Bevacizumab as a treatment option for radiation necrosis after cranial radiation therapy: a retrospective monocentric analysis.

BACKGROUND AND PURPOSE: Radiation necrosis is a possible adverse event after cranial radiation therapy and can cause severe symptoms, such as an increased intracranial pressure or neurological deterioration. The vascular endothelial growth factor (VEGF) inhibitor bevacizumab (BEV) has been shown to be a feasible therapeutic option for symptomatic radiation necrosis, either when traditional antiedematous steroid treatment fails, or as an alternative to steroid treatment. However, to the best of our knowledge, only one randomized study with a rather small cohort exists to prove a beneficial effect in this setting. Therefore, further real-life data are needed. This retrospective monocentric case study evaluates patients who received BEV due to radiation necrosis, with a specific focus on the respective clinical course. METHODS: Using the internal database for pharmaceutical products, all patients who received BEV in our department were identified. Only patients who received BEV as symptomatic treatment for radiation necrosis were included. Patient characteristics, symptoms before, during, and after treatment, and the use of dexamethasone were evaluated using medical reports and systematic internal documentation. The symptoms were graded using CTCAE version 5.0 for general neurological symptoms. Symptoms were graded directly before each cycle and after the treatment (approximately 6 weeks). Additionally, the daily steroid dose was collected at these timepoints. Patients who either improved in symptoms, received less dexamethasone after treatment, or both were considered to have a benefit from the treatment. RESULTS: Twenty-one patients who received BEV due to radiation necrosis were identified. For 10 patients (47.6%) symptoms improved and 11 patients (52.4%) remained clinically stable during the treatment. In 14 patients (66.7%) the dexamethasone dose could be reduced during therapy, 5 patients (23.8%) received the same dose of dexamethasone before and after the treatment, and 2 patients (9.5%) received a higher dose at the end of the treatment. According to this analysis, overall, 19 patients (90.5%) benefited from the treatment with BEV. No severe adverse effects were reported. CONCLUSION: BEV might be an effective and safe therapeutic option for patients with radiation necrosis as a complication after cranial radiation therapy. Patients seem to benefit from this treatment by improving symptomatically or through reduction of dexamethasone.

Differential localization of A-Raf regulates MST2-mediated apoptosis during epithelial differentiation.

A-Raf belongs to the family of oncogenic Raf kinases that are involved in mitogenic signaling by activating the MEK-ERK pathway. Low kinase activity of A-Raf toward MEK suggested that A-Raf might have alternative functions. We recently identified A-Raf as a potent inhibitor of the proapoptotic mammalian sterile 20-like kinase (MST2) tumor suppressor pathway in several cancer entities including head and neck, colon, and breast. Independent of kinase activity, A-Raf binds to MST2 thereby efficiently inhibiting apoptosis. Here, we show that the interaction of A-Raf with the MST2 pathway is regulated by subcellular compartmentalization. Although in proliferating normal cells and tumor cells A-Raf localizes to the mitochondria, differentiated non-carcinogenic cells of head and neck epithelia, which express A-Raf at the plasma membrane. The constitutive or induced re-localization of A-Raf to the plasma membrane compromises its ability to efficiently sequester and inactivate MST2, thus rendering cells susceptible to apoptosis. Physiologically, A-Raf re-localizes to the plasma membrane upon epithelial differentiation in vivo. This re-distribution is regulated by the scaffold protein kinase suppressor of Ras 2 (KSR2). Downregulation of KSR2 during mammary epithelial cell differentiation or siRNA-mediated knockdown re-localizes A-Raf to the plasma membrane causing the release of MST2. By using the MCF7 cell differentiation system, we could demonstrate that overexpression of A-Raf in MCF7 cells, which induces differentiation. Our findings offer a new paradigm to understand how differential localization of Raf complexes affects diverse signaling functions in normal cells and carcinomas.

Antiphospholipid antibody-mediated effects in an arterial model of thrombosis are dependent on Toll-like receptor 4.

