Applying enhancement filters in the pre-processing of images of lymphoma

Lymphoma is a type of cancer that affects the immune system, and is classified as Hodgkin or non-Hodgkin. It is one of the ten types of cancer that are the most common on earth. Among all malignant neoplasms diagnosed in the world, lymphoma ranges from three to four percent of them. Our work presents a study of some filters devoted to enhancing images of lymphoma at the pre-processing step. Here the enhancement is useful for removing noise from the digital images. We have analysed the noise caused by different sources like room vibration, scraps and defocusing, and in the following classes of lymphoma: follicular, mantle cell and B-cell chronic lymphocytic leukemia. The filters Gaussian, Median and Mean-Shift were applied to different colour models (RGB, Lab and HSV). Afterwards, we performed a quantitative analysis of the images by means of the Structural Similarity Index. This was done in order to evaluate the similarity between the images. In all cases we have obtained a certainty of at least 75%, which rises to 99% if one considers only HSV. Namely, we have concluded that HSV is an important choice of colour model at pre-processing histological images of lymphoma, because in this case the resulting image will get the best enhancement.

Organisation and tyrosine hydroxylase and calretinin immunoreactivity in the main olfactory bulb of paca (cuniculus paca): a large caviomorph rodent

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mapping of clay, iron oxide and adsorbed phosphate in Oxisols using diffuse reflectance spectroscopy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The role of clouds in improving the regression model for hourly values of diffuse solar radiation

The study introduces a new regression model developed to estimate the hourly values of diffuse solar radiation at the surface. The model is based on the clearness index and diffuse fraction relationship, and includes the effects of cloud (cloudiness and cloud type), traditional meteorological variables (air temperature, relative humidity and atmospheric pressure observed at the surface) and air pollution (concentration of particulate matter observed at the surface). The new model is capable of predicting hourly values of diffuse solar radiation better than the previously developed ones (R-2 = 0.93 and RMSE = 0.085). A simple version with a large applicability is proposed that takes into consideration cloud effects only (cloudiness and cloud height) and shows a R-2 = 0.92. (C) 2011 Elsevier Ltd. All rights reserved.

Simultaneous Occurrence of Biphenotypic T Cell/Myeloid Lesions Involving t(12;13)(p13;q14) in a Pediatric Patient

This paper chronicles a 2-year-old girl who presented with acute leukemia/lymphoma syndrome of the T cell immuno-phenotype. At this time, the cytogenetic analysis of her bone marrow cells showed a reciprocal translocation between the short arm of chromosome 12 and the long arm of chromosome 13, t(12;13)(p13;q14). The immunophenotyping of bone marrow blast cells by flow cytometry revealed a population of cells positive for CD56, CD117, CD45, partial CD33, partial HLA-DR, CD13, CD7, CD2 and CD5. Therefore, a diagnosis of acute leukemia with a mixed T cell/myeloid phenotype was made. The patient had a poor response to classic T cell acute lymphocytic leukemia/lymphoma therapy; thus, her treatment was changed to a myeloid leukemia protocol, which produced a good response. She underwent a successful cord blood transplantation from an unrelated HLA partially matched donor. The coexistence of these two phenotypes prompts questions about the existence of clonal instability, which might influence the choice of therapy. The rarity of the t(12;13)(p13;q14) and the coexistence of T cell/myeloid markers suggest a nonrandom association. To the best of our knowledge, this is the first reported case in which a cell clone bearing a t(12;13)(p13;q14) translocation in a mixed T cell/myeloid lesion was detected. Copyright (C) 2012 S. Karger AG, Basel

Fluctuation of Anti-Endothelial Cell Antibody Titers in Mixed Connective Tissue Disease

