Aberrant splicing of the tumor suppressor CYLD promotes the development of chronic lymphocytic leukemia via sustained NF-κB signaling.

The pathogenesis of chronic lymphocytic leukemia (CLL) has been linked to constitutive NF-κB activation but the underlying mechanisms are poorly understood. Here we show that alternative splicing of the negative regulator of NF-κB and tumor suppressor gene CYLD regulates the pool of CD5+ B cells through sustained canonical NF-κB signaling. Reinforced canonical NF-κB activity leads to the development of B1 cell-associated tumor formation in aging mice by promoting survival and proliferation of CD5+ B cells, highly reminiscent of human B-CLL. We show that a substantial number of CLL patient samples express sCYLD, strongly implicating a role for it in human B-CLL. We propose that our new CLL-like mouse model represents an appropriate tool for studying ubiquitination-driven canonical NF-κB activation in CLL. Thus, inhibition of alternative splicing of this negative regulator is essential for preventing NF-κB-driven clonal CD5+ B-cell expansion and ultimately CLL-like disease.

Is BAC transgenesis obsolete? State of the art in the era of designer nucleases.

DNA constructs based on bacterial artificial chromosomes (BACs) are frequently used to generate transgenic animals as they reduce the influence of position effects and allow predictable expression patterns for genes whose regulatory sequences are not fully identified. Despite these advantages BAC transgenics suffer from drawbacks such as complicated vector construction, low efficiency of transgenesis, and some remaining expression variegation. The recent development of transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs) has resulted in new transgenic techniques which do not have the drawbacks associated with BAC transgenesis. Initial reports indicate that such designer nucleases (DNs) allow the targeted insertion of transgenes into endogenous loci by direct injection of the targeting vector and mRNA/DNA encoding the predesigned nucleases into oocytes. This results in the transgene being inserted at a specific locus in the mouse genome, thus circumventing the drawbacks associated with BAC transgenesis.

Comparative cytotoxicity of carmustine (BCNU), nimustine (ACNU) and elmustine (HeCNU) after depletion of O6-alkylguanine-DNA alkyltransferase (O6-AGT).

BCNU was reported to have about a 6- to 8- fold lower cytotoxic potency than ACNU in cell lines naturally deficient in O6-AGT. In seven tumor cell lines with an O6-AGT activity ranging from 40 to 360 fmol/mg the cytotoxic potency of BCNU, ACNU and HeCNU, without and after O6-AGT depletion by O6-BG, was determined. Without O6-AGT depletion, BCNU was superior to both other drugs in tumor cells with high O6-AGT activity. After O6-AGT depletion, the cytotoxic potency (comparison of IC50 values) of ACNU was higher than that of BCNU (p=0.016) or that of HeCNU (p=0.016) in all tumor cell lines. We conclude that (without O6-AGT depletion) BCNU is the drug of choice especially in tumor cells with high transferase activity. The higher cytotoxic potency of ACNU after O6-AGT depletion as compared to BCNU after O6-AGT depletion is countered by the higher toxicity of ACNU in patients necessitating a clinical dose reduction as compared to BCNU. Thus, we would not expect superiority of ACNU + O6-BG over BCNU+ O6-BG after systemic administration.

Steroid hormone receptors and long term survival in invasive ovarian cancer.