Patients with antiphospholipid syndrome (APS) produce antiphospholipid antibodies (aPL) and develop vascular thrombosis that may occur in large or small vessels in the arterial or venous beds. On the other hand, many individuals produce aPL and yet never develop thrombotic events. Toll-like receptor 4 (TLR4) appears to be necessary for aPL-mediated prothrombotic effects in venous and microvascular models of thrombosis, but its role in arterial thrombosis has not been studied. Here, we propose that aPL alone are insufficient to cause thrombotic events in an arterial model of APS, and that a concomitant trigger of innate immunity (e.g. TLR4 activation) is required. We show specifically that anti-ß2-glycoprotein I (anti-ß2GPI) antibodies, a subset of aPL, accelerated thrombus formation in C57BL/6 wild-type, but not TLR4-deficient, mice in a ferric chloride-induced carotid artery injury model. These aPL bound to arterial and venous endothelial cells, particularly in the presence of ß2GPI, and to human TLR4 by enzyme-linked immunoassay. Arterial endothelium from aPL-treated mice had enhanced leukocyte adhesion, compared to control IgG-treated mice. In addition, aPL treatment of mice enhanced expression of tissue factor (TF) in leukocytes induced by the TLR4 ligand lipopolysaccharide (LPS). aPL also enhanced LPS-induced TF expression in human leukocytes in vitro. Our findings support a mechanism in which aPL enhance TF expression by leukocytes, as well as augment adhesion of leukocytes to the arterial endothelium. The activation of TLR4 in aPL-positive individuals may be required to trigger thrombotic events.

The dual role of innate immunity in antiphospholipid syndrome and systemic lupus erythematosus.

Antiphospholipid syndrome (APS), as a primary disease or a secondary syndrome in systemic lupus erythematosus (SLE), is characterized by the presence of antiphospholipid antibodies (aPL) and a clinical event. It is likely that both genetic and environmental factors lead to the development of aPL and progression to disease. However, the precise mechanisms are not known. We hypothesize that innate immune activation plays a dual role in APS and SLE, both in the production of aPL (i.e. "initiation" phase) and in the development of a clinical event (i.e. "effector" phase). We have shown that mice immunized with certain phospholipid-binding proteins (e.g. ß2-glycoprotein I (ß2GPI)), plus a concomitant trigger of innate immunity (e.g. a toll-like receptor 4 (TLR4) ligand), produce a strong ß2GPI-reactive T cell response, resulting in high levels of aPL as well as other SLE autoantibodies. We propose that ß2GPI, through its interaction with apoptotic cells, permits B cell epitope spread to multiple SLE autoantibodies. Innate immune activation is also implicated in a murine model of aPL-enhanced thrombus formation. This dual role of innate immune activation provides new insight into the mechanisms involved in the initiation of aPL and other SLE-related autoantibodies, as well as the development of aPL-mediated disease.

[Systemic therapy and hyperthermia for locally advanced soft tissue sarcoma]. / Systemische Therapie und Hyperthermie beim lokal fortgeschrittenen Weichteilsarkom.

Patients with high-risk soft tissue sarcomas (FNCLCC grades 2-3, > 5 cm and deep lying) are at a high risk of local recurrence or distant metastases despite optimal surgical tumor resection. Therefore, multimodal treatment should be considered for this difficult to treat patient group. Besides surgery, radiation therapy and chemotherapy, hyperthermia has become a valid, complementary treatment option within multimodal treatment concepts. Hyperthermia in this context means the selective heating of the tumor region to temperatures of 40-43 °C for 60 min by microwave radiation in addition to simultaneous chemotherapy or radiation therapy. A randomized phase III study demonstrated that the addition of hyperthermia to neoadjuvant chemotherapy improved tumor response and was associated with a minimal risk of early disease progression as compared to chemotherapy alone. The addition of hyperthermia to a multimodal treatment regimen for high-risk soft tissue sarcoma consisting of surgery, radiation therapy and chemotherapy, either in the neoadjuvant or adjuvant setting after incomplete or marginal tumor resection, significantly improved local progression-free and disease-free survival. Based on these results and due to the generally good tolerability of hyperthermia, this treatment method in combination with chemotherapy should be considered as a standard treatment option within multimodal treatment approaches for locally advanced high-risk soft tissue sarcoma.