Background: Antibodies directed against endothelial cell surface antigens have been described in many disorders and have been associated with disease activity. Since the most prominent histopathologic feature in mixed connective tissue disease (MCTD) is the widespread and unique proliferative vascular lesion, our aim was to evaluate the frequency of anti-endothelial cell antibodies (AECA) in this condition. Objectives: To evaluate the frequency of AECA in this disease and assess its clinical and laboratory associations. Methods: Seventy-three sera from 35 patients with MCTD (Kasukawa's criteria), collected during a 7 year period, were tested for immunoglobulins G and M (IgG and IgM) AECA by cellular ELISA, using HUVEC (human umbilical vein endothelial cells). Sera from 37 patients with systemic lupus erythematosus (SLE), 22 with systemic sclerosis (SSc) and 36 sera from normal healthy individuals were used as controls. A cellular ELISA using HeLa cells was also performed as a laboratory control method. Results: IgG-AECA was detected in 77% of MCTD patients, 54% of SLE patients, 36% of SSc patients and 6% of normal controls. In MCTD, IgG-AECA was associated with vasculitic manifestations, disease activity and lymphopenia, and was also a predictor of constant disease activity. Immunosuppressive drugs were shown to reduce IgG-AECA titers. Since antibodies directed to HeLa cell surface were negative, AECA was apparently unrelated to common epitopes present on epithelial cell lines. Conclusions: AECA are present in a large proportion of patients with MCTD and these antibodies decrease after immunosuppressive treatment. IMAJ 2012; 14:84-87

Large-Scale Population Analysis Challenges the Current Criteria for the Molecular Diagnosis of Fascioscapulohumeral Muscular Dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a common hereditary myopathy causally linked to reduced numbers (<= 8) of 3.3 kilobase D4Z4 tandem repeats at 4q35. However, because individuals carrying D4Z4-reduced alleles and no FSHD and patients with FSHD and no short allele have been observed, additional markers have been proposed to support an FSHD molecular diagnosis. In particular a reduction in the number of D4Z4 elements combined with the 4A(159/161/168)PAS haplotype (which provides the possibility of expressing DUX4) is currently used as the genetic signature uniquely associated with FSHD. Here, we analyzed these DNA elements in more than 800 Italian and Brazilian samples of normal individuals unrelated to any FSHD patients. We find that 3% of healthy subjects carry alleles with a reduced number (4-8) of D4Z4 repeats on chromosome 4q and that one-third of these alleles, 1.3%, occur in combination with the 4A161PAS haplotype. We also systematically characterized the 4q35 haplotype in 253 unrelated FSHD patients. We find that only 127 of them (50.1%) carry alleles with 1-8 D4Z4 repeats associated with 4A161PAS, whereas the remaining FSHD probands carry different haplotypes or alleles with a greater number of D4Z4 repeats. The present study shows that the current genetic signature of FSHD is a common polymorphism and that only half of FSHD probands carry this molecular signature. Our results suggest that the genetic basis of FSHD, which is remarkably heterogeneous, should be revisited, because this has important implications for genetic counseling and prenatal diagnosis of at-risk families.

Quantitative proteomic analysis and functional studies reveal that nucleophosmin is involved in cell death in glioblastoma cell line transfected with siRNA

Previously, we reported that nucleophosmin (NPM) was increased in glioblastoma multiforme (GBM). NPM is a phosphoprotein related to apoptosis, ribosome biogenesis, mitosis, and DNA repair, but details about its function remain unclear. We treated U87MG and A172 cells with small interference RNA (siRNA) and obtained a reduction of 80% in NPM1 expression. Knockdown at the protein level was evident after the 4th day and was maintained until the 7th day of transfection that was investigated by quantitative proteomic analysis using isobaric tags. The comparison of proteomic analysis of NPM1-siRNA against controls allowed the identification of 14 proteins, two proteins showed increase and 12 presented a reduction of expression levels. Gene ontology assigned most of the hypoexpressed proteins to apoptosis regulation, including GRP78. NPM1 silencing did not impair cell proliferation until the 7th day after transfection, but sensitized U87MG cells to temozolomide (TMZ), culminating with an increase in cell death and provoking at a later period a reduction of colony formation. In a large data set of GBM patients, both GRP78 and NPM1 genes were upregulated and presented a tendency to shorter overall survival time. In conclusion, NPM proved to participate in the apoptotic process, sensitizing TMZ-treated U87MG and A172 cells to cell death, and in association with upregulation of GRP78 may be helpful as a predictive factor of poor prognosis in GBM patients.

