Genotyping of circulating tumor DNA in cholangiocarcinoma reveals diagnostic and prognostic information.

Diagnosis of Cholangiocarcinoma (CCA) is difficult, thus a noninvasive approach towards (i) assessing and (ii) monitoring the tumor-specific mutational profile is desirable to improve diagnosis and tailor treatment. Tumor tissue and corresponding ctDNA samples were collected from patients with CCA prior to and during chemotherapy and were subjected to deep sequencing of 15 genes frequently mutated in CCA. A set of ctDNA samples was also submitted for 710 gene oncopanel sequencing to identify progression signatures. The blood/tissue concordance was 74% overall and 92% for intrahepatic tumors only. Variant allele frequency (VAF) in ctDNA correlated with tumor load and in the group of intrahepatic CCA with PFS. 63% of therapy naive patients had their mutational profile changed during chemotherapy. A set of 76 potential progression driver genes was identified among 710 candidates. The molecular landscape of CCA is accessible via ctDNA. This could be helpful to facilitate diagnosis and personalize and adapt therapeutic strategies.

[H3F3A mutated multicentric giant cell tumor of bone : A very rare primary bone disease]. / H3F3A-mutierter multifokaler Riesenzelltumor des Knochens : Eine äußerst seltene primär ossäre Erkrankung.

This article presents the case of a metachronic multicentric giant cell tumor of bone (GCTB). The patient obtained his first diagnosis of GCTB in the left humerus at the age of 47 years. Furthermore, he suffered from a GCTB in the head of his 4th left metacarpal bone and from a recurrence of the latter. All tumors carried the characteristic H3F3A mutation, which was proven by Sanger sequencing and a mutation specific antibody. The case is the first description of a multicentric H3F3A mutated GCTB.

FTO deficiency induces UCP-1 expression and mitochondrial uncoupling in adipocytes.

Variants in the fat mass- and obesity-associated (FTO) gene are associated with obesity and body fat mass in genome-wide association studies. However, the mechanism by which FTO predisposes individuals to obesity is not clear so far. First mechanistic evidence was shown in Fto-negative mice. These mice are resistant to obesity due to enhanced energy expenditure, whereas the mass of brown adipose tissue remains unchanged. We hypothesize that FTO is involved in the induction of white adipose tissue browning, which leads to mitochondrial uncoupling and increases energy expenditure. Uncoupling protein 1 (Ucp-1) was significantly higher expressed in both gonadal and inguinal adipose depots of Fto(-/-) compared with Fto(+/+) littermates accompanied by the appearance of multivacuolar, Ucp-1-positive adipocytes in these tissues. By using lentiviral short hairpin RNA constructs, we established FTO-deficient human preadipocytes and adipocytes and analyzed key metabolic processes. FTO-deficient adipocytes showed an adipogenic differentiation rate comparable with control cells but exhibited a reduced de novo lipogenesis despite unchanged glucose uptake. In agreement with the mouse data, FTO-deficient adipocytes exhibited 4-fold higher expression of UCP-1 in mitochondria compared with control cells. The up-regulation of UCP-1 in FTO-deficient adipocytes resulted in enhanced mitochondrial uncoupling. We conclude that FTO deficiency leads to the induction of a brown adipocyte phenotype, thereby enhancing energy expenditure. Further understanding of the signaling pathway connecting FTO with UCP-1 expression might lead to new options for obesity and overweight treatment.

Yersinia enterocolitica leads to transient induction of TNF-alpha and activates NF-kappaB in synovial fibroblasts.

