Trigeminal endonasal perception - an outcome predictor for septoplasty.

BACKGROUND: No adequate test exists to predict outcome after septoplasty. Despite adequate surgery, patients still might experience nasal breathing impairment. The aim of this study was to determine if pre-operative trigeminal sensitivity can predict satisfaction after septoplasty. METHODS: Single centre prospective cohort study in tertiary referral centre with follow-up time of 6 weeks postoperatively. Patients scheduled for septoplasty or septorhinoplasty with turbinoplasty were consecutively selected the day before surgery. Standard preoperative examinations (acoustic rhinometry and Sniffin’ Sticks 12 test), the evaluation of nasal obstruction on a visual analogue scale (VAS) and the trigeminal lateralisation task were performed before and 6 weeks after surgery. Biopsies were taken during surgery and TRPV1 mRNA expression was measured by PCR. RESULTS: Thirty patients were included with a median age of 29 years and equal gender distribution. Trigeminal perception and sensation of nasal obstruction showed a significant correlation: preoperative lateralisation test scores, representing endonasal trigeminal sensitivity, correlated significantly with the mean VAS change scores, which demonstrate subjective improvement. A lateralisation test score of 31.5 and more had a sensitivity of 88% to predict an improvement of more than 3 VAS points. Additionally, high TRPV1 mRNA expression was linked with good postoperative VAS scores. CONCLUSION: The preoperative evaluation of the trigeminal sensitivity could improve patients’ selection for septoplasty with a higher rate of satisfaction. Endonasal trigeminal sensitivity is directly linked with subjective outcome. Therefore, patients with low trigeminal sensitivity should undergo septoplasty only after thorough counselling.

The cell adhesion molecule EphA4 is involved in circadian clock functions.

Circadian (∼24 h) rhythms of cellular network plasticity in the central circadian clock, the suprachiasmatic nucleus (SCN), have been described. The neuronal network in the SCN regulates photic resetting of the circadian clock as well as stability of the circadian system during both entrained and constant conditions. EphA4, a cell adhesion molecule regulating synaptic plasticity by controlling connections of neurons and astrocytes, is expressed in the SCN. To address whether EphA4 plays a role in circadian photoreception and influences the neuronal network of the SCN, we have analyzed circadian wheel-running behavior of EphA4 knockout (EphA4-/- ) mice under different light conditions and upon photic resetting, as well as their light-induced protein response in the SCN. EphA4-/- mice exhibited reduced wheel-running activity, longer endogenous periods under constant darkness and shorter periods under constant light conditions, suggesting an effect of EphA4 on SCN function. Moreover, EphA4-/- mice exhibited suppressed phase delays of their wheel-running activity following a light pulse during the beginning of the subjective night (CT15). Accordingly, light-induced c-FOS (FBJ murine osteosarcoma viral oncogene homolog) expression was diminished. Our results suggest a circadian role for EphA4 in the SCN neuronal network, affecting the circadian system and contributing to the circadian response to light.

BH3-only proteins in cell death initiation, malignant disease and anticancer therapy.

Induction of apoptosis in tumour cells, either by direct activation of the death receptor pathway using agonistic antibodies or recombinant ligands, or direct triggering of the Bcl-2-regulated intrinsic apoptosis pathway by small molecule drugs, carries high hopes to overcome the shortcomings of current anticancer therapies. The latter therapy concept builds on a more detailed understanding of how Bcl-2-like molecules maintain mitochondrial integrity and how BH3-only proteins and Bax/Bak-like molecules can undermine it. Means to unleash the apoptotic potential of BH3-only proteins in tumour cells, or bypass the need for BH3-only proteins by blocking possible interactions of Bcl-2-like prosurvival molecules with Bax and/or Bak allowing their direct activation, constitute interesting options for the design of novel anticancer therapies.

Cell survival and proliferation in Drosophila S2 cells following apoptotic stress in the absence of the APAF-1 homolog, ARK, or downstream caspases.

In Drosophila, the APAF-1 homolog ARK is required for the activation of the initiator caspase DRONC, which in turn cleaves the effector caspases DRICE and DCP-1. While the function of ARK is important in stress-induced apoptosis in Drosophila S2 cells, as its removal completely suppresses cell death, the decision to undergo apoptosis appears to be regulated at the level of caspase activation, which is controlled by the IAP proteins, particularly DIAP1. Here, we further dissect the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. We found that the induction of apoptosis was dependent in each case on expression of ARK and DRONC and surviving cells continued to proliferate. We noted a difference in the effects of silencing the executioner caspases DCP-1 and DRICE; knock-down of either or both of these had dramatic effects to sustain cell survival following depletion of DIAP1, but had only minor effects following cellular stress. Our results suggest that the executioner caspases are essential for death following DIAP1 knock-down, indicating that the initiator caspase DRONC may lack executioner functions. The apparent absence of mitochondrial outer membrane permeabilization (MOMP) in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted.

