Cytomegalovirus infection in intensive care unit patients with hematological malignancies: Characteristics and clinical outcomes.

BACKGROUND: Cytomegalovirus (CMV) infection is associated with poor outcome in ICU patients. However, data on immunocompromised patients are scarce. This study aims to describe characteristics and outcomes of critically ill hematological patients and CMV infection. CMV disease characteristics and relationship between CMV viral load, CMV disease, coinfections by other pathogens and outcomes are described. METHODS: Retrospective single center study (Jan 2010-Dec 2017). Adult patients, admitted to the ICU, having underlying hematological malignancy and CMV infection were included. Results are reported as median (interquartile) or n (%). Factors associated with hospital mortality or CMV disease were analysed using logistic regression. RESULTS: 178 patients were included (median age 55y [42-64], 69.1% male). Hospital mortality was 53% (n = 95). Median viral load was 2.7 Log [2.3-3.5]. CMV disease occurred in 44 (24.7%) patients. Coinfections concerned 159 patients (89.3%). After adjustment for confounders, need for vasopressors (OR 2.53; 95%CI 1.11-5.97) and viral load (OR 1.88 per Log; 95%CI 1.29-2.85) were associated with hospital mortality. However, neither CMV disease nor treatment were associated with outcomes. Allogeneic stem cell transplantation (OR 2.55; 95%CI 1.05-6.16), mechanical ventilation (OR 4.11; OR 1.77-10.54) and viral load (OR 1.77 per Log; 95%CI 1.23-2.61) were independently associated with CMV disease. Coinfections were not associated with CMV disease or hospital mortality. CONCLUSION: In critically-ill hematological patients, CMV viral load is independently associated with hospital mortality. Conversely, neither CMV disease nor treatment was associated with outcome suggesting viral load to be a surrogate for immune status rather than a cause of poor outcome.

Ultrastructural localization of binding sites for PNA and VVA-B(4) lectins in human breast cancer cell lines detected by confocal fluorescence microscopy.

Three cancer cell lines (MCF-7, HBL-100, MDA-MB 231) and subnormal breast epithelial cell line MCF-10A were labeled with FITC-conjugated VVA-B4 lectin, specific for D-GalNAcalpha-O-ser/thr, matching the structure of Tn antigen sugar residues, and with RTIC-conjugated PNA lectin, specific for DGalbeta1-3GalNAc-O-ser/thr, corresponding to the structure of T antigen. Simultaneous expression of Tn and T antigens on the same cells (but in widely differing proportions) led to their large heterogeneity and occurrence of numerous cell subpopulations within each of the studied cell lines. This observation proved that the changes leading to the formation of Tn antigen are not caused by an irreversible genetic mutation of beta1-3-galactosyltransferase. Expression of Tn antigen on MCF-10A cells with normal (or subnormal) karyotype suggests that the process of malignant transformation of the cell begins with the changes in molecular structure of glycoconjugates.

Determination of BRCA2 oncosuppressor protein expression in human mammary cells by affinity perfusion high-performance chromatography.

In this work, we report a method for the determination of BRCA2 oncosuppressor protein in human mammary cells by affinity perfusion chromatography. This method involves labeling proteins with [(35)S]-methionine. The isolation and purification of DNA-binding proteins was performed by affinity chromatography on Heparin POROS 20HE. BRCA2 proteins, known to act in the transcriptional control and in DNA repair activity, are included in the DNA-binding proteins. Specific immunoprecipitation was performed with anti-BRCA2 antibodies, and the immune complex [(35)S-BRCA2 proteins/anti-BRCA2 antibodies] was isolated by affinity chromatography on a Protein A POROS column. This procedure allows the determination of the percentage of BRCA2 among the DNA-binding proteins and the quantitation of the difference of expression of BRCA2 oncosuppressor protein in breast carcinoma cells and in normal breast cells treated or untreated with phytoestrogens, such as daidzein or genistein.

Characterization of anti-BRCA2 antibodies in cell lines by Western blot analysis.

BRCA2 is a tumor suppressor gene associated with familial predisposition to breast and ovarian cancer. BRCA2 has been implicated in response to DNA damage, cell cycle control and transcription. However, the mechanisms by which the BRCA2 protein suppresses tumor cell growth are largely unknown. To begin to understand the contribution of BRCA2 protein to tumorigenesis, we evaluated the specificity of 4 anti-BRCA2 antibodies directed against several different epitopes using immunoblotting techniques. The two monoclonal antibodies (3E6 and 5F6) detected a specific 384-kDa protein in human breast cancer cell lines (MCF7 and MDA-MB 231) and in a human colon carcinoma cell line (CCL 221). The two polyclonal antibodies (9433 and 9434) recognized the 384-kDa BRCA2 protein respectively in MCF7 and in CCL 221 cells, but both BRCA2 polyclonal antibodies also cross-reacted with smaller proteins.

Localization of human BRCA1 and BRCA2 in non-inherited colorectal carcinomas and matched normal mucosas.