Changes in the expression of proteins associated with aerobic glycolysis and cell migration are involved in tumorigenic ability of two glioma cell lines

Background: The most frequent and malignant brain cancer is glioblastoma multiforme (GBM). In gliomas, tumor progression and poor prognosis are associated with the tumorigenic ability of the cells. U87MG cells (wild-type p53) are known to be tumorigenic in nude mice, but T98G cells (mutant p53) are not tumorigenic. We investigated the proteomic profiling of these two cell lines in order to gain new insights into the mechanisms that may be involved in tumorigenesis. Results: We found 24 differentially expressed proteins between T98G and U87MG cells. Gene Ontology supports the notion that over-representation of differentially expressed proteins is involved in glycolysis, cell migration and stress oxidative response. Among those associated with the glycolysis pathway, TPIS and LDHB are up-regulated in U87MG cells. Measurement of glucose consumption and lactate production suggests that glycolysis is more effective in U87MG cells. On the other hand, G6PD expression was 3-fold higher in T98G cells and this may indicate a shift to the pentose-phosphate pathway. Moreover, GRP78 expression was also three-fold higher in T98G than in U87MG cells. Under thapsigargin treatment both cell lines showed increased GRP78 expression and the effect of this agent was inversely correlated to cell migration. Quantitative RT-PCR and immunohistochemistry of GRP78 in patient samples indicated a higher level of expression of GRP78 in grade IV tumors compared to grade I and non-neoplastic tissues, respectively. Conclusions: Taken together, these results suggest an important role of proteins involved in key functions such as glycolysis and cell migration that may explain the difference in tumorigenic ability between these two glioma cell lines and that may be extrapolated to the differential aggressiveness of glioma tumors.

In Vitro Development and Cell Allocation After Aggregation of Syngeneic Wild Type and Fluorescence-Expressing Bovine Cloned Embryos

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the first third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may influence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells. Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the. 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student's test (P < 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were similar between one another. Cloned GFP embryos had lower in vitro development to the blastocyst stage than WT embryos, which had more TCN than parthenote or aggregated chimeric WT/GFP embryos. Aggregated GFP embryos had fewer cells than the other embryo groups. Discussion: The in vitro development of GFP cloned embryos was lower than WT embryos, with no effects on cell allocation in resulting blastocysts. Differences in blastocyst rate between groups were likely due to lower GFP-expressing cell viability, as GFP donor cells were at high population cell doublings when used for cloning. On a per embryo basis, embryo aggregation on Day 1 resulted in blastocyst development similar to non-aggregated embryos on Day 7, with no differences in cell proportion between groups. The use of GFP-expressing cells was proven a promising strategy for the study of cell allocation during embryo development, which may assist in the elucidation of mechanisms of abnormalities after in vitro embryo manipulations, leading to the development of improved protocols for the in vitro production (IVP) of bovine embryos.

Central Granular Cell Odontogenic Tumor of the Maxilla

The central granular cell odontogenic tumor (CGCOT) is a rare benign odontogenic neoplasm composed of varying amounts of large eosinophilic granular cells and apparently inactive odontogenic epithelium. It tends to occur as a small asymptomatic swelling in the posterior region of the mandible with nonaggressive appearance. We report an unusual case of CGCOT in the maxillary region with clinical features of malignancy. The patient underwent surgical treatment, and the 2-year follow-up revealed no signs of recurrence. Central granular cell odontogenic tumor is a very rare condition with few cases reported, especially in the maxillary region. This case highlights the possibility of aggressive behavior by these lesions.

Disseminated histoplasmosis in a juvenile lupus erythematosus patient

Introduction: Histoplasmosis is an infection caused by dimorphic fungus, Histoplasma capsulatum, and it has not been reported in juvenile systemic lupus erythematosus (JSLE) patients, particularly progressive disseminated histoplasmosis (PDH) subtype. Case report: We reported herein a 14-year old girl who was diagnosed with JSLE. Six months later, she had abdominal distension and received prednisone, hydroxychloroquine and azathioprine. Computer tomography evidenced hepatosplenomegaly and multiple mesenteric, mediastinal and retroperitoneal enlarged lymph nodes, forming large conglomerates at the mesentery, suggestive of lymphoproliferative disorder. After 10 days, she had acute surgical abdominal, and underwent a laparotomy and intestinal perforation and conglomerates of lymph nodes were observed. The jejunum biopsy showed perforated acute enteritis with hemorrhage and necrosis, and Grocott staining identified Histoplasma sp. and the culture showed a heavy growth of Histoplasma capsulatum. At that moment liposomal amphotericin B (1.0 mg/Kg/day) was introduced. Despite this treatment she died due to septic shock eight days later. Diffuse Histoplasma capsulatum was evidenced at autopsy. Conclusion: We reported a severe opportunistic infection in JSLE patient with adenopathy and multiple intestinal perforations. This study reinforces the importance of early diagnosis and antifungal therapy, especially in patients with these uncommon clinical manifestations.