Phospholipid-binding proteins differ in their capacity to induce autoantibodies and murine systemic lupus erythematosus.

We have previously shown that immunization of nonautoimmune mice with the phospholipid-binding protein ß2-glycoprotein I (ß2GPI), in combination with lipopolysaccharide (LPS), induces a murine model of systemic lupus erythematosus (SLE), with sequential emergence of autoantibodies and glomerulonephritis. Here, we determine whether the paradigm for induction of murine SLE extends to other phospholipid-binding proteins. Mice were immunized with a phospholipid-binding protein (prothrombin (PT), protein S, or ß2GPI), or a nonphospholipid-binding protein (glu-plasminogen), in the presence of LPS. The breadth and degree of the autoantibody response, and the frequency of glomerulonephritis, varied among the three proteins, with ß2GPI being the most effective in inducing SLE-like disease. The phospholipid-binding proteins also differed in the pattern of serum cytokines they elicited. The most apparent difference between ß2GPI and the other phospholipid-binding proteins was in their ability to bind to LPS: ß2GPI bound to LPS, while PT and protein S did not. Our data suggest that binding to phospholipid(s) is a necessary, but not sufficient, condition for full induction of murine SLE. We propose that other properties, such as physiologic function, avidity for anionic phospholipids, and degree of interaction with other cell surface and/or circulating molecules (particularly LPS) may determine the range and severity of disease.

Evidence for a magnocellular disadvantage in early-onset schizophrenic patients: a source analysis of the N80 visual-evoked component.

BACKGROUND: Visual impairments in schizophrenia have been suggested to be partly caused by early processing deficits of the magnocellular (M) pathway. This might include disturbed interactions between the M and parvocellular (P) pathways and especially impaired M priming, which can disturb highlighting of relevant information. Such disorders may result from neurodevelopmental irregularities, which are assumed to be substantially involved in schizophrenia. This study sought to test the hypothesis that M priming is impaired in schizophrenia. In order to elucidate this neurodevelopmental aspect, we investigated patients with different ages of schizophrenia onset. This provided a useful design to integrate visual information processing in a neurodevelopmental model of schizophrenia. METHOD: Nine stimulus conditions were used to investigate the M- and P-pathways and their interaction in a pattern reversal VEP paradigm. N80 generators were analyzed using source localization (Brain Electrical Source Analysis software: BESA). Forty schizophrenia patients (early-onset=19; adult-onset=21) were compared with age- and gender-matched healthy controls (early-onset controls=19; adult-onset controls=21). Hypotheses were tested using a bootstrap resampling procedure. RESULTS: The N80 component was represented by a single dipole located in the occipital visual cortex. The bootstrap analysis yielded significant differences between early-onset schizophrenia patients and controls. We found lower amplitudes in response to mixed M-P conditions and normal amplitudes in response to isolated P- and M-biased stimulation. Concerning the latencies, significant differences were found between adult-onset subjects and their controls, with prolonged latencies for schizophrenia patients. CONCLUSIONS: The early VEP component N80 evoked by mixed M-P conditions is assumed to be a correlate of M priming and showed reduced amplitude in early-onset schizophrenic patients but not in adult-onset patients. These findings point towards an M priming deficit in early-onset patients and are compatible with a neurodevelopmental hypothesis of schizophrenia, probably reflecting asynchronies in brain maturational abnormalities occurring at different ages of illness onset.

Bowtie nano-aperture as interface between near-fields and a single-mode fiber.

We present the development and study of a single bowtie nano-aperture (BNA) at the end of a monomode optical fiber as an interface between near-fields/nano-optical objects and the fiber mode. To optimize energy conversion between BNA and the single fiber mode, the BNA is opened at the apex of a specially designed polymer fiber tip which acts as an efficient mediator (like a horn optical antenna) between the two systems. As a first application, we propose to use our device as polarizing electric-field nanocollector for scanning near-field optical microscopy (SNOM). However, this BNA-on-fiber probe may also find applications in nanolithography, addressing and telecommunications as well as in situ biological and chemical probing and trapping.