Differential effects of deoxycholic acid and taurodeoxycholic acid on NF-kappa B signal transduction and IL-8 gene expression in colonic epithelial cells.

Several effects of bile acids (BAs) on colonic epithelial cells (CECs) have been described, including induction of proliferation and apoptosis. Some of these effects are mediated through activation of the NF-kappa B transcriptional system. In this study, we investigated the molecular mechanisms underlying the BA-induced gene expression in CECs. The human CEC line HT-29 and primary human CECs were treated with dilutions of salts of deoxycholic acid (DCA) and taurodeoxycholic acid (TDCA). NF-kappa B binding activity was analyzed with EMSA, RelA translocation with immunofluorescence, and I kappa B alpha- and RelA-phosphorylation with Western blot analysis. IL-8 mRNA and protein expression were assessed by quantitative PCR and ELISA. Functional impact of NF-kappa B activation was determined by blocking the proteasome activity with MG132 or by preventing IKK activity with a dominant-negative IKK beta delivered by adenoviral dominant-negative (dn) IKK beta (Ad5dnIKK beta). DCA and TDCA induced IL-8 expression in a dose- and time-dependent manner. It is interesting that DCA but not TDCA induced I kappa B alpha-phosphorylation, RelA translocation, and NF-kappa B binding activity. Accordingly, the proteasome inhibitor MG132 blocked DCA- but not TDCA-induced IL-8 gene expression. In contrast, TDCA-induced IL-8 gene expression correlated with enhanced RelA phosphorylation, which was blocked by Ad5dnIKK beta. Our data suggest that DCA-induced signal transduction mainly utilized the I kappa B degradation and RelA nuclear translocation pathway, whereas TDCA primarily induced IL-8 gene expression through RelA phosphorylation. These differences may have implications for the understanding of the pathophysiology of inflammation and carcinogenesis in the gut.

Toll-like receptors 2 and 4 are up-regulated during intestinal inflammation.

BACKGROUND & AIMS: Bacterial wall products play an important role in the activation of immune and nonimmune cells of the intestinal mucosa. Toll-like receptors (TLRs) TLR2 and TLR4 have been identified as signaling receptors activated by bacterial wall components. METHODS: Expression of TLRs in human intestinal mucosa obtained by endoscopy and surgery was analyzed by immunohistochemistry. Intestinal macrophages were isolated by immunomagnetic beads armed with a CD33 antibody. Reverse-transcription polymerase chain reaction was performed for TLR1-5. Results were confirmed by Northern blot and flow cytometry. Interleukin-1beta messenger RNA (mRNA) was quantified by a polymerase chain reaction-enzyme-linked immunosorbent assay-kit. RESULTS: Immunohistochemistry revealed a significant increase in the TLR2 and TLR4 antigen expression on submucosal cells in inflamed intestinal mucosa compared with non-inflamed mucosa. TLR expression was localized in intestinal macrophages by double-labeling techniques. No TLR-polymerase chain reaction product could be obtained with mRNA from CD33-positive macrophages from normal mucosa. We observed an induction of mRNA for TLR2, TLR4, and TLR5 in inflammation-associated macrophages. TLR1 and TLR3 were only detectable in blood monocytes. Monocytes reacted to lipopolysaccharide stimulation with a 3-fold and in vitro differentiated macrophages with a 16-fold increase of cellular interleukin-1beta mRNA. Macrophages from normal mucosa did not respond to lipopolysaccharide showing the functional relevance of TLR expression. CONCLUSIONS: This study shows the inflammation-dependent induction of TLR2 and TLR4 expression in intestinal macrophages. The absence of TLRs abolishes the reactivity of mucosal macrophages to bacterial wall products. Presence of TLRs may thereby contribute to the inflammatory process.

Interstitial nephritis, hepatic failure, and systemic eosinophilia after minocycline treatment.