Accumulation of the SET protein in HEK293T cells and mild oxidative stress: cell survival or death signaling

SET protein (I2PP2A) is an inhibitor of PP2A, which regulates the phosphorylated Akt (protein kinase B) levels. We assessed the effects of SET overexpression in HEK293T cells, both in the presence and the absence of mild oxidative stress induced by 50 mu M tert-butyl hydroperoxide. Immunoblotting assays demonstrated that SET accumulated in HEK293T cells and increased the levels of phosphorylated Akt and PTEN; in addition, SET decreased glutathione antioxidant defense of cell and increased expression of genes encoding antioxidant defense proteins. Immunofluorescence analysis demonstrated that accumulated SET was equally distributed in cytoplasm and nucleus; however, in cells that had been exposed to oxidative stress, SET was found in large aggregates in the cytoplasm. SET accumulation in HEK293T cells correlated with inhibition of basal apoptosis as evidenced by a decrease in annexin V staining and activity of caspases; under mild oxidative stress, SET accumulation correlated with caspase-independent cell death, as evidenced by increased PI and annexin V/PI double staining. The results suggest that accumulated SET could act via Akt/PTEN either as cell survival signal or as oxidative stress sensor for cell death.

Synthetic phosphoethanolamine a precursor of membrane phospholipids reduce tumor growth in mice bearing melanoma B16-F10 and in vitro induce apoptosis and arrest in G2/M phase

Phosphoethanolamine (Pho-s) is a compound involved in phospholipid turnover, acting as a substrate for many phospholipids of the cell membranes, especially phosphatidylcholine. We recently reported that synthetic Pho-s has potent effects on a wide variety of tumor cells. To determine if Pho-s has a potential antitumor activity, in this study we evaluated the activity of Pho-s against the B16-F10 melanoma both in vitro and in mice bearing a dorsal tumor. The treatment of B16F10 cells with Pho-s resulted in a dose-dependent inhibition of cell proliferation. At low concentrations, this activity appears to be involved in the arrest of the cell cycle at G2/M, while at high concentrations Pho-s induces apoptosis. In accordance with these results, the loss of mitochondrial potential and increased caspase-3 activity suggest that Phos has dual antitumor effects; i.e. it induces apoptosis at high concentrations and modulates the cell cycle at lower concentrations. In vivo, we evaluated the effect of Pho-s in mice bearing B16-F10 melanoma. The results show that Pho-s reduces the tumoral volume increasing survival rate. Furthermore, the tumor doubling time and tumor delays were substantially reduced when compared with untreated mice. Histological analyses reveal that Pho-s induces changes in cell morphology, typical characteristics of apoptosis, in addition the large areas of necrosis correlating with a reduction of tumor size. The results presented here support the hypothesis that Pho-s has antitumor effects by the induction of apoptosis as well as the inhibition of cell proliferation by arrest at G2/M. Thus, Pho-s can be regarded as a promising agent for the treatment of melanoma. Published by Elsevier Masson SAS.

Absence of Functional LIN28B Mutations in a Large Cohort of Patients with Idiopathic Central Precocious Puberty

Aim: To investigate LIN28B gene variants in children with idiopathic central precocious puberty (CPP). Patients and Methods: We studied 178 Brazilian children with CPP (171 girls, 16.8% familial cases). A large multiethnic group (1,599 subjects; Multiethnic Cohort, MEC) was used as control. DNA analysis and biochemical in vitro studies were performed. Results: A heterozygous LIN28B variant, p. H199R, was identified in a girl who developed CPP at 5.2 years. This variant was absent in 310 Brazilian control individuals, but it was found in the same allele frequency in women from the MEC cohort, independent of the age of menarche. Functional studies revealed that when ectopically expressed in cells, the mutant protein was capable of binding pre-let-7 microRNA and inhibiting let-7 expression to the same extent as wild-type Lin28B protein. Other rare LIN28B variants (p.P173P, c.198+32_33delCT, g.9575731A>C and c.-11C>T) were identified in CPP patients and controls. Therefore, no functional mutation was identified. Conclusion: In vitro studies revealed that the rare LIN28B p.H199R variant identified in a girl with CPP does not affect the Lin28B function in the regulation of let-7 expression. Although LIN28B SNPs were associated with normal pubertal timing, rare variations in this gene do not seem to be commonly involved in the molecular pathogenesis of CPP. Copyright (C) 2012 S. Karger AG, Basel