Primary orbital reconstruction with selective laser melted core patient-specific implants: overview of 100 patients.

Contemporary advances in technology have enabled the transfer of industrial laser melting technology to surgery, and its use can improve the accuracy of orbital restoration. The aim of this study therefore was to evaluate the accuracy of primary orbital reconstruction with the use of selective laser melted, patient-specific implants and navigation. A total of 100 patients with complex orbital fractures were included. Planned orbital volumes were compared with those achieved, and angles were compared with the unaffected side. Analysis included the overlay of postoperative on planned images (iPlan® 3.0.5, Brainlab). The mean (SD) orbital volume of the unaffected side was 27.2 (2.8)ml in men and 25.0 (2.6)ml in women. Fractures that involved the posterior third of the orbital floor and comminuted fractures showed significant orbital enlargement (p=0.026). The mean (SD) reconstructed orbital volume was 26.9 (2.7)ml in men and 24.26 (2.5)ml in women. Three-dimensional analysis of the colour mapping showed minor deviations when compared with the unaffected side. The results suggest that a high degree of accuracy can be routinely achieved in these complex cases.

Hyperactivation of mTORC1 and mTORC2 by multiple oncogenic events causes addiction to eIF4E-dependent mRNA translation in T-cell leukemia.

High activation of the PI3K-AKT-mTOR pathway is characteristic for T-cell acute lymphoblastic leukemia (T-ALL). The activity of the master regulator of this pathway, PTEN, is often impaired in T-ALL. However, experimental evidence suggests that input from receptor tyrosine kinases (RTKs) is required for sustained mTOR activation, even in the absence of PTEN. We previously reported the expression of Neurotrophin receptor tyrosine kinases (TRKs) and their respective ligands in primary human leukemia samples. In the present study we aimed to dissect the downstream signaling cascades of TRK-induced T-ALL in a murine model and show that T-ALLs induced by deregulated receptor tyrosine kinase signaling acquire activating mutations in Notch1 and lose PTEN during clonal evolution. Some clones additionally lost one allele of the homeodomain transcription factor Cux1. All events independently led to a gradual hyperactivation of both mTORC1 and mTORC2 signaling. We dissected the role of the individual mTOR complexes by shRNA knockdown and found that the separate depletion of mTORC1 or mTORC2 reduced the growth of T-ALL blasts, but was not sufficient to induce apoptosis. In contrast, knockdown of the mTOR downstream effector eIF4E caused a striking cytotoxic effect, demonstrating a critical addiction to cap-dependent mRNA-translation. Although high mTORC2-AKT activation is commonly associated with drug-resistance, we demonstrate that T-ALL displaying a strong mTORC2-AKT activation were specifically susceptible to 4EGI-1, an inhibitor of the eIF4E-eIF4G interaction. To decipher the mechanism of 4EGI-1, we performed a genome-wide analysis of mRNAs that are translationally regulated by 4EGI-1 in T-ALL. 4EGI-1 effectively reduced the ribosomal occupancy of mRNAs that were strongly upregulated in T-ALL blasts compared with normal thymocytes including transcripts important for translation, mitochondria and cell cycle progression, such as cyclins and ribosomal proteins. These data suggest that disrupting the eIF4E-eIF4G interaction constitutes a promising therapy strategy in mTOR-deregulated T-cell leukemia.

[Management of the patient with Meniere's disease. Clear diagnosis but vertigo-provoking perspective]. / Patientenführung bei Morbus Menière. Klare Diagnose, meist schwindelerregende Perspektiven.

Meniere's disease is characterised by recurrent attacks of vertigo, sensory hearing loss and tinnitus. Meniere's attacks can lead to additional bouts of dizziness. While diagnosis in recurrent spells is easily secured, managing the patients is as difficult as the often unclear development of this -- mostly -- progressive disease. The underlying principle for therapy of this frequently changing disease is a reliable patient-physician relation, which should be based on extensive knowledge of the disease and not only include ENT findings but also broad medical counselling.

The MAPK kinase kinase TAK1 plays a central role in coupling the interleukin-1 receptor to both transcriptional and RNA-targeted mechanisms of gene regulation.