The dual role of innate immunity in the antiphospholipid syndrome.

The antiphospholipid syndrome (APS), as both a primary syndrome and a syndrome in association with systemic lupus erythematosus (SLE), can be a devastating disease. It is unclear what factors (genetic and/or environmental) lead to the generation of antiphospholipid antibodies (aPL). It is equally unclear why only certain individuals with aPL develop clinical events. We hypothesize that innate immune activation plays a critical role at two distinct stages of APS, namely, the initiation phase, in which aPL first appear, and the effector phase, in which aPL precipitate a thrombotic event. According to the model we propose, aPL alone are insufficient to cause thrombosis and a concomitant trigger of innate immunity, e.g. a toll-like receptor (TLR) ligand, must be present for thrombosis to occur. Here, we discuss our findings that mice immunized with beta(2)-glycoprotein I (beta(2)GPI) and lipopolysaccharide (LPS), a TLR ligand, produce high levels of aPL and other SLE-associated autoantibodies, and develop lupus-like glomerulonephritis. We also discuss our data showing that autoantibodies to heat shock protein 60 (HSP60), an 'endogenous TLR ligand', promote thrombus generation in a murine model of arterial injury. Thus, both pathogen-derived TLR ligands (e.g. LPS) and endogenous TLR ligands (e.g. HSP60) may contribute to the pathogenesis of APS. This putative dual role of innate immunity provides new insight into the generation of aPL as well as the enigma of why some individuals with aPL develop APS, while others do not.

[DASH data of non-clinical versus clinical groups of persons--a comparative study of T-norms for clinical use]. / DASH-Daten nicht-klinischer versus klinischer Personengruppen--eine Vergleichsstudie mit T-Normen für die klinische Praxis.

AIM: The aim of this study was to determine a) data for the disabilities of arm, shoulder and hand (DASH) score of a non-clinical group as well as to establish b) a comparison of DASH data between different groups of people. PATIENTS AND METHODS: After collection of 716 DASH questionnaires from healthy, working individuals (non-clinical sample) and 795 questionnaires from patients with injuries and conditions of the upper extremity (clinical sample), the data were assessed according to age, gender, manually or non-manually working into DASH total, DASH activity, DASH impairment, DASH music/sport and DASH work scores. In order to establish a comparability, these data were transformed using T-norms. RESULTS: Median DASH data of the non-clinical sample showed a functional impairment, since they deviated from 0. Men, aged 50-65, who were working manually showed the highest DASH score. Age did not have a significant effect on the DASH score of the clinical sample. The median overall DASH score of the clinical sample was higher than the scores seen with the non-clinical sample. CONCLUSION: Using T-norms a reference framework has been established for DASH scores, thus making it possible to interpret and compare individual scores between patients.

Autoantibodies to heat shock protein 60 promote thrombus formation in a murine model of arterial thrombosis.

BACKGROUND AND OBJECTIVES: Anti-heat shock protein (HSP)60 autoantibodies are associated with atherosclerosis and are known to affect endothelial cells in vitro. However, their role in thrombus formation remains unclear. We hypothesized that anti-HSP60 autoantibodies could potentiate thrombosis, and evaluated the effect of anti-murine HSP60 antibodies in a ferric chloride (FeCl3)-induced murine model of carotid artery injury. METHODS: Anti-HSP60, or control, IgG was administered to BALB/c mice 48 h prior to inducing carotid artery injury, and blood flow was monitored using an ultrasound probe. RESULTS: Thrombus formation was more rapid and stable in anti-HSP60 IGG-treated mice than in controls (blood flow=1.7%+/-0.6% vs. 34%+/-12.6%, P=0.0157). Occlusion was complete in all anti-HSP60 IgG-treated mice (13/13), with no reperfusion being observed. In contrast, 64% (9/14) of control mice had complete occlusion, with reperfusion occurring in 6/9 mice. Thrombi were significantly larger in anti-HSP60 IgG-treated mice (P=0.0001), and contained four-fold more inflammatory cells (P=0.0281) than in controls. Non-injured contralateral arteries of anti-HSP60 IgG-treated mice were also affected, exhibiting abnormal endothelial cell morphology and significantly greater von Willebrand factor (VWF) and P-selectin expression than control mice (P=0.0024 and P=0.001, respectively). CONCLUSIONS: In summary, the presence of circulating anti-HSP60 autoantibodies resulted in increased P-selectin and VWF expression and altered cell morphology in endothelial cells lining uninjured carotid arteries, and promoted thrombosis and inflammatory cell recruitment in FeCl3-injured carotid arteries. These findings suggest that anti-HSP60 autoantibodies may constitute an important prothrombotic risk factor in cardiovascular disease in human vascular disease.