This report describes a 15-year-old white boy who presented with fever, back pain, a disseminated exanthematous rash, renal failure, and hepatopathy 3 weeks after the initiation of oral minocycline therapy for facial acne. Marked peripheral and urine eosinophilia were noted. A bone marrow aspiration showed more than 50% eosinophils without any evidence of malignancy, and a simultaneous kidney biopsy showed acute interstitial nephritis (AIN). The patient's symptoms and laboratory findings improved after high-dose steroid therapy was initiated, worsened when it was withheld, and improved again after it was reinitiated in view of the biopsy findings. The patient recovered completely, and steroids were tapered to discontinuation over 3 months. Over a year later, the patient's peripheral blood mononuclear cells (PBMCs) were cultured for 2 weeks in the presence or absence of minocycline ex vivo, and minocycline was found to induce the emergence of CD4(+) cells after 1 week in culture. In conclusion, this article shows for the first time several new aspects of minocycline-induced morbidity: renal and hepatic failure can occur together, and AIN and elevated blood eosinophil counts can be accompanied by marked bone marrow eosinophilia, suggesting a systemic allergic response as the underlying pathomechanism. Furthermore, the initial phase of such a response appears to involve CD4(+) T cells detectable ex vivo. Lastly, high-dose treatment with corticosteroids appears to be beneficial in this setting.

A novel locus of enterocyte effacement (LEE) pathogenicity island inserted at pheV in bovine Shiga toxin-producing Escherichia coli strain O103:H2.

We describe a locus of enterocyte effacement (LEE) which is part of a new pathogenicity island (PAI) detected in the bovine Shiga toxin-producing Escherichia coli strain RW1374 (O103:H2). This PAI is at least 80 kb in size and inserted in the vicinity of the pheV tRNA gene at 67 min of the E. coli chromosome. Furthermore, the PAI differs from the previously described LEEs by unique flanking regions at both sides, which harbor one copy each of an insertion element in an inverted orientation that is 96% identical to insertion site (IS)629. In addition, a 5-kb PAI-specific sequence downstream of the LEE core region and adjacent to the E. coli K12 region is duplicated upstream of the LEE core region as well. The duplicated sequences are more than 80% identical to each other and consist partially of prophage sequences.

GRO-alpha in normal and pathological thyroid tissues and its regulation in thyroid-derived cells.

Thyroid glands affected by Graves' disease (GD) show striking leukocytic infiltration, mainly by T-cells. The mechanisms by which the various leukocytes are maintained in the thyroid are unknown. Growth-regulated oncogene-alpha (GRO-alpha) in interaction with its receptor CXCR2 is a chemoattractant for both T-cells and neutrophils and may be one of the chemokines involved in the cell maintenance. GRO-alpha and CD18 mRNA as a marker of leukocytic infiltration were quantified in thyroid tissue using competitive RT-PCR. We found very high GRO-alpha mRNA levels in all thyroid tissues. In GD patients (n=16), the GRO-alpha mRNA did not correlate with the CD18 mRNA level or thyroid peroxidase and TSH-receptor antibodies in patients' sera. In thyroid autonomy (n=10), the GRO-alpha mRNA levels were significantly lower in autonomous single adenomas compared with the corresponding normal tissue. In order to define the cellular source of GRO-alpha mRNA and protein, we examined various thyroid-derived cells. Thyrocytes, thyroid-derived leukocytes and fibroblasts showed basal GRO-alpha mRNA and protein expression, which was remarkably upregulated by different stimuli in vitro. The expression of GRO-alpha by thyroid carcinoma cell lines confirms that thyrocytes may actually produce GRO-alpha. As shown by flow cytometry and immunohistology, CD68+ monocytes/macrophages are the only cell population strongly expressing CXCR2 in the thyroid.

Reduced expression of stromal-derived factor 1 in autonomous thyroid adenomas and its regulation in thyroid-derived cells.

Stromal-derived factor 1 (SDF-1) and CXCR4 comprise a unique chemokine/chemokine receptor pair, exhibiting important functions in morphogenesis and growth regulation as well as attractant properties on T lymphocytes. No data are available on SDF-1 and CXCR4 in normal or pathological thyroid tissues. SDF-1, CXCR4, and CD18 messenger ribonucleic acid (mRNA) as a marker of leukocytic infiltration were quantified in tissues affected by thyroid adenoma (n = 11) and Graves' disease (GD; n = 16) using competitive RT-PCR. SDF-1 mRNA levels differed significantly between autonomous adenomas and the corresponding normal tissue, but not in GD between patients with low or high leukocyte infiltration, thyroid peroxidase, and TSH receptor autoantibodies, respectively. We found a strong correlation between CXCR4 and CD18 mRNA, which indicates CXCR4 expression by leukocytes. To define the cellular source of SDF-1 and CXCR4 in thyroid tissue, we examined various thyroid-derived cells. Fibroblasts are the most potent producers of SDF-1, although thyrocytes also secrete SDF-1 in vitro. Leukocytes showed very weak SDF-1 mRNA levels and no secretion of the chemokine. Immunohistology confirmed and extended these results; SDF-1 expression was found in fibroblasts, but not or very weakly in CD45(+) leukocytes and thyrocytes. Only leukocytes were CXCR4(+). As examined by flow cytometry, the number of CD3(+) T cells expressing CXCR4 is significantly higher in the thyroid than in peripheral blood. SDF-1 seems to be involved in thyroid tissue homeostasis in thyroid adenoma, but not in the maintenance of lymphocytic infiltration in GD.