Mechanisms of fulminant gene induction during an inflammatory response were investigated using expression of the chemoattractant cytokine interleukin-8 (IL-8) as a model. Recently we found that coordinate activation of NF-kappaB and c-Jun N-terminal protein kinase (JNK) is required for strong IL-8 transcription, whereas the p38 MAP kinase (MAPK) pathway stabilizes the IL-8 mRNA. It is unclear how these pathways are coupled to the receptor for IL-1, an important physiological inducer of IL-8. Expression of the MAP kinase kinase kinase (MAPKKK) TAK1 together with its coactivator TAB1 in HeLa cells activated all three pathways and was sufficient to induce IL-8 formation, NF-kappaB + JNK2-mediated transcription from a minimal IL-8 promoter, and p38 MAPK-mediated stabilization of a reporter mRNA containing IL-8-derived regulatory mRNA sequences. Expression of a kinase-inactive mutant of TAK1 largely blocked IL-1-induced transcription and mRNA stabilization, as well as formation of endogenous IL-8. Truncated TAB1, lacking the TAK1 binding domain, or a TAK1-derived peptide containing a TAK1 autoinhibitory domain were also efficient in inhibition. These data indicate that the previously described three-pathway model of IL-8 induction is operative in response to a physiological stimulus, IL-1, and that the MAPKKK TAK1 couples the IL-1 receptor to both transcriptional and RNA-targeted mechanisms mediated by the three pathways.

The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism.

Stabilization of mRNAs contributes to the strong and rapid induction of genes in the inflammatory response. The signaling mechanisms involved were investigated using a tetracycline-controlled expression system to determine the half-lives of interleukin (IL)-6 and IL-8 mRNAs. Transcript stability was low in untreated HeLa cells, but increased in cells expressing a constitutively active form of the MAP kinase kinase kinase MEKK1. Destabilization and signal-induced stabilization was transferred to the stable beta-globin mRNA by a 161-nucleotide fragment of IL-8 mRNA which contains an AU-rich region, as well as by defined AU-rich elements (AREs) of the c-fos and GM-CSF mRNAs. Of the different MEKK1-activated signaling pathways, no significant effects on mRNA degradation were observed for the SAPK/JNK, extracellular regulated kinase and NF-kappaB pathways. Selective activation of the p38 MAP kinase (=SAPK2) pathway by MAP kinase kinase 6 induced mRNA stabilization. A dominant-negative mutant of p38 MAP kinase interfered with MEKK1 and also IL-1-induced stabilization. Furthermore, an active form of the p38 MAP kinase-activated protein kinase (MAPKAP K2 or MK2) induced mRNA stabilization, whereas a negative interfering MK2 mutant interfered with MAP kinase kinase 6-induced stabilization. These findings indicate that the p38 MAP kinase pathway contributes to cytokine/stress-induced gene expression by stabilizing mRNAs through an MK2-dependent, ARE-targeted mechanism.

Induction of interleukin-8 synthesis integrates effects on transcription and mRNA degradation from at least three different cytokine- or stress-activated signal transduction pathways.

A hallmark of inflammation is the burst-like formation of certain proteins, initiated by cellular stress and proinflammatory cytokines like interleukin 1 (IL-1) and tumor necrosis factor, stimuli which simultaneously activate different mitogen-activated protein (MAP) kinases and NF-kappaB. Cooperation of these signaling pathways to induce formation of IL-8, a prototype chemokine which causes leukocyte migration and activation, was investigated by expressing active and inactive forms of protein kinases. Constitutively active MAP kinase kinase 7 (MKK7), an activator of the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathway, induced IL-8 synthesis and transcription from a minimal IL-8 promoter. Furthermore, MKK7 synergized in both effects with NF-kappaB-inducing kinase (NIK). Activation of the IL-8 promoter by either of the kinases required functional NF-kappaB and AP-1 sites. While NIK and MKK7 did not affect degradation of IL-8 mRNA, an active form of MKK6, which selectively activates p38 MAP kinase, induced marked stabilization of the transcript and further increased IL-8 protein formation induced by NIK plus MKK7. Consistently, the MAP kinase kinase kinase MEKK1, which can activate NF-kappaB, SAPK/JNK, and p38 MAP kinases, most potently induced IL-8 formation. These results provide evidence that maximal IL-8 gene expression requires the coordinate action of at least three different signal transduction pathways which cooperate to induce mRNA synthesis and suppress mRNA degradation.

Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally.

Homozygous targeted disruption of the mouse Caspase 8 (Casp8) gene was found to be lethal in utero. The Caspase 8 null embryos exhibited impaired heart muscle development and congested accumulation of erythrocytes. Recovery of hematopoietic colony-forming cells from the embryos was very low. In fibroblast strains derived from these embryos, the TNF receptors, Fas/Apo1, and DR3 were able to activate the Jun N-terminal kinase and to trigger IkappaB alpha phosphorylation and degradation. They failed, however, to induce cell death, while doing so effectively in wild-type fibroblasts. These findings indicate that Caspase 8 plays a necessary and nonredundant role in death induction by several receptors of the TNF/NGF family and serves a vital role in embryonal development.

