Proteomics-based identification of cancer-associated proteins in chronic lymphocytic leukaemia

BACKGROUND: Chronic lymphocytic leukaemia (CLL) is a neoplasm of B-cells characterized by variable prognosis. Exploring the proteome of CLL cells may provide insights into the disease. Therefore, eleven proteomics experiments were conducted on eleven primary CLL samples. RESULTS: We reported a CLL proteome consisting of 919 proteins (false discovery rate (FDR) 1%) whose identification was based on the sequencing of two or more distinct peptides (FDR of peptide sequencing 1%). Mass spectrometry-based protein identification was validated for four different proteins using Western blotting and specific antibodies in different CLL samples. Small sizes of nucleolin (~57 kDa and ~68 kDa) showed a potential association with good prognosis CLL cells (n = 8, p < 0.01). Compared with normal B-cells, CLL cells over-expressed thyroid hormone receptor-associated protein 3 (THRAP3; n = 9; p = 0.00007), which is implicated in cell proliferation; and heterochromatin protein 1-binding protein 3 (HP1BP3; n = 10; p = 0.0002), which promotes cell survival and tumourogenesis. A smaller form of HP1BP3, which may correspond to HP1BP3 isoform-2, was specifically identified in normal B-cells (n = 10; p = 0.0001). HP1BP3 and THRAP3 predicted poor prognosis of CLL (p 0.05). Consistently, THRAP3 and HP1BP3 were found to be associated with cancer-related pathways (p 0.05). CONCLUSIONS: Our findings add to the known proteome of CLL and confirm the prognostic importance of two novel cancer-associated proteins in this disease.

Immunoexpression of TS, p53, COX2, EGFR, MSH6 and MLH1 biomarkers and its correlation with degree of differentiation, tumor staging and prognostic factors in colorectal adenocarcinoma: a retrospective longitudinal study

ABSTRACT BACKGROUND: There are cases of colorectal tumors that, although small, show more aggressive evolution than large tumors. This motivated us to study whether there are any proteins capable of alerting about these changes. The aim here was to correlate the immunoexpression of the TS, p53, COX2, EGFR, MSH6 and MLH1 biomarkers in tumors in patients with colorectal adenocarcinoma, with the degree of cell differentiation, tumor staging and clinical-pathological prognostic factors. DESIGN AND SETTING: Retrospective observational study at a public tertiary-level hospital. METHODS: We analyzed tissue-microarray paraffin blocks of tumor tissues that had been resected from 107 patients. We used Fisher's exact test to study associations between tumor differentiation/staging and the immunoexpression of biomarkers. We also used Kaplan-Meier estimation, the log-rank test and the adjusted Cox regression model to investigate the patients' overall survival (in months) according to biomarkers and disease-free interval. RESULTS: The degree of tumor differentiation and tumor staging were not associated with the biomarkers, except in cases of patients in stages III or IV, in which there was a correlation with MLH1 expression (P=0.021). Patient survival and disease-free interval were not associated with the biomarkers. CONCLUSION: There were no associations between the degree of tumor differentiation, staging, length of survival or disease-free interval and the immunoexpression of the TS, p53, COX2, EGFR or MSH6 tumor markers. In advanced cases of colorectal adenocarcinoma (stages III and IV), there was a higher percentage of MLH1-negative results.

Characterization of reactive stroma in prostate cancer: involvement of growth factors, metalloproteinase matrix, sexual hormones receptors and prostatic stem cells

ABSTRACT Introduction and Objectives: Reactive Stroma (RStr) is observed in many human cancers and is related to carcinogenesis. The objectives of the present study were to stablish a relationship of the RStr microenvironment with prostate cancer (Pca) through a morphological and molecular characterization, and to identify a possible relationship between RStr with worse prognosis factors and occurrence of malignant prostatic stem cells. Materials and Methods: Forty prostatic samples were selected from men with Pca diagnosis submitted to radical prostatectomy; they were divided in two groups: Group-1 (n=20): samples without reactive stroma; Group-2 (n=20): samples of PCa with intense stroma reaction. Prostatic samples were evaluated for RStr intensity by Masson Trichromic stain and posteriorly submitted to histopathological and immunohistochemistry analysis for antigens: α-actin, vimentin, IGF-1, MMP-2, FGF-2, C-Myc, PSCA, AR, Erα and ERβ. Results: Reactive stroma with intense desmoplastic reactivity was significantly more frequent in intermediate (Gleason 7, 3+4) and high grade tumors (Gleason 7, 4+3). The group with intense stromal reactivity showed significant higher levels of Vimentin, IGF-1, MMP-2, FGF-2, C-Myc, PSCA and ERα. Conclusions: It can be concluded that RStr may be a predictive marker of Pca progression, since it was associated with increase of growth factors, imbalance of androgen and estrogen receptors and presence of malign prostatic stem cells.

