Functional characterisation of osteosarcoma cell lines and identification of mRNAs and miRNAs associated with aggressive cancer phenotypes.

BACKGROUND: Osteosarcoma is the most common primary malignant bone tumour, predominantly affecting children and adolescents. Cancer cell line models are required to understand the underlying mechanisms of tumour progression and for preclinical investigations. METHODS: To identify cell lines that are well suited for studies of critical cancer-related phenotypes, such as tumour initiation, growth and metastasis, we have evaluated 22 osteosarcoma cell lines for in vivo tumorigenicity, in vitro colony-forming ability, invasive/migratory potential and proliferation capacity. Importantly, we have also identified mRNA and microRNA (miRNA) gene expression patterns associated with these phenotypes by expression profiling. RESULTS: The cell lines exhibited a wide range of cancer-related phenotypes, from rather indolent to very aggressive. Several mRNAs were differentially expressed in highly aggressive osteosarcoma cell lines compared with non-aggressive cell lines, including RUNX2, several S100 genes, collagen genes and genes encoding proteins involved in growth factor binding, cell adhesion and extracellular matrix remodelling. Most notably, four genes-COL1A2, KYNU, ACTG2 and NPPB-were differentially expressed in high and non-aggressive cell lines for all the cancer-related phenotypes investigated, suggesting that they might have important roles in the process of osteosarcoma tumorigenesis. At the miRNA level, miR-199b-5p and mir-100-3p were downregulated in the highly aggressive cell lines, whereas miR-155-5p, miR-135b-5p and miR-146a-5p were upregulated. miR-135b-5p and miR-146a-5p were further predicted to be linked to the metastatic capacity of the disease. INTERPRETATION: The detailed characterisation of cell line phenotypes will support the selection of models to use for specific preclinical investigations. The differentially expressed mRNAs and miRNAs identified in this study may represent good candidates for future therapeutic targets. To our knowledge, this is the first time that expression profiles are associated with functional characteristics of osteosarcoma cell lines.

HPV16 activates the AIM2 inflammasome in keratinocytes.

Human papillomaviruses (HPV) are double-stranded DNA viruses, which selectively infect keratinocytes in stratified epithelia. After an initial infection, many patients clear HPV. In some patients, however, HPV persist, and dysfunctional innate immune responses to HPV infection could be involved in the ineffective clearing of these viruses. In this study, the mechanisms of HPV-induced immune responses in keratinocytes were investigated. Binding of viral DNA leads to AIM2 inflammasome activation and IL-1ß release, while IFI16 activation results in IFN-ß release. Using immunohistochemistry, AIM2 and IFI16-two recently identified sensors for cytosolic DNA-were also detected in HPV positive skin lesions. CISH stainings further confirmed the presence of cytosolic HPV16 DNA in biopsy samples. Moreover, active IL-1ß and cleaved caspase-1 were detected in HPV infected skin, suggesting inflammasome activation by viral DNA. In subsequent functional studies, HPV16 DNA triggered IL-1ß and IL-18 release via the AIM2 inflammasome in normal human keratinocytes. Although HPV DNA did not induce IFN-ß in keratinocytes, IFN-ß secretion was observed when AIM2 was blocked. Meanwhile, blocking of IFI16 increased HPV16 DNA-induced IL-1ß, but not IL-18, secretion. These findings suggest crosstalk between IFI16 and AIM2 in the immune response to HPV DNA. In sum, novel aspects concerning HPV-induced innate immune responses were identified. Eventually, understanding the mechanisms of HPV-induced inflammasome activation could lead to the development of novel strategies for the prevention and treatment of HPV infections.

Translocation t(14;18) and gain of chromosome 18/BCL2: effects on BCL2 expression and apoptosis in B-cell non-Hodgkin's lymphomas.

Gain of chromosome 18q and translocation t(14;18) are] frequently found in B-cell non-Hodgkin's lymphomas (B-NHL). Increased BCL2 transcription and BCL2 protein expression have been suggested to be the result of the gain. We utilized FISH, PCR and array CGH to study BCL2 and chromosome 18 copy number changes and rearrangements in 93 cases of B-NHL. BCL2 protein was expressed in >75% of the tumor cells in 92% of the cases by immunohistochemistry. Gain of BCL2 was associated with a 25% increase in BCL2 expression levels (immunoblotting), whereas t(14;18) resulted in a 55% increase in BCL2 levels compared to cases without BCL2 alterations. The tumor cell (spontaneous) apoptotic fractions were similar for the cases with different BCL2 genotypes. However, the normal cell apoptotic fractions were higher for the tumors with t(14;18) compared to the tumors without BCL2 alterations, while the tumors with gain of BCL2 only showed intermediate levels. Low-level gains of parts of chromosome 18 were found in 14 of the 38 B-NHL cases with t(14;18), with a consensus region 18pter-q21.33 that did not include the BCL2 gene. The 11 cases with 18q gain only showed a consensus region encompassing 18q21.2-18q21.32 and 18q21.33, which contain PMAIP1/MALT1 and BCL2, respectively.