BACKGROUND: Steroid hormone receptors are important determinants of prognosis and predictive behavior in tumor tissues of several origins. Since their role in ovarian cancer is still controversial, we investigated the prevalence and prognostic impact of the estrogen (ER) and progesterone (PR) receptors and combinations (ER+PR+, ER+PR-, ER-PR+, and ER-PR-) in a comparably large number of patients with a long clinical follow-up. METHODS: The present analysis included 186 patients with invasive ovarian carcinomas treated at the Department of Obstetrics and Gynecology of the Justus-Liebig-University Giessen between 1982 and 1996, the follow-up lasting up to 15.8 years (median 2.4 yrs). The expression of ER and PR was assessed by immunohistochemistry using alkaline phosphatase antialkaline phosphatase in microwave pretreated, formalin fixed, and paraffin embedded specimens of the primary tumors and was evaluated semiquantitatively using a standardized immunoreactive scoring system. Receptor expression and combinations were compared to clinical, histologic and prognostic factors, the tumor proliferation, and the clinical outcome. RESULTS: Kaplan-Meier survival analyses supported the favorable prognostic value of PR and its level of expression in ovarian carcinomas. Especially the ER-PR+ combination, which accounted for 10.2% of all tumors, showed a significantly superior prognosis when compared with all other combinations (survivors 15 of 19 vs. 67 of 167, log rank P = 0.009) and was associated with early stage, low ascites quantity, and higher tumor differentiation. Five-year survival rates were 13/16 (81.3%) for ER-PR+ tumors versus 58/128 (45.3%) for all other steroid hormone receptor combinations. Residual analysis proved the results. CONCLUSIONS: The determination of steroid hormone receptor status offers additional prognostic information in ovarian carcinomas. Especially the ER-PR+ phenotype predicts a favorable tumor biology and long term survival, probably reflecting functional effects on tumor proliferation, differentiation, and cellular apoptosis.

Epitope mapping and reactivity of autoantibodies to the E2 component of 2-oxoglutarate dehydrogenase complex in primary biliary cirrhosis using recombinant 2-oxoglutarate dehydrogenase complex.

Five different target mitochondrial autoantigens recognized by sera from patients with primary biliary cirrhosis (PBC) have been identified as subunits of the following 2-oxo acid dehydrogenase complexes: the pyruvate dehydrogenase complex (PDC), the branched chain 2-oxo acid dehydrogenase complex (BCOADC), and the 2-oxoglutarate dehydrogenase complex (OGDC). Unlike the E2 subunits of PDC (PDC-E2) and BCOADC (BCOADC-E2), the E2 subunits of OGDC (OGDC-E2) reactivity of PBC sera and the reactive epitope of OGDC-D2 have not hitherto been studied in detail. In this report, we took advantage of a recombinant fusion protein for OGDC-E2 to address these issues. Eighty of 268 (29.9%) PBC patient sera but none of the 45 controls reacted with recombinant OGDC-E2. The recombinant OGDC-E2 was judged to express the immunodominant epitope, because when sera from patients with PBC were preabsorbed with the recombinant fusion protein, such sera were depleted of reactivity against 48 kD OGDC-E2 when probed on beef heart mitochondria (BHM) but retained reactivity toward PDC-E2 and/or BCOADC-E2. Furthermore, affinity-purified PBC sera against recombinant OGDC-E2 reacted only with native OGDC-E2 and not with any other enzyme components of the 2-oxo acid dehydrogenase complex. Antimitochondrial autoantibodies (AMA) against OGDC-E2 included immunoglobulin (Ig)G2, IgG3 and IgM and the relative titers were as follows: IgG2 > IgG3 > IgM. Finally, using overlapping recombinant polypeptides, it was determined that a minimum of 81 amino acids (residues 67-147) corresponding to the lipoyl domain of OGDC-E2 are necessary for reactivity, suggesting that a conformational autoepitope is recognized by AMA. These data suggest that each of the 2-oxo acid dehydrogenase enzymes has distinct antigenicity despite their similarities in structure and function. The availability of recombinant OGDC-E2 autoantigen will allow the design of additional studies to further our understanding of the role of mitochondrial autoantigens in the pathogenesis of PBC.

The nursing care recording system. A preliminary study of a system for assessment of nursing care demands in the ICU.

A new system, Nursing Care Recording (NCR), for the recording of nursing care in a general ICU is presented. NCR classifies ICU patients according to their need for intensive nursing care. Comparing the NCR with the Therapeutic Intervention Scoring System (TISS), a correlation coefficient of 0.60 was found. The main difference between the two systems was related to recording procedures allowing changes in nursing intensity within a 24-h period, reflecting patient improvement due to therapy, which was detected by NCR but not by TISS. NCR can be used to estimate nursing capacity during different shifts and may be useful in the assessment of the total nursing staff necessary for a given ICU. It is suggested that NCR will allow detection of changes in the nursing care work load, whether this change is due to new activities in the unit or to alterations in the individual patient care.