Pasteurization of milk proteins promotes allergic sensitization by enhancing uptake through Peyer's patches.

BACKGROUND: The underlying mechanisms responsible for allergic sensitization to food proteins remain elusive. To investigate the intrinsic properties (as well as the effect of pasteurization) of the milk proteins alpha-lactalbumin, beta-lactoglobulin and casein that promote the induction of milk allergy. METHODS: Alteration of structure and immune-reactivity of native and pasteurized proteins was assessed by gel filtration and ELISA. Uptake of these proteins was compared in vitro and in vivo. The biological effect was assessed by orally sensitizing C3H/HeJ mice with milk proteins followed by a graded oral challenge. Required dose to induce anaphylaxis, symptoms and mean body temperature was recorded. Antigen-specific antibodies and cytokine production by splenocytes were analyzed. RESULTS: Soluble beta-lactoglobulin and alpha-lactalbumin but not insoluble casein were readily transcytosed through enterocytes in vitro and in vivo. Pasteurization caused aggregation of beta-lactoglobulin and alpha-lactalbumin inhibiting uptake by intestinal epithelial cells in vitro and in vivo. Furthermore, aggregation redirected uptake to Peyer's patches, which promoted significantly higher Th2-associated antibody and cytokine production in mice than their native counterparts. Despite this only the soluble forms of beta-lactoglobulin and alpha-lactalbumin elicited anaphylaxis (following priming) when allergens were administered orally. Aggregated beta-lactoglobulin and alpha-lactalbumin as well as casein required systemic administration to induce anaphylaxis. CONCLUSIONS: These results indicate that triggering of an anaphylactic response requires two phases (1) sensitization by aggregates through Peyer's patches and (2) efficient transfer of soluble protein across the epithelial barrier. As the majority of common food allergens tend to form aggregates, this may be of clinical importance.

Correlation between thermal diffusion and solvent self-diffusion in semidilute and concentrated polymer solutions.

We have performed measurements of thermal diffusion coefficients DT and solvent self-diffusion coefficients Dss in semidilute to concentrated polymer solutions. Solutes of different glass transition temperatures and solvents of different solvent qualities have been used. The investigated systems are in detail: poly(dimethyl-siloxane) in toluene, tristyrene in toluene, polystyrene in toluene, polystyrene in tetrahydrofuran, polystyrene in benzene, and polystyrene in cyclohexane. The thermal diffusion data are compared to our data and literature data for solvent self-diffusion coefficients. In all systems the concentration dependence of DT closely parallels the one of Dss which may be viewed as a local probe for friction on a length scale of the size of one polymer segment. This identifies local friction as the dominating parameter determining the concentration dependence of DT. Solvent quality, in contrast, has no influence on DT.

Thermal diffusion of dilute polymer solutions: the role of solvent viscosity.

We have performed measurements of the thermal diffusion coefficient D(T) in the dilute limit on polystyrene in cyclo-octane, cyclohexane, benzene, toluene, tetrahydrofuran, ethyl acetate, and methyl ethyl ketone and of poly(dimethyl-siloxane) in toluene. These data have been combined with literature data to test various theoretical predictions. The viscosity is identified as the dominating and only relevant solvent parameter. On the polymer side, the size or mass of an effective correlated segment determines the strength of the Soret effect. Large and heavy effective segments, as found in stiffer chains, lead to higher D(T).