OBJECTIVE: The importance of the presence of bacterial antigen or even living bacteria for the pathogenesis of reactive arthritis has been discussed increasingly ever since bacterial antigen was found in inflamed joints. Bacteria may persist in the body and drive the local immune response, maintaining arthritis. Cytokines, in particular tumor necrosis factor-alpha (TNF-alpha) are essential for bacterial elimination. In reactive arthritis, the course of the disease is influenced by several cytokines, including TNF-alpha. TNF-alpha expression can be mediated by transcription factor nuclear factor-kappa B (NF-kappaB). Moreover, TNF-alpha is also one of the strongest activators of NF-kappaB. METHODS: In vitro expression of TNF-alpha and activation of NF-kappaB in synovial fibroblasts after infection with Yersinia enterocolitica or Salmonella enteritidis was analysed by electrophoretic mobility shift assay, Western blot assay and real-time PCR. RESULTS: We found that infection of synovial fibroblasts with yersinia and salmonellae lead to the transient expression of TNF-alpha mRNA and induction of NF-kappaB. CONCLUSION: Induction of TNF-alpha in synovial fibroblasts after infection with yersiniae or salmonellae might be insufficient to eliminate bacteria, and this could allow the intracellular persistence of these bacteria. Our results therefore support the hypothesis that a permissive cytokine pattern might contribute to the pathogenesis of reactive arthritis.

Signal-specific and phosphorylation-dependent RelB degradation: a potential mechanism of NF-kappaB control.

RelB is an unusual member of the Rel/NF-kappaB family of transcription factors which are involved in oncogenic processes. Due to a relaxed control by the IkappaBs, the cytosolic NF-kappaB inhibitors, RelB is constitutively expressed in the nuclei of lymphoid cells. We show here that RelB is inducibly degraded upon activation of T cells in a fashion similar to the IkappaBs. However, RelB degradation differs from that of IkappaBs since it is not induced by TNFalpha but only by T cell receptor or TPA/ionomycin stimulation. Moreover, RelB degradation occurs in three steps: (i) after stimulation RelB is rapidly phosphorylated at amino acids Thr84 and Ser552 followed by (ii) an N-terminal cut and, finally, (iii) the complete degradation in the proteasomes. Since mutation of the two phosphoacceptor sites to non-acceptor sites abolished RelB phosphorylation in vivo and led to the stabilization of the mutated RelB(DM), site-specific phosphorylation appears to be a necessary prerequisite for RelB degradation. RelB is a crucial regulator of NF-kappaB-dependent gene expression. Thus, the signal-induced degradation of RelB should be an important control mechanism of NF-kappaB activity.

Induction of nuclear factor-kappa B during primary B cell differentiation.

We have investigated activation of nuclear factor-kappa B (NF-kappa B) in the process of primary B cell differentiation in vitro. In this system, NF-kappa B is strongly induced when B cells develop from the pre-B cell to the immature B cell stage. Unlike the typical NF-kappa B activation in response to exogenous stimuli, induction proceeds with a slow time course. NF-kappa B induction is only observed in B cells that undergo differentiation, not in Rag2-deficient cells. Nuclear DNA binding complexes predominantly comprise p50/RelA heterodimers and, to a lesser extent, c-Rel-containing dimers. The increase in NF-kappa B binding activity is accompanied by a slow and steady decrease in I kappa B beta protein levels. Interestingly, absolute RelA protein levels remain unaffected, whereas RelB and c-Rel synthesis is induced. The reason for preferential nuclear translocation of RelA complexes appears to be selective inhibition by the I kappa B beta protein. I kappa B beta can efficiently inhibit p50/RelA complexes, but has a much reduced ability to interfere with p50/c-Rel DNA binding both in vitro and in vivo. Interestingly, p50/RelB complexes are not at all targeted by I kappa B beta, and coimmunoprecipitation experiments show no evidence for an association of I kappa B beta and RelB in vivo. Consistent with these observations, I kappa B beta cotransfection can inhibit p50/RelA-mediated trans-activation, but barely affects p50/RelB mediated trans-activation.

Cyclosporin A interferes with the inducible degradation of NF-kappa B inhibitors, but not with the processing of p105/NF-kappa B1 in T cells.

