Estradiol-dependent collagenolytic enzyme activity in long-term organ culture of human breast cancer.

An organ culture method suitable for the maintenance of viable human breast cancer for at least 14 days has been described. This method was applied to a total of 94 breast cancer specimens. It allowed good survival of "soft" tumors of various histological types, with loose connective stroma even in hormone-free medium. In contrast, "scirrhous" cancers showed poor survival in hormone-free medium; viable cells were maintained only at the very periphery of the explants. Supplementation of the medium with insulin (10 mug/ml), ovine prolactin (5 mug/ml), and hydrocortisone (1 mug/ml) in various combinations seemed to induce enlargement of viable cancer cells and moderate loosening of the stroma in some cases. However, it did not improve the survival of central tumor cords in scirrhous explants. Further supplementation of the medium with 17 beta-estradiol (minimum effective dose, 0.1 to 10 ng/ml), although it did not affect soft tumors, markedly improved survival of the cancer cells of scirrhous tumors throughout the whole explants, with evidence of collagen digestion around the neoplastic cells. This was observed in 18 of 20 scirrhous cancers subjected to this treatment. Estradiol need not be present during the whole culture period; the results at 14 days were identical in explants treated with estradiol for the first 7 days only or for the entire period. Addition of purified collagenase during the first 24 or 48 hr of culture resulted in complete dissolution of the collage. After such treatment, culture under the usual conditions resulted in excellent survival of the explants without improvement from hormone supplementation; thus, while estradiol was necessary when collagen was present, it was not longer required after collagen digestion. It can be concluded that breast cancer cells in organ culture are only slightly, or not at all, hormone dependent for survival, provided that they are not restrained by a dense collagen barrier. The estrogen-induced changes allowing survival inside the scirrhous explants strongly suggest the presence of an estrogen-dependent collagenolytic enzyme system in the collagen-rich breast cancers. This system could represent an important component of the hormone dependency of human breast cancer growth.     

Lactobacillus casei reduces CD8+ T cell-mediated skin inflammation

Probiotics, including Lactobacilli, have been postulated to alleviate allergic and inflammatory diseases, but evidence that they exert an anti-inflammatory effect by immune modulation of pathogenic T cell effectors is still lacking. The aim of this study was to examine whether L. casei could affect antigen-specific T cell-mediated skin inflammation. To this end, we used contact hypersensitivity to the hapten 2,4-dinitrofluorobenzene, a model of allergic contact dermatitis mediated by CD8+ CTL and controlled by CD4+ regulatory T cells. Daily oral administration of fermented milk containing L. casei or L. casei alone decreased skin inflammation by inhibiting the priming/expansion of hapten-specific IFN-gamma-producing CD8+ effector T cells. The down-regulatory effect of the probiotics required the presence of CD4+ T cells, which control the size of the hapten-specific CD8+ T cell pool primed by skin sensitization. L. casei cell wall was as efficient as live L. casei to regulate both the CHS response and the hapten-specific CD8+ T cell response, suggesting that cell wall components contribute to the immunomodulatory effect of L. casei. This study provides the first evidence that oral administration of L. casei can reduce antigen-specific skin inflammation by controlling the size of the CD8+ effector pool.     

Detection of Epstein-Barr viral genomes in lymph nodes of Hodgkin's disease patients

Previous studies using Southern blot analysis or in situ hybridization have shown that approximately 20% of patients with Hodgkin's disease have Epstein-Barr virus (EBV) in involved tissues. We used the more sensitive polymerase chain reaction (PCR) technique to determine if a higher percentage of EBV could be detected. Of the 16 Hodgkin's disease patients studied, the PCR technique detected EBV in eight (50%). No prognostic significance was associated with the presence of EBV in the eight EBV-positive patients, and the presence of EBV was not associated with B cell monoclonality.     