We characterized the expression of BRCA1 and BRCA2 in 38 sporadic colorectal carcinomas and matched normal mucosas with 9 anti-BRCA1 antibodies and 4 anti-BRCA2 antibodies, raised against several different epitopes, using immunohistochemical technique. We demonstrated an increased BRCA1 and BRCA2 staining in the apical cell pole of epithelial malignant cells and we also revealed a significant increase in BRCA1 and BRCA2 nuclear foci in tumor colorectal specimens in comparison with corresponding normal tissues. These increases in BRCA1 and BRCA2 expression may be explained by the fact that colorectal tissue is subject to very active proliferation and differentiation.

BRCA1 and BRCA2 protein expressions in an ovotestis of a 46, XX true hermaphrodite.

BRCA1 and BRCA2 breast cancer susceptibility genes encode proteins, the normal cellular functions of which are complex and multiple, and germ-line mutations in individuals predispose both to breast and to ovarian cancer. There is nevertheless substantial evidence linking BRCA1 and BRCA2 to homologous recombination and DNA repair, to transcriptional control and to tissue proliferation. There is controversy regarding the localization of BRCA1 and BRCA2 proteins to either nucleus or cytoplasm and whether the expression is present in premeiotic germ cells or can still be expressed in mitotic spermatogonia. We report herein an immunohistochemical study of BRCA1 and BRCA2 distribution in a rather unusual tissue (an ovotestis), which addresses this issue.

Brca1 and Brca2 protein expression patterns in different tissues of murine origin.

We have analyzed by immunohistochemistry Brca1 and Brca2 protein expression in mouse during embryonic development, in adult tissues, and during postnatal mammary gland development. Our observations confirm previous localization of Brca1 and Brca2 mRNA on frozen sections by in situ hybridization, and demonstrate that Brca1 and Brca2 proteins are expressed in rapidly proliferating cell types undergoing differentiation. These results imply that Brca1 and Brca2 proteins are involved in the process of proliferation and differentiation in multiple tissues, notably in the mammary gland during pregnancy and lactation.

Cyclosporine A inhibition of prolactin-dependent up-regulation of BRCA1 protein expression in human breast cell lines.

BACKGROUND: Previously, we reported experimental evidence that BRCA1, breast and ovarian cancer susceptibility gene is up-regulated in response to Prolactin stimulation. In this work, we studied the effects of Cyclosporine A and the competition with Prolactin on BRCA1 protein expression in vitro. METHODS: The expression of BRCA1 was monitored in a human breast cancer cell line (MCF7) by comparison with a normal breast epithelial one (MCF10a) treated with Cyclosporine A and ovine Prolactin. The amount of BRCA1 protein expression was quantified using a strategy of two successive affinity perfusion chromatographies. RESULTS AND CONCLUSION: We showed that Prolactin in presence of Cyclosporine A has no effect on BRCA1 protein expression in human breast cell lines. This emphasized the hypothesis that BRCA1 may be stimulated by Prolactin.

Real-time PCR quantification of full-length and exon 11 spliced BRCA1 transcripts in human breast cancer cell lines.

Germline alterations of the BRCA1 tumor suppressor gene have been implicated at least in half of familial breast cancers. Nevertheless, in sporadic breast cancer no mutation of this gene has been characterized to date. In sporadic breast tumors, other BRCA1 gene loss of function mechanisms, such as down-regulation of gene expression, have been suggested. In an effort to better understand the relationship between BRCA1 expression and malignant transformation, we have adapted the new real-time quantitative PCR method based on a 5' nuclease assay and the use of doubly labeled fluorescent TaqMan probes to quantify BRCA1 mRNA. We have compared expression of BRCA1 mRNA with or without exon 11 in the normal breast epithelial cell line MCF10a and in three cancer cell lines (MCF-7, MDA-MB231 and HBL100) by comparing two methods of quantification: the comparative C(T) and the standard curve. We found that the full length BRCA1 mRNA, which encodes the functional nuclear protein, was down-regulated in tumor cells when compared with MCF10a cells.

BRCA2 protein expression in sporadic breast carcinoma with or without allelic loss of BRCA2.

To elucidate the cellular role of BRCA2 in sporadic breast tumors, we studied the cellular localization and the expression of BRCA2 in carcinomas presenting or not allelic loss of BRCA2. The breast tumors were first classified with or without allelic loss of BRCA2 and then immunohistochemical staining was performed on tumors and matched normal tissues using antibodies raised against BRCA2. We showed that BRCA2 is found either in the nucleus or in perinuclear compartments such as the endoplasmic reticulum and the Golgi vesicles. We have earlier demonstrated the presence of BRCA1 as an exocrine secretion in the lumen of ductules in the normal mammary gland, as well as BRCA1 and BRCA2 in milk-fat globules inside mammary-gland ductules during lactation. Here we show that BRCA2 is present within the lumina of breast ductules, indicating that BRCA2 protein may also be secreted in the mammary gland. No correlation was found in breast tumors between the expression of BRCA2 protein and allelic loss of BRCA2.