Isolation of genomic DNA sequences that bind vitamin D receptor complexes.

Vitamin D signaling is believed to be transduced by a heterodimeric receptor complex that binds to specific sequences of DNA termed vitamin D response elements (VDREs) in the promoter regions of target genes. However, recent studies have suggested that considerable flexibility exists in the types of binding sites the vitamin D receptor (VDR) is capable of recognizing, including some that bind VDR homodimers. In this report, a screening method involving immunoselection and PCR amplification was utilized to examine genomic binding sites for the receptor. Four individual fragments ranging in size from ca. 250-320 bp were nominally isolated from the amplified pool of captured fragments for further analysis. Each of the four sequences was capable of forming specific, unique VDR complexes using recombinant human VDR (rhVDR) alone or rhVDR heteromers formed in conjunction with the addition of recombinant human retinoid X receptor alpha (rhRXRalpha). Two of these fragments exhibited significant hormone-dependent repression of luciferase activity when linked to a thymidine kinase driven reporter vector. DNaseI footprinting revealed specific binding over DR+3 or related half-site sequences found within both of these DNA fragments. The results from this study demonstrate that specific, functional binding sites for the VDR can be successfully isolated from genomic DNA and should aid in the discovery of genes regulated by the steroid hormone.

Apoptotic signaling during initiation of detachment-induced apoptosis ("anoikis") of primary human intestinal epithelial cells.

Apoptosis after the loss of cell anchorage--"anoikis"--plays an important role in the life cycle of adherent cells. Furthermore, loss of anchorage dependency is believed to be a critical step in metastatic transformation. The aim of this study was to further characterize the sequence of intracellular events during anoikis in a nontransformed population of human intestinal epithelial cells (IECs). Purified human IECs were kept in suspension to induce anoikis in over 90% of IECs within 3 h. Two initiator caspases, caspase-2 and -9, are activated within 15 min, followed by the hierarchical activation of downstream caspases within 1 h. The activation of the caspase FLICE (caspase-8) does not contribute to the initiation of anoikis, and massive release of cytochrome c from mitochondria cannot be detected before 60 min, indicating that cytochrome c release does not play a role during initiation of anoikis. This study delineates the signaling cascade during anoikis of nontransformed cells. Future studies may identify alterations of this cascade in neoplastic cells, thereby possibly gaining insight into carcinogenesis and metastatic transformation.

Developing national epidemiologic capacity to meet the challenges of emerging infections in Germany.

In January 1996, the Robert Koch Institute, Germany's national public health institute, began strengthening its epidemiologic capacity to respond to emerging and other infectious diseases. Six integrated strategies were initiated: developing employee training, outbreak investigation, and epidemiologic research programs; strengthening surveillance systems; improving communications to program partners and constituents; and building international collaborations. By December 1999, five employees had completed a 2-year applied epidemiology training program, 186 health department personnel had completed a 2-week training course, 27 outbreak investigations had been completed, eight short-term research projects had been initiated, major surveillance and epidemiologic research efforts for foodborne and nosocomial infections had begun, and 16 scientific manuscripts had been published or were in press. The German experience indicates that, with a concerted effort, considerable progress in building a national applied infectious disease program can be achieved in a short time frame.

Cloning of bovine RANTES mRNA and its expression and regulation in ovaries in the periovulatory period.

RANTES may be one of the chemoattractants involved in stimulating eosinophils and macrophages to migrate selectively into bovine dominant follicles and into developing corpora lutea. We sequenced a 736 bp fragment of the bovine RANTES mRNA encoding the complete protein and defined the ovarian source of RANTES mRNA. As demonstrated by competitive RT-PCR, follicle-derived macrophages showed a 100-1000 times higher RANTES mRNA level compared to unpurified granulosa cells or follicle-derived fibroblasts. By means of in situ hybridization, RANTES mRNA positive macrophages were located in the former thecal layer of the developing corpora lutea.