Evidence for complete surface wave band gap in a piezoelectric phononic crystal.

A complete surface acoustic wave band gap is found experimentally in a two-dimensional square-lattice piezoelectric phononic crystal etched in lithium niobate. Propagation in the phononic crystal is studied by direct generation and detection of surface waves using interdigital transducers. The complete band gap extends from 203 to 226 MHZ, in good agreement with theoretical predictions. Near the upper edge of the complete band gap, it is observed that radiation to the bulk of the substrate dominates. This observation is explained by introducing the concept of the sound line.

Molecular characterization of the tumor-associated antigen AAA-TOB3.

In search for new valuable tumor-associated antigens using the AMIDA technique, we identified the KIAA 1273-AAA-TOB3 protein. KIAA 1273 and AAA-TOB3 were considered synonyms for the atad3B gene product. We show that the atad3b gene encodes two distinct proteins, both overexpressed in head and neck carcinomas and required for correct cell division. Both products differ within the N terminus, are generated upon distinct transcription initiation sites, and have been termed AAA-TOB3s and AAA-TOB3l. Both isoforms are early targets of c-myc and are located in mitochondria. A previous report suggested pro-apoptotic properties of the murine homolog of AAA-TOB3l. Here, we did not observe any pro-apoptotic effects in human cell lines, overexpressing h-AAA-TOB3s or h-AAA-TOB3l. By contrast, the specific knock-down of both mRNAs resulted in polynuclear cells and decreased proliferation, along with dysfunctional cell division followed by increased apoptosis. Thus, the present data suggest a role for AAA-TOB3s/l in tumor progression.

The persistence of anticardiolipin antibodies is associated with an increased risk of the presence of lupus anticoagulant and anti-beta2-glycoprotein I antibodies.

OBJECTIVE: We studied antiphospholipid antibodies (aPL) in blood samples from a cohort of individuals followed for thrombosis to determine whether the persistent presence of anticardiolipin antibodies (aCL) is associated with a greater likelihood of having lupus anticoagulant and/or anti-beta2-glycoprotein I antibodies (LA/abeta2GPI). METHODS: Blood samples from 353 individuals who had been tested for aCL on at least two occasions were tested for abeta2GPI and LA. Two groups were defined: aCL-persistent, who tested aCL-positive on at least two occasions, and aCL non-persistent, who tested aCL-positive on fewer than two occasions. Multivariate logistic regressions were performed using LA/abeta2GPI, LA and abeta2GPI as outcome variables and the percentage of aCL-positive tests as the predictor variable, adjusted for age, gender, family history of cardiovascular disease (CVD), systemic lupus erythematosus (SLE), smoking and number of venous (VT) and arterial thromboses (AT). RESULTS: Sixty-eight (19%) individuals were aCL persistent and 285 (81%) were aCL non-persistent. LA/abeta2GPI was found in 36 (53%) of the aCL persistent group and 38 (13%) of the aCL non-persistent group. The two groups were similar for age, gender and smoking. Family history of CVD, SLE, VT and AT were more frequent in the aCL persistent group. Multivariate analyses revealed that odds ratios for LA/abeta2GPI, LA and abeta2GPI were 1.34 [95% confidence interval (CI) = 1.22-1.47], 1.36 (95% CI = 1.24-1.50) and 1.47 (95% CI = 1.31-1.65) respectively for each 10% increase in aCL-positive tests vs 0% positive tests. CONCLUSION: Persistence of aCL positivity is associated with an increased risk of LA/abeta2GPI.

Antiphospholipid antibodies are associated with enhanced oxidative stress, decreased plasma nitric oxide and paraoxonase activity in an experimental mouse model.