Beneficial effects montelukast, cysteinyl-leukotriene receptor antagonist, on renal damage after unilateral ureteral obstruction in rats

Introductıon Ureteral obstruction is a common pathology and caused kidney fibrosis and dysfunction at late period. In this present, we investigated the antifibrotic and antiinflammatory effects of montelukast which is cysteinyl leukotriene receptor antagonist, on kidney damage after unilateral ureteral obstruction(UUO) in rats. Mateirıals and Methods 32 rats divided four groups. Group 1 was control, group 2 was sham, group 3 was rats with UUO and group 4 was rats with UUO which were given montelukast sodium (oral 10 mg/kg/day). After 14 days, rats were killed and their kidneys were taken and blood analysis was performed. Tubular necrosis, mononuclear cell infiltration and interstitial fibrosis scoring were determined histopathologically in a part of kidneys; nitric oxide(NO), malondialdehyde(MDA) and reduced glutathione(GSH) levels were determined in the other part of kidneys. Urea-creatinine levels were investigated at blood analysis. Statistical analyses were made by the Chi-square test and one-way analysis of variance (ANOVA). Results There was no difference significantly for urea-creatinine levels between groups. Pathologically, there was serious tubular necrosis and fibrosis in group 3 and there was significantly decreasing for tubular necrosis and fibrosis in group 4(p<0.005). Also, there was significantly increasing for NO and MDA levels; decreasing for GSH levels in group 3 compared the other groups(p<0.005). Conclusıon We can say that montelukast prevent kidney damage with antioxidant effect, independently of NO. .

International cooperation and health policy implementation in a post-conflict situation: the case of East Timor / Cooperação internacional e formulação de políticas de saúde em situação pós-conflito: o caso do Timor-Leste

This study centers on relationships among national and international actors in preparation of the first health policy document for East Timor, under the United Nations transitional administration, between 1999 and 2002. International cooperation support for the health system rehabilitation process during the post-conflict period is analyzed as part of reconstruction of the State in parallel with construction of the country's political and institutional framework. Knowledge, ideas, "ways of doing," and induced and accepted practices permeate an interplay of power relationships that condition both national political alliance-building and the architecture of international aid, pointing to input to a discussion of how these mechanisms interact at different conjunctures and times in different negotiating frameworks. .

Dedica-se, aqui, às relações entre diferentes atores na elaboração do primeiro documento de política de saúde para o Timor-Leste, sob a administração transitória das Nações Unidas, de 1999 a 2002. O apoio da cooperação internacional no processo de reabilitação do sistema de saúde no período pós-conflito é analisado como parte da reconstrução do Estado e concomitante à construção do arcabouço político e institucional no país. Conhecimentos, ideias, "modos de fazer" e práticas induzidas e aceitas entremeiam um jogo de relações de poder que condiciona tanto a articulação política nacional quanto a arquitetura da ajuda externa, apontando elementos para a discussão de como esses mecanismos se organizam em conjunturas diferentes de negociação.

Prognostic Value of Immunohistochemical Biomarkers at Different Cut-off Values in Patients with Diffuse Large B-cell Lymphoma Treated with CHOP Chemotherapy

Many predictive models have been proposed for better stratification of diffuse large B-cell lymphoma (DLBCL). Hans' algorithm has been widely used as standard to sub-classify DLBCL into germinal center B-cell (GCB) and non-GCB origins. However, there have been disagreements in the literature regarding its prognostic significance. Here, we retrospectively analyzed Hans' algorithm and the individual immunohistochemical biomarkers at different cut-off values (5%, 30%, 50% or 75%) in 94 Korean patients with DLBCL treated with combination chemotherapy with cyclophosphamide, daunorubicin, vincristine, and prednisone. No significant differences were observed between the GCB (18 patients, 19.1%) and non-GCB (76, 80.9%) groups. Among individual biomarkers, CD10 negativity (cut point: 30%) and bcl-6 positivity (cut point: 5%) were independent good prognostic markers in progression-free survival (PFS), whereas bcl-6 (cut point: 5%) positivity was an independent good prognostic marker in overall survival irrelevant of international prognostic index. The present study showed the lack of predictability of Hans' algorithm in DLBCL patients, and that CD10, Bcl-6 may have diverse prognostic significance at different cut-off values. Our results suggest that the proposed cut-off value may not be applied universally, and that the optimal cut-off value may need to be optimized for individual laboratory.