Copy number variations in DCC/18q and ERBB2/17q are associated with disease‐free survival in microsatellite stable colon cancer

We conducted a prospective study to assess the prognostic impact of selected copy number variations (CNVs) in Stage II–III microsatellite stable (MSS) colon cancer. A total of 401 patients were included from 01/2004 to 01/2009. The CNVs in 8 selected target genes, DCC/18q, EGFR/7p, TP53/17p, BLK/8p, MYC/8q, APC/5q, ERBB2/17q and STK6/20q, were detected using a quantitative multiplex polymerase chain reaction of short fluorescent fragment (QMPSF) method. The primary end‐point was the impact of the CNVs on the 4‐year disease‐free survival (DFS). The recurrence rate at 4 years was 20.9%, corresponding to 14% Stage II patients versus 31% Stage III patients (p < 0.0001). The 4‐year DFS was significantly decreased in patients with a loss at DCC/18q (p = 0.012) and a gain at ERBB2/17q (p = 0.041). The multivariate analysis demonstrated that Stage III, a loss at DCC/18q and a gain at ERBB2/17q were independent factors associated with DFS. A combination of DCC/18q and ERBB2/17q was also associated with relapse, with the hazard ratio increasing from 1 to 2.4 (95% confidence interval (CI), 1.5–4.1) and 3.1 (95% CI, 1.2–8.4) in the presence of 0, 1 or 2 alterations, respectively (p = 0.0013). CNVs in DCC/18q and ERBB2/17q are significantly associated with DFS in Stage II–III MSS colon cancer.     

B cell depletion reduces the development of atherosclerosis in mice

B cell depletion significantly reduces the burden of several immune-mediated diseases. However, B cell activation has been until now associated with a protection against atherosclerosis, suggesting that B cell–depleting therapies would enhance cardiovascular risk. We unexpectedly show that mature B cell depletion using a CD20-specific monoclonal antibody induces a significant reduction of atherosclerosis in various mouse models of the disease. This treatment preserves the production of natural and potentially protective anti–oxidized low-density lipoprotein (oxLDL) IgM autoantibodies over IgG type anti-oxLDL antibodies, and markedly reduces pathogenic T cell activation. B cell depletion diminished T cell–derived IFN-γ secretion and enhanced production of IL-17; neutralization of the latter abrogated CD20 antibody–mediated atheroprotection. These results challenge the current paradigm that B cell activation plays an overall protective role in atherogenesis and identify new antiatherogenic strategies based on B cell modulation.Atherosclerosis-related cardiovascular diseases are the leading cause of mortality worldwide. Immune-mediated reactions initiated in response to multiple potential antigens, including oxidatively modified lipoproteins and phospholipids, play prominent roles in atherosclerotic lesion development, progression, and complications (Binder et al., 2002; Hansson and Libby, 2006; Tedgui and Mallat, 2006). Besides the critical requirement for monocytes/macrophages (Smith et al., 1995), adaptive immunity substantially contributes to the perpetuation of the immunoinflammatory response, further promoting vascular inflammation and lesion development (Binder et al., 2002; Hansson and Libby, 2006; Tedgui and Mallat, 2006). Mice on a severe combined immunodeficiency or Rag-deficient background show reduced susceptibility to atherosclerosis under moderate cholesterol overload (Dansky et al., 1997; Daugherty et al., 1997; Zhou et al., 2000). Resupplementation of these mice with purified T lymphocytes accelerates lesion development (Zhou et al., 2000), even though it does not fully recapitulate lesion development of the immunocompetent mice. The proatherogenic T cells are related to the Th1 lineage (Gupta et al., 1997; Buono et al., 2005), and are counterregulated by both Th2 (Binder et al., 2004; Miller et al., 2008) and T reg cell responses (Ait-Oufella et al., 2006; Tedgui and Mallat, 2006).The development of atherosclerosis is also associated with signs of B cell activation, particularly manifested by enhanced production of natural IgM type and adaptive IgG type anti–oxidized low-density lipoprotein (oxLDL) autoantibodies (Shaw et al., 2000; Caligiuri et al., 2002). However, in contrast to other immune-mediated diseases, i.e., rheumatoid arthritis and systemic lupus erythematosus, B cells have been assigned a protective role in atherosclerosis (Caligiuri et al., 2002; Major et al., 2002; Binder et al., 2004; Miller et al., 2008). Although IgG type anti-oxLDL antibodies show variable association with vascular risk, circulating levels of IgM type anti-oxLDL antibodies have been more frequently linked with reduced vascular risk in humans (Karvonen et al., 2003; Tsimikas et al., 2007). In mice, IL-5– and IL-33–mediated atheroprotective effects have been indirectly associated with specific B1 cell activation and enhanced production of natural IgM type anti-oxLDL antibodies (Binder et al., 2004; Miller et al., 2008). On the other hand, splenectomy (Caligiuri et al., 2002) or transfer of μMT-deficient (B cell–deficient) bone marrow (Major et al., 2002) into lethally irradiated atherosclerosis-susceptible mice resulted in profound reduction of IgG (Caligiuri et al., 2002) or total (Major et al., 2002) anti-oxLDL antibody production, and was associated with acceleration of lesion development. These studies led to the current paradigm that overall B cell activation is atheroprotective. Surprisingly, however, whether mature B cell depletion accelerates atherosclerotic lesion development in immunocompetent mice, as expected from previous studies, is still unexplored. This is a critical question given the potentially important risk of cardiovascular complications that might arise from the clinical use of B cell–depleting CD20-targeted immune therapy in patients with severe rheumatoid arthritis or systemic lupus erythematosus, who are at particularly high risk of cardiovascular diseases (for review see Roman et al., 2001). We have therefore designed a series of experiment to address this important question.     