Influence of renal replacement therapy on outcome of patients with acute renal failure.

There are many controversial results about the influence of acute renal failure (ARF) and renal replacement therapy (RRT) on patient outcome in intensive care units. This retrospective study compared demographics. severity, course, and prognosis of ARF during 36 months (period 1, 1991 through 1993; 128 cases) and 18 months (period 2, 1994 through 1995; 141 cases). Compared with period 1, during period 2 there was a markedly increased incidence of ARF. There were no significant differences in patient demographics or etiology of renal failure, but the therapeutic approach to ARF was quite different. During period 2, RRT was started at earlier stages of renal insufficiency (that is, less elevated creatinine serum concentrations or reduced diuresis). Additionally, there was a significant increase in the numbers of continuous RRT (CRRT) replacing the discontinuous mode of dialysis treatment. Compared with period 1, mortality was reduced from 78.9 to 59.6% during period 2 (P < 0.001). There were no differences in mortality between the patients from internal and surgical wards. Mortality in patients treated with CRRT was in period 1 and in period 2 higher than mortality in patients treated with intermittent RRT, but these results are biased by a preferred use of CRRT in severely ill patients with an unstable circulatory system. These data suggest that the early onset of RRT reduces the mortality of intensive care unit patients with ARF independent of underlying diseases. An influence of the method of RRT, sex, and age on outcome of patients with ARF could not be proven.

[Contact cyclophotocoagulation with the continuous wave Nd:YAG laser with quartz fiber. Optimizing coagulation parameters]. / Kontaktzyklophotokoagulation mit dem Dauerstrich-Nd:YAG-Laser über Quarzfaser. Optimierung der Koagulationsparameter.

Transscleral cyclo-photocoagulation with a cw-Nd:YAG-laser was studied in enucleated porcine eyes with application times of between 10 ms and 1.5 s. Contact coagulation via a quartz fiber (core 600 microns) required about 32% less power to create visible ciliary body coagulation compared to non-contact coagulation via a focusing handpiece. Application of focusing fiber tips led to a further reduction in the coagulation threshold by a factor of 0.6. The energy per laser application required for ciliary body coagulation increased with application time. During contact coagulation using plane fiber tips a pronounced temperature rise was sometimes observed at the fiber tip due to deposits of carbonized tissue with the subsequent risk of scleral damage. In a pilot study five eyes with secondary glaucoma were treated under coagulation conditions found to be optimal (8 W at 0.2 s).

Thermal side reactions during in vitro contact cyclophotocoagulation with the continuous wave Nd:YAG laser.

We compared the effects of continuous wave Nd:YAG laser irradiation on the ciliary body and sclera of enucleated porcine eyes produced by two means of transmission: contact coagulations via a quartz fiber (core 600 microns), and noncontact coagulations via a focussing handpiece. Application times ranged from 10 ms to 1.5 seconds. During contact coagulations using plane fiber tips the temperature at the fiber tip, due to deposits of carbonized tissue, sometimes increased markedly, increasing the risk of perforation. Clinical application of the contact method using plain fiber tips is not recommended.

[Kidney changes in patients with proliferative diabetic retinopathy]. / Nierenveränderungen bei Patienten mit proliferativer diabetischer Retinopathie.

On 42 patients (25 males, 17 females) at the age of 46.1 +/- 13.4 years with a proliferative diabetic retinopathy clinical and laboratory examinations were performed for the proof of a renal lesion. This disease was found in 59.5% of the cases. In the foreground of the pathological findings were a proteinuria, a restriction of the creatinine clearance and of the concentration power of the kidneys as well as the hypertension. The diabetic nephropathy had its peak of frequency between the 50th and 60th year of age and showed significant relations to the duration of diabetes as well as to be early age of manifestation. Close ophthalmological and nephrological examinations, particularly of the juvenile diabetics, should render possible an early recognition and treatment of the diabetic microangiopathy.