Apoptose, proliferação e histomorfometria do baço de ratas adultas com hipofunção tireoidiana e ovariana / Apoptosis, proliferation and spleen histomorphometry of adult female rats with thyroid and ovarian hypofunction

Apoptose, proliferação e histomorfometria do baço foram investigados em ratas Wistar adultas ovariectomizadas e não-ovariectomizadas, mantidas em hipotireoidismo induzido pela administração diária de propiltiouracil (PTU) por 120 dias. Dois grupos eutireóideos ovariectomizados e não-ovariectomizados serviram como controle. Foi colhido o plasma para dosagem de T4 livre e o baço para análise da histomorfometria, do índice apoptótico e da expressão imunohistoquímica de caspase 3 e CDC47. Valores de T4 livre foram menores nas ratas tratadas com PTU (p < 0,05). Nos grupos hipotireóideos houve redução do peso do baço, do número e do tamanho dos folículos linfóides e aumento do índice apoptótico e da expressão de caspase 3 (p < 0,05). Porém, o baço de ratas hipotireóideas ovariectomizadas apresentou aumento menos acentuado do índice apoptótico e da expressão de caspase 3 do que o baço de ratas hipotireóideas não-ovariectomizadas (p < 0,05). O grupo eutireóideo ovariectomizado apresentou hiperplasia da polpa branca em relação ao grupo eutireóideo não-ovariectomizado. Não houve diferença na expressão de CDC47 entre os grupos. Conclui-se que a hipofunção tireoidiana e gonadal apresentam efeitos distintos no baço e que na associação hipotireoidismo-hipogonadismo há aumento menos acentuado do índice apoptótico e da expressão de caspase-3 esplênica do que no hipotireoidismo isolado.

Apoptosis, proliferation and histomorphometry of spleen were investigated in ovariectomized and non-ovariectomized adult Wistar rats maintained in hypothyroidism induced by daily administration of propylthiouracil (PTU) during 120 days. Two groups ovariectomized euthyroid and non-ovariectomized euthyroid were used as controls. Plasma was collected for free T4 dosage and the spleen for histomorphometry analysis, apoptosis index and the immunohistochemistry expression of caspase 3 and CDC47. Values of free T4 were lower in rats treated with PTU (p<0.05). In the hypothyroid groups there was some decrease in the spleen weight as well as the number and size of lymphoid follicles and there was some increase in the apoptotic index and the caspase 3 expression (p<0.05). However, the increase in the apoptosis index and the expression of caspase 3 in ovariectomized hypothyroid rats spleen was less accentuated than non-ovariectomized hypothyroid ones (p<0.05). The ovariectomized euthyroid group presented white pulp hyperplasia in comparison to the non-ovariectomized euthyroid group. There was no difference in the CDC47 expression between groups. It was concluded that the thyroid and ovarian hypofunction have distinct effects on the spleen and that in the hypothyroidism-hypogonadism association, the increase in the apoptosis index and in the expression of splenic caspase 3 is not as much as in isolated hypothyroidism.

Cultivation of human corneal limbal stem cells in Mebiol gel--A thermo-reversible gelation polymer.

BACKGROUND & OBJECTIVES: Cultivated limbal stem cell transplantation is being used as a current treatment modality for limbal stem cell deficiency. However, use of allogenic biological material as substrate is associated with risks of transmission of certain diseases and allograft rejection. Therefore development of non-toxic biodegradable synthetic polymers is important. We undertook this study to evaluate the use of a synthetic polymer Mebiol gel as a substrate for the growth of limbal phenotype cells and cornea phenotype cells from limbal explants. METHODS: Human cadaveric limbal explants cells were cultivated on Mebiol gel. The proliferative capacity of cultivated cells was analyzed with thymidine incorporation studies. Immunostaining for presumed limbal stem cell association markers and cornea differentiation markers was performed and confirmed with reverse transcription (RT-PCR). RESULTS: The limbal explants underwent proliferation in vitro. The cultivated cells expressed the presumed limbal stem cell association markers (ABCG2 and p63), the transient amplifying cell markers (connexin 43, integrin alpha9) and the cornea differentiation marker (K3). RT PCR confirmed the immunohistochemical data. INTERPRETATION & CONCLUSION: Our findings showed that the synthetic polymer Mebiol gel was able to support limbal explant proliferation. The cultured cells expressed presumed limbal stem cell association markers, transient amplifying cells and cornea phenotype markers. Mebiol Gel can be used as a scaffold for growing limbal explants.

