Serum levels of anti-heat shock protein 27 antibodies in patients with chronic liver disease

Heat shock protein 27 (HSP27), an intracellular molecular chaperone, is involved in the pathogenesis of cancer by promoting both tumor cell proliferation and resistance to therapy. HSP27 is also present in the circulation and circulating HSP27 (sHSP27) can elicit an autoimmune response with production of antibodies. Levels of sHSP27 are enhanced in patients with hepatocellular carcinoma (HCC); it is, however, unknown whether changes in HSP27 antibody levels occur in patients with HCC and can be exploited as a circulating biomarker of HCC. Our aim was to assess the potential association between newly diagnosed HCC and serum anti-HSP27 antibody levels. In this cross-sectional study, anti-HSP27 antibody levels were measured in serum samples from 71 HCC patients, 80 subjects with chronic liver disease, and 38 control subjects by immunoenzymatic assay. Anti-HSP27 antibody levels did not differ significantly among groups. However, in patients with chronic active hepatitis/cirrhosis, anti-HSP27 levels were significantly higher in subjects with a positive history of alcoholism (p = 0.03). Our data do not support the hypothesis that anti-HSP27 antibody levels may help identify patients with HCC among subjects with chronic liver disease. However, our finding that alcohol-related liver disease is associated with higher anti-HSP27 levels is novel and deserves further investigations.     

Modeling human osteosarcoma in mice through 3AB-OS cancer stem cell xenografts

Osteosarcoma is the second leading cause of cancer‐related death for children and young adults. In this study, we have subcutaneously injected—with and without matrigel—athymic mice (Fox1nu/nu) with human osteosarcoma 3AB‐OS pluripotent cancer stem cells (CSCs), which we previously isolated from human osteosarcoma MG63 cells. Engrafted 3AB‐OS cells were highly tumorigenic and matrigel greatly accelerated both tumor engraftment and growth rate. 3AB‐OS CSC xenografts lacked crucial regulators of beta‐catenin levels (E‐cadherin, APC, and GSK‐3beta), and crucial factors to restrain proliferation, resulting therefore in a strong proliferation potential. During the first weeks of engraftment 3AB‐OS‐derived tumors expressed high levels of pAKT, beta1‐integrin and pFAK, nuclear beta‐catenin, c‐Myc, cyclin D2, along with high levels of hyperphosphorylated‐inactive pRb and anti‐apoptotic proteins such as Bcl‐2 and XIAP, and matrigel increased the expression of proliferative markers. Thereafter 3AB‐OS tumor xenografts obtained with matrigel co‐injection showed decreased proliferative potential and AKT levels, and undetectable hyperphosphorylated pRb, whereas beta1‐integrin and pFAK levels still increased. Engrafted tumor cells also showed multilineage commitment with matrigel particularly favoring the mesenchymal lineage. Concomitantly, many blood vessels and muscle fibers appeared in the tumor mass. Our findings suggest that matrigel might regulate 3AB‐OS cell behavior providing adequate cues for transducing proliferation and differentiation signals triggered by pAKT, beta1‐integrin, and pFAK and addressed by pRb protein. Our results provide for the first time a mouse model that recapitulates in vivo crucial features of human osteosarcoma CSCs that could be used to test and predict the efficacy in vivo of novel therapeutic treatments. J. Cell. Biochem. 113: 3380–3392, 2012. © 2012 Wiley Periodicals, Inc.     

Characterization and molecular cloning of Bifidobacterium longum cryptic plasmid pMB1

The small cryptic plasmid pMB1 (1.9 kb), previously isolated from Bifidobacterium longum, has been characterized by physical mapping. Two cloning vectors, pMR3 and pDG7, carrying chloramphenicol and ampicillin resistances derived from pJH101, have been electroporated in Escherichia coli.     

Dynamics of Vaginal Bacterial Communities in Women Developing Bacterial Vaginosis, Candidiasis, or No Infection, Analyzed by PCR-Denaturing Gradient Gel Electrophoresis and Real-Time PCR

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.     

Growth of choroid plexus epithelium vesicles in vitro depends on secretory activity

Although a number of models have been used to study choroid plexus epithelium (CPe) function, analysis in physiological conditions of this polarised epithelium which produces the majority of the cerebrospinal fluid (CSF) and is one of the key barriers between blood and CSF in the brain remains challenging. As CPe cells form polarised CPe vesicles when cultured in Matrigel, we have assessed their behaviour and potential use for pharmacological studies. Like CPe cells in vivo, CPe vesicles express transthyretin, E2f5, Fox-j1 and p73, and contain tight junctions, as indicated by ZO-1 expression and electron microscopy analysis. Time-lapse microscopy shows that CPe cells plated in Matrigel are highly migratory and rapidly form homotypic cell aggregates, which then reorganise to form vesicles whose size increases linearly overtime. Neither aggregate nor vesicle size is affected by AraC treatment, though this inhibitor significantly reduces proliferation in CPe monolayers. Increase in size of vesicles, which have reached a growth plateau is observed following addition of fluorescently-labelled CPe cells, which become incorporated into the vesicle walls. Significantly, treatment with secretion inhibitors blocks vesicle formation and their expansion. These results show that secretion, rather than cell division, controls vesicle growth, consistent with low levels of proliferation and thinning of the CPe observed both in growing vesicles and during CPe development. Therefore, changes in vesicle size can be used to evaluate the effect of putative molecules involved in the regulation of secretion.     

Soluble TNF-like cytokine (TL1A) production by immune complexes stimulated monocytes in rheumatoid arthritis

TNF-like cytokine (TL1A) is a newly identified member of the TNF superfamily of ligands that is important for T cell costimulation and Th1 polarization. However, despite increasing information about its functions, very little is known about expression of TL1A in normal or pathological states. In this study, we report that mononuclear phagocytes appear to be a major source of TL1A in rheumatoid arthritis (RA), as revealed by their strong TL1A expression in either synovial fluids or synovial tissue of rheumatoid factor (RF)-seropositive RA patients, but not RF-/RA patients. Accordingly, in vitro experiments revealed that human monocytes express and release significant amounts of soluble TL1A when stimulated with insoluble immune complexes (IC), polyethylene glycol precipitates from the serum of RF+/RA patients, or with insoluble ICs purified from RA synovial fluids. Monocyte-derived soluble TL1A was biologically active as determined by its capacity to induce apoptosis of the human erythroleukemic cell line TF-1, as well as to cooperate with IL-12 and IL-18 in inducing the production of IFN-gamma by CD4(+) T cells. Because RA is a chronic inflammatory disease with autoimmune etiology, in which ICs, autoantibodies (including RF), and various cytokines contribute to its pathology, our data suggest that TL1A could be involved in its pathogenesis and contribute to the severity of RA disease that is typical of RF+/RA patients.