["Open" versus laparoscopic appendectomy]. / "Offene" versus laparoskopische Appendektomie.

A total of 701 patients operated on with the suspected diagnosis of acute appendicitis were included in a prospective study. Three hundred and nine patients were treated conventionally and 387 laparoscopically. In 14 patients the operation had to be converted. Operating time was 44 min in the laparoscopic group and 54 min in the conventional group. The advantages of the laparoscopic technique were better visualisation of the abdominal cavity, fewer complications, shorter hospital stay and better cosmetic result. Complications occurred in 2.9% of the patients in the laparoscopic group and in 13.6% of the patients in the conventional group. All complications following laparoscopic procedures could be treated laparoscopically.

Low-dose recombinant interleukin-2 therapy in advanced multiple myeloma.

In vitro data have demonstrated autologous T-lymphocytes with anti-tumour activity in multiple myeloma (MM). Therefore a phase I/II trial was conducted to study the feasibility, the effect on several immunological parameters, and the tumour response induction of low-dose recombinant interleukin-2 (rIL-2) in MM patients. 18 MM patients of advanced stages in progress, who had failed on standard chemotherapy received 9 x 10(6) IU/m2 rIL-2 twice daily on days 1 and 2 and 0.9 x 10(6) IU/m2 twice daily for 5 subsequent days per week subcutaneously from days 3 to 56 (repeated every 12 weeks until progression). Patients were treated for between 8 and 1086 + d (mean 241 d) without serious side-effects. 6/17 patients experienced tumour response (2/17 objective tumour mass reduction, 4/17 long-lasting stable disease following tumour progression before initiation of rIL-2 treatment). During therapy the number of eosinophils increased 15-fold, CD4+ T lymphocytes were activated as demonstrated by enhanced CD25 antigen expression, and CD56+ NK cells expanded in the peripheral blood. Furthermore, a diminished pre-treatment ratio of CD4+/CD8+ lymphocytes was normalized during rIL-2 treatment. NK cell activity and lymphokine activated killer (LAK) cell activity was significantly enhanced. Endogenous IL-2 production and elevated soluble IL-2 receptor serum concentrations were induced. Low-dose rIL-2 can stimulate immune enhancement in MM despite the characteristic tumour-induced immunodeficiency. The treatment has proven though limited efficacy in advanced MM. Because most of the responders experienced termination of tumour progression rather than tumour regression, rIL-2 maintenance of chemotherapy-induced remissions should be investigated.

Immunoglobulin allotypes are not associated with HLA-antigens, autoantibodies and clinical symptoms in systemic lupus erythematosus. Members of the SLE Study Group.

Immunoglobulin heavy chain (G1m, G2m, G3m, A2m) and kappa light chain (Km) allotype and phenotype frequencies of 323 central European Caucasian patients with systemic lupus erythematosus (SLE) were examined and correlated with various genetic, serologic and clinical markers of SLE. No significant associations were found between immunoglobulin allotypes or phenotypes and all 20 parameters tested (nephritis, vasculitis, arthralgias, photosensitivity, discoid lesions, central nervous system disease, Raynaud's phenomenon, sex, anti-Ro, anti-La, anti-nRNP, HLA-DR1-DR7, HLA phenotypes B8-DR3, B7-DR2). It could therefore be assumed that Gm, A2m and Km allotypes were not associated with HLA-antigens and had no influence on the serologic and clinical expression of SLE.

[Long-term stability of enzymes in solution]. / Untersuchungen zur Langzeitstabilität von Enzymen in Lösung.

Two proteases (thermitase, a thermostable serine protease from Thermoactinomyces vulgaris and subtilisin Carlsberg) and one non-proteolytic enzyme (urate oxidase from Penicillium spp.) were used for revealing the main influences leading to the inactivation of enzyme preparations during their long-time storage at low temperatures. The temperature dependences (0 degree C-60 degrees C) of inactivation resulted in a linear Arrhenius-plot for each of the three native enzymes as well as in the presence of all stabilizing substances tested. Therefore, a method is available which shortens the time considerably needed for the experiments aimed at the discovery of substances stabilizing enzymes under storage conditions (i.e. long time at low temperatures). Because of the linear Arrhenius-plots potential stabilizers can be tested experimentally at suitable higher temperatures and one can extrapolate on their influence on the enzyme at storage temperatures (0 degree C-10 degrees C). By using this method effective combinations of stabilizers for urate oxidase were found, and possible reasons of their stabilizing influence on the enzyme are discussed.