The transcription factor NF-kappa B controls the induction of numerous cytokine promoters during the activation of T lymphocytes. Inhibition of T cell activation by the immunosuppressants cyclosporin A (CsA) and FK506 exerts a suppressive effect on the induction of these NF-kappa B-controlled cytokine promoters. We show for human Jurkat T leukemia cells, as well as human and mouse primary T lymphocytes, that this inhibitory effect is accompanied by an impaired nuclear translocation of the Rel proteins c-Rel, RelA/p65 and NF-kappa B1/p50, whereas the nuclear appearance of RelB remains unaffected. CsA does not interfere with the synthesis of Rel proteins, but prevents the inducible degradation of cytosolic NF-kappa B inhibitors I kappa B alpha and I kappa B beta upon T cell activation. CsA neither inhibits the processing of the NF-kappa B1 precursor p105 to p50, nor does it "stabilize" the C-terminal portion of p105, I kappa B gamma, which is degraded during p105 processing to mature p50. These results indicate that CsA interferes with a specific event in the signal-induced degradation of I kappa B alpha and I kappa B beta, but does not affect the processing of NF-kappa B1/p105 to p50.

Cloning of chicken interferon regulatory factor-2 (IRF-2) cDNA: expression and mapping of the IRF-2 gene.

A cDNA clone encoding a member of the avian interferon regulatory factor (IRF) family homologous to mammalian IRF-2 was isolated from cDNA library from poly[rI:rC]-induced chicken embryo fibroblasts (CEF). The deduced amino acid sequence shows a characteristic DNA binding domain of 124 amino acids at the amino-terminal end with 96.8% identity to human and 96% to mouse IRF-2. Identities in the C-terminal part are 77.5% and 77%, respectively. Identity to all other known members of the chicken IRF (Ch-IRF) family is distinctly lower. In C32 cells, an IRF-2 mRNA of 2.4 kb is constitutively expressed in very low amounts but is inducible by Ch-IFN in the absence or presence of cycloheximide. The Ch-IRF-2 gene is a single copy gene and was mapped by fluorescence in situ hybridization to the long arm of chromosome 4.

Rel/NF-kappa B transcription factors and cancer.

The transcription factors of the Rel/NF-kappa B family play a central role in immune regulation. In addition, they may be involved in the pathogenesis of certain tumors, especially those of hematopoietic origin. In this review evidence is summarized indicating an association between dysregulated NF-kappa B-dependent gene expression and tumorigenesis. Furthermore, possible molecular mechanisms through which NF-kappa B might exert these effects, such as disturbance of cell cycle control or apoptosis, are outlined. Potential therapeutic approaches based on these findings are also described. Rel/NF-kappa B factors are presented here as an important example of the growing number of transcription factors possibly involved in oncogenesis.

Prolonged hospitalization because of inappropriate delay of warfarin therapy in deep venous thrombosis.

Current anticoagulation practices of physicians in an academic medical center were examined by retrospective review of records of 26 patients admitted for uncomplicated deep venous thrombosis (DVT) between 1978 and 1982. Patients received intravenous heparin for an average of 11.5 +/- 2.8 days. Warfarin therapy was started on day 8.3 +/- 3.1 and therapeutic oral anticoagulation was achieved by day 14.5 +/- 4.5. Total hospital stay averaged 16.8 +/- 5.1 days. No patient received concurrent initiation of heparin and warfarin therapy and only two patients received warfarin before day 5. These findings are in contrast to anticoagulation practices in Great Britain and Scandinavia where concurrent initiation of heparin and warfarin has been common practice for many years. Such a regimen is safe and usually requires fewer days of hospitalization for DVT. We conclude that failure to start warfarin therapy on the first hospital day resulted in a costly and unnecessarily prolonged hospital stay.

Adult respiratory distress syndrome secondary to tularemia pneumonia.

Three patients with tularemia pneumonia developed adult respiratory distress syndrome and required positive end-expiratory pressure for adequate oxygenation. Rapid improvement was noted following appropriate antibiotic therapy, and mechanical ventilation was successfully discontinued after six to eight days.