OBJECTIVE: Oxidative stress contributes to atherosclerosis, and evidence of enhanced oxidative stress exists in antiphospholipid syndrome (APS). In a non-lupus murine model, we evaluated whether anticardiolipin (aCL) antibodies could affect the oxidant/antioxidant balance as an early biochemical step of APS. METHODS: Hybridomas producing human and murine aCL and anti-beta(2)-glycoprotein I (abeta2-GPI) monoclonal antibodies were injected into three groups of five female BALB/c severe combined immunodeficiency (SCID) mice. Corresponding hybridomas secreting non-antiphospholipid antibodies of the same isotype were employed as controls. Sera and organs were collected after 30 days. Paraoxonase (PON) activity, peroxynitrite, superoxide, nitric oxide (NO) and nitrotyrosine were measured in plasma. Expression of endothelial nitric oxide synthase and inducible nitric oxide synthase (iNOS) was assessed by western blot and immunohistochemistry. RESULTS: PON activity and NO (sum of nitrate and nitrite) levels were reduced in the human aCL IgG group (P<0.002 and P<0.04, respectively), whilst peroxynitrite and superoxide and expression of total antioxidant capacity of plasma were increased (P<0.01). PON and NO were decreased in the murine abeta2-GPI IgG and IgM aCL groups (P<0.03 and P<0.05, respectively). Nitrotyrosine was elevated in the human aCL IgG group (P<0.03). Western blotting showed reduced iNOS expression in the hearts of the IgG aCL group, confirmed by immunostaining. PON inversely correlated with IgG aCL titres (P<0.001), superoxide (P<0.008) and peroxynitrite levels (P<0.0009). Peroxynitrite and total IgG aCL were independent predictors of PON (P<0.0009 and P<0.02, respectively). Superoxide was the only independent predictor of NO (P<0.008) and of nitrotyrosine (P<0.002). CONCLUSION: aCL antibodies are associated with the decreased PON activity and reduced NO that may occur in the preclinical phase of APS.

Profile identification of disease-associated humoral antigens using AMIDA, a novel proteomics-based technology.

We describe AMIDA (autoantibody-mediated identification of antigens), a novel target identification technology based on the immunoprecipitation of disease-specific antigens by autologous serum antibodies followed by two-dimensional electrophoretic separation, and their identification via mass spectrometry. Twenty-seven potential carcinoma antigens were identified including proteins of hitherto unknown function. Validation of one of the identified antigens, cytokeratin 8, revealed its de novo expression in hyperplastic tissue, gradual overexpression with increasing malignancy, and ectopic localization on the cell surface. Furthermore, a strong prevalence of CK8-specific antibodies occurred in the serum of cancer patients already at early disease stages. In situ hybridization for one marker of unknown function, KIAA1273/TOB3, demonstrated its strong overexpression in head and neck carcinomas, thus making it a likely tumor antigen candidate. Eventually, AMIDA could foster significant improvements for the diagnosis and therapy of human diseases eliciting a humoral immune response, and allows for the rapid identification of new target molecules.

Automated microaxial tomography of cell nuclei after specific labelling by fluorescence in situ hybridisation.

Microaxial tomography provides a good means for microscopic image acquisition of cells or sub-cellular components like cell nuclei with an improved resolution, because shortcomings of spatial resolution anisotropy in optical microscopy can be overcome. Thus, spatial information of the object can be obtained without the necessity of confocal imaging. Since the very early developments of microaxial tomography, a considerable drawback of this method was a complicated image acquisition and processing procedure that requires much operator time. In order to solve this problem the Heidelberg 2pi-tilting device has been mounted on the Brno high-resolution cytometer as an attempt to bring together advanced microscopy and fast automated computer image acquisition and analysis. A special software module that drives all hardware components required for automated microaxial tomography and performs image acquisition and processing has been developed. First, a general image acquisition strategy is presented. Then the procedure for automation of axial tomography and the developed software module are described. The rotation precision has been experimentally proved followed by experiments with a specific biological example. For this application, also a method for the preparation of cell nuclei attached to glass fibres has been developed that allows for the first time imaging of three-dimensionally conserved, fluorescence in situ hybridisation-stained cell nuclei fixed to a glass fibre.