Stress-activated protein kinase/Jun N-terminal kinase is required for interleukin (IL)-1-induced IL-6 and IL-8 gene expression in the human epidermal carcinoma cell line KB.

The cytokine interleukin-1 (IL-1) is a major inflammatory hormone which activates a broad range of genes during inflammation. The signaling mechanisms triggered by IL-1 include activation of several distinct protein kinase systems. The stress-activated protein kinase (SAPK), also termed Jun N-terminal kinase (JNK), is activated particularly strongly by the cytokine. In an attempt to delineate its role in activation of gene expression by IL-1, we inhibited the IL-1-induced SAPK/JNK activity by stable overexpression of either a catalytically inactive mutant of SAPKbeta (SAPKbeta(K-R)) or antisense RNA to SAPKbeta in human epidermal carcinoma cells. A detailed analysis of signal transduction in those cells showed that activation of neither NFkappaB nor p38 mitogen-activated protein kinase was affected, suggesting that we achieved specific blockade of the SAPK/JNK. In untransfected and vector-transfected KB cells, IL-1 induced a strong increase in expression of IL-6 and IL-8 mRNA, along with the synthesis of high amounts of the proteins. In two KB cell clones stably overexpressing the mutant SAPKbeta(K-R), and three clones stably overexpressing antisense RNA to SAPKbeta, expression of IL-6 and IL-8 in response to IL-1 was strongly reduced at both the mRNA and protein level. These data indicate that the SAPK/JNK pathway provides an indispensable signal for IL-1-induced expression of IL-6 and IL-8.

Protein binding regions of the mRNAs for the 55 kDa tumor necrosis factor receptor and the glucose transporter 1: sequence homology and competition for cellular proteins.

Gene expression is influenced by mechanisms regulating mRNA degradation. Knowledge on regulatory RNA elements involved and on proteins interacting with them is still limited. A 33 nucleotide (nt) region of the 55 kDa tumor necrosis factor receptor (TNFR-55) mRNA, previously reported by us to engage in such interaction with proteins from U-937 cells, exhibits homology to a 38 nt regulatory region of the glucose transporter GLUT-1 mRNA. Labeled RNA fragments comprising these two regions bind similar sets of proteins. Upon phorbol ester-induced differentiation into macrophage-like cells, protein binding to both fragments is changed similarly. Furthermore, both compete with each other for protein binding. This suggests that GLUT-1 and TNFR-55 RNA share a novel protein binding RNA motif involved in regulation of their half life.

Studies of persistent infection by Chlamydia trachomatis serovar K in TPA-differentiated U937 cells and the role of IFN-gamma.

Inoculation of phorbol ester-differentiated U937 cells as a model for human macrophages with Chlamydia trachomatis of the urogenital serovar K resulted in a persistent infection, with maximal growth at day 7, until day 10 post-infection. At these times inclusion bodies were present in 0.5-2% of the cells. Typical inclusion bodies containing elementary bodies and reticulate bodies were observed by electron microscopy. Furthermore, single chlamydial particles resembling atypical elementary or intermediate bodies were identified in the cytoplasm in > 80% of the host cells. IFN-gamma exerts antichlamydial activity in epithelial and fibroblastoid cells, but the infection of U937 cells by C. trachomatis was not affected by IFN-gamma. The activity of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) was not detected in untreated or in IFN-gamma-treated or chlamydiae-infected or mock-infected U937 cells. The presence of atypical persisting chlamydiae and the lack of IDO expression in U937 cells indicates that the development of these atypical bacteria is independent from IFN-gamma-mediated tryptophan deprivation and other IFN-gamma-mediated effects. Evaluation of persistently infected cells revealed that the expression of the chlamydial major outer-membrane protein, heat-shock protein (hsp60) and lipopolysaccharide (LPS) antigens was not significantly altered in the course of the culture. An intense staining of the LPS on the surface of the host cells was demonstrated by immunofluorescence. The data show that phorbol ester-differentiated U937 cells restrict chlamydial growth strongly but not completely through a mechanism distinct from IDO-mediated tryptophan deprivation. The mechanisms of persistence of chlamydiae in monocytes, which differ considerably from those described for other cells, require further investigation.

TNF receptors--how they function and interact.