Strategy for Rapid Detection of Carbapenemase-Producing Enterobacteriaceae

A prospective survey was conducted on 862 Enterobacteriaceae isolates with reduced susceptibility to carbapenems. The Carba NP test, UV spectrophotometry, and a DNA microarray were used to detect carbapenemase producers, and the results were compared to those from PCR and sequencing. The 172 carbapenemase producers were detected using the Carba NP test and UV spectrophotometry, whereas the DNA microarray failed to detect IMI producers. The use of the Carba NP test as a first screening, followed by the use of molecular techniques, has been determined to be an efficient strategy for identifying carbapenemase-producing Enterobacteriaceae.     

UPLC MS/MS method for quantification of mycophenolic acid and metabolites in human plasma: Application to pharmacokinetic study

BackgroundWe described the development and full validation of a rapid, high throughput sensible and accurate UPLC method using tandem mass spectrometry detection for mycophenolate acid (MPA) and its metabolites, MPA glucuronide (MPAG) and acyl MPA glucuronide (AcMPAG) concentration determination with MPA-D3 as internal standard in human plasma.MethodsPlasma pretreatment involved a one-step protein precipitation with acetonitrile. The separation was performed by reverse-phase chromatography on a Waters BEH HSST3 100 mm*2.1 mm*1.8 μm column. The multiple reaction monitoring transitions used for quantification were m/z 321.04 → 303.02 for MPA, 524.09 → 303.02 for AcMPAG and MPAG and 324.03 → 306.04 for MPA-D3 in the electrospray positive ionization mode.ResultsThe method was linear over the concentration range of 0.1–20 mg/L for MPA and AcMPAG and 1–200 mg/L for MPAG respectively. The intra- and inter-day precision values were below 14% and accuracy was from 94.0 to 103.3% at all quality control levels. The lower LOQ was 0.1 mg/L for MPA and AcMPAG, 1 mg/L for MPAG.ConclusionSample analysis time was reduced to 7 min including sample preparation. The present method was successfully applied to a pharmacokinetic study following oral administration of enterocoated sodium mycophenolate in de novo renal transplantation.