A nonradioactive method for detecting DNA-binding activity of nuclear transcription factors / 华中科技大学学报（医学）（英德文版）

To determine the feasibility of a nonradioactive electrophoresis mobility shift assay for detecting nuclear transcription factor, double-stranded oligonucleotides encoding the consensus target sequence of NF-kappa B were labelled with DIG by terminal transferase. After nuclear protein stimulated with phorbol 12-myristate 13-acetate (PMA) or PMA and pyrrolidine dithiocarbamate (PDTC) electrophoresed on 8% nondenaturing poliacrylamide gel together with oligeonucleotide probe, they were electro-blotted nylon membrane positively charged. Anti-DIG-AP antibody catalyzed chemiluminescent substrate CSPD to image on X-film. The results showed that nuclear proteins binded specifically to the NF-kappa B consensus sequence in the EMSA by chemiluminescent technique method and the activity of NF-kappa B in PMA group was more than that in PMA + PDTC group. It is suggested that detection of NF-kappa B by EMSA with chemiluminescent technique is feasible and simple, which can be performed in ordinary laboratories.

Is Immunohistochemistry for MLH1 and MSH2 Proteins a Useful Method for Detection of Microsatellite Instability in Sporadic Colorectal Cancer? / 대한소화기학회지

BACKGROUND/AIMS: In order to identify microsatellite instability (MSI), the test based on the polymerase chain reaction (PCR) can be used. However, PCR is not routinely performed in all hospital laboratories. Recently, immunohistochemistry (IHC) for MLH1 and MSH2 proteins has been reported as a rapid and useful method for MSI. However, the efficacy of IHC in the detection of the MSI has not been well established. The aim of this study was to evaluate the usefulness of IHC in the detection of the MSI by comparing it with the test results using PCR in colorectal cancer (CRC). METHODS: Paraffin-embedded normal and tumor tissues from seventy-five patients who underwent surgical resection of CRC were used. Abnormal expression of MLH1 and MSH2 protein was determined by IHC using MLH1 and MSH2 antibodies. Normal and tumor DNAs were obtained from thirty CRC tissues that showed abnormal expression of MLH1 and MSH2 proteins by IHC. The MSI status was confirmed by PCR using five markers. RESULTS: Thirty tumors showed abnormal expression of MLH1 and MSH2 proteins by IHC, but only three tumors out of them were confirmed to have MSI by PCR. CONCLUSIONS: This result suggests that IHC with MLH1 and MSH2 antibodies does not seem to be a useful method to identify MSI in CRC, therefore PCR is required for detection of the MSI.

Morphological and biochemical analysis of anti-nuclear matrix protein antibodies in human sera

Autoimmune sera have been used in the diagnosis of autoimmune diseases as well as the analysis of nuclear substructures. In an attempt to study the biological characteristics of the nuclear matrix, we screened human sera using immunofluorescent staining and immunoblot. We detected antibodies against nuclear matrix (NM), a remnant nonchromatin protein compartment after the treatment of detergent, salt and nuclease, in 212 out of 284 tested sera (74.6%) by immunoblot. Peptides with molecular weights of 70 kDa, 50 kDa and 25 kDa were detected in the order of frequency. Clinical informations of 198 out of 212 cases were available and went as follows: 38 cases were autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis; 132 non-autoimmune and non-neoplastic diseases; 16 neoplastic diseases and 12 cases unclassified. The immunofluorescent staining intensity by anti-nuclear matrix protein (NMP) antibodies decreased variably, but fibrillogranular, speckled and nucleolar immunolocalization patterns were retained after in situ fractionation. Ku70 and La protein were detected by anti-NMP antibodies. Immunolocalization by anti-NMP antibodies indicates that the NMPs constitute a variety of characteristic nuclear substructures and may serve as autoantigens in diverse human diseases. In addition, the presence of Ku70 and La protein as NMPs suggests that the NM can be functionally active in association with DNA or RNA.