Tumor necrosis factor (TNF) is a pleiotropic cytokine regulating various immune and inflammatory reactions. It induces cellular responses upon binding to specific cell surface receptors of two distinct molecular species--p55 and p75 TNF receptors (TNF-Rs). Cloning of the respective cDNAs and obtaining the receptor-specific molecular tools such a agonistic and blocking monoclonal antibodies, TNF muteins with exclusive ability to bind to only one receptor type, as well as generation of TNF-R mutants by site-directed mutagenesis enabled researchers to answer key questions of the biology of initial steps in the cascade of TNF signal transduction. The presented mini-review describes the mode of function of the two receptors and discusses the possible ways of interaction between them.

Regulation of expression of transmembrane and soluble 75 kDa tumor necrosis factor receptors by interferon-gamma and granulocyte-macrophage colony-stimulating factor involves transcriptional activation.

The receptors for tumor necrosis factor (TNF) play an important role in the response to this cytokine, both as signal transducing molecules and, in their shed forms, as regulators of TNF availability. Expression of the receptors was studied in the human monocytic leukemia line THP-1. Within two days of incubation, the proinflammatory cytokines, interferon (IFN)-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF), each induced a slight increase in cell surface expression of the 75 kDa TNF receptors (TNF-R75), and a more pronounced increase in the generation of soluble TNF-R75. Similarly, receptor mRNA levels were increased in response to both cytokines. GM-CSF and IFN-gamma in combination induced a much stronger increase in cell surface and soluble receptors as well as in receptor mRNA. Expression of the 55 kDa TNF receptor and its mRNA was largely unaffected by the two cytokines. Experiments using TNF-neutralizing antibodies indicate that the changes in TNF-R75 expression occurred independently of endogenously-produced TNF. The half life of TNF-R75 mRNA in cells exposed to GM-CSF + IFN-gamma did not differ significantly from that in untreated cells. According to nuclear run-on assays the synthesis of TNF-R75 mRNA in cells treated with GM-CSF + IFN-gamma, as well as with the phorbol ester TPA, was markedly increased compared to untreated cells, indicating that the observed changes in receptor expression primarily involve altered transcription of the gene. The results suggest that in inflammatory processes, GM-CSF and IFN-gamma contribute to increased synthesis of TNF-R75 by monocytic cells, a prerequisite for the formation of large amounts of soluble receptors.

Interaction between the mRNA of the 55-kDa tumor necrosis factor receptor and cellular proteins. Possible involvement in post-transcriptional regulation of receptor expression.

Numerous effects of tumor necrosis factor are signaled by its 55-kDa receptors. Studying their expression we found that the level of receptor mRNA was decreased during the phorbol ester-induced differentiation of myelomonocytic cell lines. While only minor changes in transcription were noted, the half-life of receptor mRNA in the differentiated cells was markedly decreased, indicating the involvement of post-transcriptional regulation. In an electrophoretic mobility shift assay, formation of complexes between radiolabeled receptor mRNA and cellular proteins was observed. The decrease in receptor mRNA levels during phorbol ester-induced differentiation was paralleled by a change in the pattern of those complexes. Protein-RNA interaction was selective, as it was not competed by unrelated RNAs. Yet, certain mRNAs that contain AU-rich sequences, known to be involved in the control of their stability, did compete with the receptor mRNA, although the latter is devoid of such sequences. A region of 18 nucleotides within its coding region was found to contain an element essential for the formation of all complexes and sufficient for the formation of those with lower molecular mass. Adjacent bases were required in addition for the formation of the complexes with higher molecular mass. The results suggest that proteins interacting with this region of the 55-kDa tumor necrosis factor receptor mRNA contribute to the regulation of its expression.

Cytokines.

The defense of the organism against pathogens involves complex processes in which many different types of cells take part. Their functions are coordinated by polypeptide mediators collectively named cytokines. The intense research devoted to these molecules in the past has yielded an insight into their molecular characteristics, mode of action, physiological function, and their deleterious role in numerous diseases. Yet therapeutic application of that knowledge--either controlled administration of cytokines or neutralization of their negative effects in certain pathological processes--despite initial achievements has remained a major challenge.

Dual role of the p75 tumor necrosis factor (TNF) receptor in TNF cytotoxicity.

