruction of limbal stem cells. The dogs of GII (n=6) only underwent destruction of stem cells (control group). Light microscopy examination of the right eye was performed on days 3, 7, 14, 30, 60, and 120 after limbal transplantation (GI), and on days 33, 37, 44, 60, 90, and 150 after limbal destruction (GII). Results showed a complete destruction of limbal stem cells with loss of corneal transparency. Limbal transplantation prevented conjunctivalization in grafted area. Corneal vascularization and a 360º corneal conjunctivalization were noted in the control dogs (GII). Corneal transparency was restored from day 60th after surgery. Histological examination did not distinguish the transition between the graft and the normal corneal epithelium at anytime. Goblet cells were found in control animals (GII) on 33, 37, 60, and 150 days, whereas a single grafted dog (GI) presented a few goblet cells on day 60th post-transplantation. Limbal autograft transplantation was effective in restoring corneal clarity with no development of ocular complications

Avaliaram-se os efeitos do transplante de células tronco autógenas do limbo esclerocórneo de cães, sobre lesões córneo-limbais. Empregaram-se 18 cães, distribuídos em dois grupos, GI e GII. Nos animais do GI (n=12), foram realizados transplantes de limbo, após 30 dias da destruição das células tronco-límbicas. Nos do GII (n=6), realizou-se apenas a destruição do limbo (controle). Aos 3, 7, 15, 30, 60 e 120 dias do transplante de limbo (GI) e aos 33, 37, 45, 60, 90 e 150 dias da destruição do limbo (GII), os olhos foram coletados por enucleação subconjuntival, para estudos em microscopia de luz. A destruição do limbo resultou em completa excisão das células tronco, com perda da transparência corneal. O transplante do limbo evitou a conjuntivalização na área em que foi realizado. Os animais do grupo-controle manifestaram conjuntivalização em 360º e vascularização corneal. Na anatomopatologia, em nenhum dos períodos foi possível distinguir o enxerto do epitélio corneal normal. As células caliciformes foram observadas nos animais do GII, nos períodos 33, 37, 60, 150 dias. No GI, apenas um cão manifestou células caliciformes de forma discreta, aos 60 dias do transplante. O transplante autógeno foi eficiente em possibilitar a melhoria da transparência córnea, sem intercorrências oculares

p63 correlates with both BRCA1 and cytokeratin 5 in invasive breast carcinomas: further evidence for the pathogenesis of the basal phenotype of breast cancer.

AIMS: To study the expression of p63, cytokeratin (CK) 5 and CK8/18 in invasive ductal carcinomas and their relationship with BRCA1 and other pathological and immunohistochemical features of clinical significance. METHODS AND RESULTS: Immunohistochemistry with the antibodies p63, CK5, CK8/18, BRCA1, oestrogen receptor, progesterone receptor, p53, c-erbB-2 and Ki67 was performed in 102 formalin-fixed paraffin-embedded samples of invasive ductal carcinomas. The CK5+ cases were submitted to a double-immunolabelling study with p63. There was a strong relationship between CK5 and p63 expression and both markers were associated with hormonal receptor-negative high-grade carcinomas with high proliferative rate. Furthermore, there was coexpression of CK5 and p63 in neoplastic cells, indicating that p63, like CK5, is a marker of the basal phenotype of breast cancer. There was a strong relationship between reduced expression of BRCA1 with both p63 and CK5 expression as well as an inverse correlation between p63 and CK8/18 expression, suggesting that loss of p63 expression is required for the transition between a basal to a luminal phenotype of breast carcinoma. CONCLUSIONS: Since p63 is thought to be a marker of stem cells and may act as an oncogene, our data support the idea that BRCA1 acts as stem cell regulator.

Implication of stem cell factor in the proliferation of choroidal melanocytes.

The tyrosine kinase receptor c-kit and its ligand stem cell factor exert a broad range of biological activities during organogenesis. It also improves normal cell development including complex biological responses involved in the differentiation and proliferation of the melanocytes. Diffuse uveal melanocytic proliferation is a rare paraneoplasic syndrome, resulting in rapid bilateral visual loss due to proliferation of melanocytes within the choroid. We have therefore investigated whether the c-kit/stem cell factor pathway regulates the proliferation of choroidal melanocytes and also if such pathway plays a role in bilateral uveal melanocytic proliferation. Normal cultured melanocytes of the choroid and paraffin-embedded sections of melanocytic proliferation were studied. C-kit expression and effects of stem cell factor were measured. Western blot assays of cell extracts demonstrated that c-kit was expressed in choroidal melanocytes. Immunocytochemical analysis on cultured melanocytes showed a cytoplasmic distribution. Immunohistochemical analysis on melanocytic proliferation showed a strong cytoplasmic distribution in the pigmented spindle-shaped melanocytes localized in the multiple focal areas of choroidal thickening. The addition of stem cell factor did not change melanocyte morphologies and was mitogenic in the presence of bFGF, isobutyl-1-methylxanthine and cholera toxin. In contrast, stem cell factor was not able to produce any significant melanin. Activation of c-kit by its ligand may contribute to the proliferation of choroidal melanocytes.

Bilateral diffuse uveal melanocytic proliferation associated with extraocular cancers: review of a process particularly associated with gynecologic cancers.

We reviewed cases of a paraneoplastic syndrome in which uveal melanocytes proliferated and led to blindness. Eighteen cases were derived from the literature, and two were taken from our institution. The average patient age at the time of the diagnosis was 63 years (range, 34-89 years). There were 13 women and 7 men. In approximately half of the cases, the ocular symptoms antedated those of the inciting tumor. Most of the inciting tumors were poorly differentiated carcinomas. The most common tumors were from the female genital tract (ovary and uterus) among the women patients and from the lung among the men. Tumors from the breast were rare (one possible case), and tumors of the prostate were conspicuously absent. All five inciting tumors whose histopathology was reviewed expressed neuron-specific enolase, but none prominently expressed antigens more specific for neuroendocrine carcinomas such as chromogranin or synaptophysin. It is our experience that many general pathologists are not aware of this unique paraneoplastic syndrome. Our report is the first to document a statistically significant association between this syndrome and gynecologic cancers.