Whereas there is ample evidence for involvement of the p55 tumor necrosis factor (TNF) receptor (p55-R) in the cytocidal effect of TNF, the role of the p75 TNF receptor (p75-R) in this effect is a matter of debate. In this study, we probed the function of p75-R in cells sensitive to the cytotoxicity of TNF using a wide panel of antibodies (Abs) against the receptor's extracellular domain. Two distinct Ab effects were observed. The Abs triggered signaling for cytotoxicity. This effect: (a) was correlated with the extent of p75-R expression by the cells; (b) was dependent on receptor cross-linking by the Abs; (c) occurred in HeLa cells, but not in A9 cells transfected with human p75-R or in HeLa cells expressing cytoplasmically truncated p75-R mutants, indicating that it involves cell-specific activities of the intracellular domain of the receptor; (d) was synergistic with the cytocidal effect of Abs against p55-R. Moreover, it seemed to reverse induced desensitization to the cytocidal effect of anti p55-R Abs, suggesting that it involves mechanisms different from those of the signaling by the p55 TNF-R. In addition, the Abs affected the response to TNF in a way that does not involve the signaling activity of p75-R. These effects: (a) could be observed also in cells in which only p55-R signaled for the cytocidal effect; (b) were not dependent on receptor cross-linking by the Abs; (c) varied according to the site at which the Abs bound to the receptor; and (d) were correlated inversely with the effects of the Abs on TNF binding to p75-R. That is, Abs binding to the membrane-distal part of the receptor's extracellular domain displaced TNF from the p75 receptor and enhanced cytocidal effect, whereas Abs that bind to the membrane-proximal part of the extracellular domain--a region at which a conformational change seems to take place upon TNF binding--decreased the dissociation of TNF from p75-R and inhibited its cytocidal effect. The above findings suggest that p75-R contributes to the cytocidal effect of TNF both by its own signaling and by regulating the access of TNF to p55-R.

Selective up-regulation of the 75-kDa tumor necrosis factor (TNF) receptor and its mRNA by TNF and IL-1.

In cells of the fibroblastoid line SV-80, rapid down-modulation of TNF binding in response to TNF itself, or to IL-1, was followed by a gradual recovery of binding, which occurred even in the continuous presence of the cytokines. Untreated cells carried mainly the 55-kDa receptor species. In cells treated with TNF or IL-1, the 55-kDa TNF-R, although increasing after initial down-modulation, remained lower than before treatment. Expression of the 75-kDa TNF-R species upon treatment with either cytokine was markedly increased. Both TNF and IL-1 also induced a strong increase of the mRNA for the 75-kDa receptors, whereas the amount of mRNA for the 55-kDa receptors decreased. The effects of TNF on cell surface expression of the TNF-R could not be blocked with antibodies to IL-1, nor could the effects of IL-1 be blocked with antibodies to TNF, indicating that each cytokine affects the cell surface expression of the receptors independently of the other. Applying both cytokines together resulted in much stronger increase in expression of the 75-kDa TNF-R than applying each alone. Similar changes in cell surface expression and mRNA levels of the two TNF-R as observed in SV-80 cells were also found in TNF and IL-1-treated human foreskin fibroblasts. It is suggested that these sustained changes in the pattern of receptor expression contribute to the adjustment of the cellular response to TNF when formation of TNF and IL-1 takes place over a prolonged period.

Selective decrease in cell surface expression and mRNA level of the 55-kDa tumor necrosis factor receptor during differentiation of HL-60 cells into macrophage-like but not granulocyte-like cells.

Expression of the two known receptors for TNF was studied in the promyelocytic leukemia cell line HL-60 before and after differentiation of the cells along the granulocyte lineage (induced by incubation with retinoic acid), or along the macrophage lineage (induced by incubation with the phorbol diester, PMA). The extent of inhibition of TNF binding by receptor-specific antisera, as well as the size of the complexes formed after cross-linking TNF to its receptors on intact cells, indicated that both receptor species were expressed on the surface of the undifferentiated HL60 cells. Differentiation into granulocyte-like cells resulted in some increase in TNF binding. The increase was apparently due to enhanced expression of the 75-kDa TNF-R, whereas the amounts of the 55-kDa TNF-R did not change significantly. In contrast, in HL-60 cells induced to differentiate into macrophage-like cells, expression of the 55-kDa TNF-R species was completely abolished. The pattern of TNF-R expression in the differentiated HL-60 cells was similar to that observed in leukocytes isolated from peripheral blood: on granulocytes, there were about equal amounts of both receptor species, whereas on monocytes the 75-kDa receptor was predominant. The loss of 55-kDa receptors during differentiation of HL-60 cells into macrophage-like cells was accompanied by a pronounced decrease in the level of the mRNA for that receptor, suggesting that at least part of the change in TNF-R expression is due to mechanisms that control the amounts of receptor mRNA. Although little is yet known regarding the functional differences between the two receptor species, marked changes in the pattern of their expression, as observed during HL-60 cell differentiation, are likely to alter the kind of response of the cells to TNF and may therefore play an important role in the coordination of TNF effects in the organism.