The repair mechanism resulting in alveolar protein degradation is largely unknown. In the alveolar space the presence of extracellular, biologically active proteasomes are recently reported [1,2]. The proteasome is involved in protein degradation and is able to degrade proteins at a high rate, possibly to minimize oncotic pressure [1]. We tested proteasome release by alveolar epithelial cells type II (AEC-II). AEC-II were isolated from 24 macroscopically tumour-free lung tissues as described previously [3]. Cells were stimulated with dibutyryl cAMP; for second part of the study after levelling with 1 µM indomethacin and 10 nM PGE2. Unstimulated AEC-II and primary isolated PBMC served as control. Extracellular proteasome concentration was measured by an established ELISA targeting the proteasome in supernatant. For statistical evaluation the Wilcoxon signed rank test was applied. Our study demonstrates for the first time that human non-tumourous AEC-II do release proteasomes. Stimulated with db-cAMP, the extracellular proteasome concentration (median unstim. 700 ng/mL) results in a wide insignificant range (median stim. 600 ng/ml). Indeed after levelling with indomethacin and PGE2 (median unstim. 400 ng/ml) the stimulation with db-cAMP. increases proteasome release highly significantly (stim. 460 ng/ml). Unchanged proteasome concentration in the alveolar fluid showed no artificial elution of the proteasomes by the pre-smoking lavage. In conclusion, AEC-II release proteasomes cAMP-dependent into the alveolar space. [1] Sixt, S.U. et al. Am J Physiol Lung Cell Mol Physiol 2007; 292:L1280-8 [2] Sixt, S.U. et al. Am J Respir Crit Care Med 2009; 179:1098-106. [3] Hoehne, K. et al. PLoS One 2012; 7:e38369.

Einleitung: Die Alveolarproteinose (AP) ist ein Füllungssyndrom mit Akkumulation von Phospholipiden und Lipoproteinen in den Alveolen. Das Proteasom ist ein Proteinkomplex, der als zentraler Regulator den intrazellulären Proteinabbau bewirkt und Oligopeptide für die Antigenpräsentation generiert. Das Proteasomsystem wurde bereits im Alveolarraum von Patienten mit ARDS, ALI und Sarkoidosis nachgewiesen. Darüber hinaus kann das Proteasom extrazellulär Albumin abbauen, um den intraalveolären onkotischen Druck zu reduzieren. Ziel dieser Studie war es, das Proteasom in der BAL bei AP nachzuweisen und seine eventuelle Rolle als Biomarker zu prüfen.

Alterations of the intracellular ubiquitin-proteasome pathway are found in neurodegenerative and inflammatory disorders of the central nervous system, as well as in its malignancies. Inhibitory substrates of the proteasomes represent promising approaches to control autoimmune inflammations and induction of apoptosis in cancer cells. Extracellular circulating proteasomes are positively correlated to outcome prognosis in hematogenic neoplasias and the outcome in critically ill patients. Previously, we reported raised levels of proteolytic active 20S proteasomes in the extracellular alveolar space in patients with acute respiratory distress syndrome (ARDS). For the cerebrospinal fluid, we assumed that extracellular circulating proteasomes with enzymatic activity can be found, too. Cerebrospinal fluid (CSF) samples of twenty-six patients (14 females, 12 males), who underwent diagnostic spinal myelography, were analyzed for leukocyte cell count, total protein content, lactate and interleukine-6 (Il-6) concentrations. CSF samples were analyzed for concentration and enzymatic activity of extracellular 20S proteasomes (fluorescenic substrate cleavage; femtokatal). Blood samples were analyzed with respect to concentration of extracellular circulating proteasomes. Choroidal plexus was harvested at autopsies and examined with immunoelectron microscopy (EM) for identification of possible transportation mechanisms. Statistical analysis was performed using SPSS (18.0.3). In all patients, extracellular proteasome was found in the CSF. The mean concentration was 24.6 ng/ml. Enzymatic activity of the 20S subunits of proteasomes was positively identified by the fluorescenic subtrate cleavage at a mean of 8.5 fkat/ml. Concentrations of extracellular proteasomes in the CSF, total protein content and Il-6 were uncorrelated. Immunoelectron microscopy revealed merging vesicles of proteasomes with the outer cell membrane suggestive of an exozytic transport mechanism. For the first time, extracellular circulating 20S proteasome in the CSF of healthy individuals is identified and its enzymatic activity detected. A possible exozytic vesicle-bond transportation mechanism is suggested by immunoelectron microscopy. The present study raises more questions on the function of extracellular proteasome in the CSF and encourages further studies on the role of extracellular protesomes in pathological conditions of the central nervous system (tumor lesions and inflammatory processes).

Background: Proteasomes are important proteolytic complexes for the processing of endogenous antigen presentation by major histocompatibility complex (MHC) class I molecules. Low molecule weight protein (LMP) 2 and LMP7 are IFN-γ-inducible and MHC-II-encoded β subunits which can incorporate into the constitutive 20S proteasome and turn it to immunoproteasomes with alterations of catalytic properties.

Background: Recent data suggest a role of the ubiquitin proteasome system in various malignancies. In patients with neoplasms, increased extracellular concentrations of circulating 20S proteasome (c-proteasome) have been detected in blood plasma. We tested the hypothesis that the plasma c-proteasome concentration is a biomarker associated with tumor stage and nodal status in patients with the primary diagnosis of non-metastatic breast cancer. Patients and Methods: Venous plasma concentration of 20S proteasome was measured by ELISA technique in 224 non-metastatic breast cancer patients and in 50 healthy volunteers. To assess the relation of proteasome expression to c-proteasome concentration, tumor specimens from 32 patients were immunohistochemically stained for 20S proteasome using an antibody directed against the core subunits of the catalytic domain of the 20S proteasome. Results: The median c-proteasome concentration was higher (p<0.0001) in breast cancer patients (397.5 ng/ml, range: 200-50,000 ng/ml) than in healthy controls (305 ng/ml, range: 140-425 ng/ml). There was no significant correlation between c-proteasome concentration and strength of proteasomal staining in tumor specimens. Neither tumor size, nor nodal status, nor any other prognostically important clinical parameter, including the presence of disseminated tumor cells in the bone marrow, correlated with high c-proteasome concentrations. Conclusion: Circulating proteasome concentrations appear to be higher in patients presenting with primary breast cancer than in healthy controls. Thus, the ubiquitin-proteasome system might represent a potential target in breast cancer treatment.

Existence of repair mechanisms resulting in bronchial and alveolar protein degradation induced by smoke exposure are largely unknown. In the alveolar space the presence of an extracellular, biologically active 20S proteasome is recently reported [1,2]. The proteasome is involved in protein degradation and is able to degrade proteins at a high rate, possibly to minimize oncotic pressure [1]. We tested whether the 20S proteasome in the bronchoalveolar space of healthy subjects is influenced by inhalation of tobacco smoke. Twelve healthy adult subjects underwent bronchoalveolar lavage (BAL) before and after cigarette smoking. To separate bronchiolar and alveolar proteasome concentration, first and second portion of lavage fluid were evaluated separately. Extracellular proteasome concentration was measured by an established in-house ELISA [2] targeting the 20S proteasome subunit a6 (HC2) in cell-free BAL fluid. For statistical evaluation the Wilcoxon signed rank test was applied. Acute smoke induces a significant decrease in extracellular 20S proteasome concentration in the bronchiolar space (94±55 to 65±50 ng/mL; p<0.05), but no change in the alveolar space (87±48 to 93±55 ng/mL). Unchanged proteasome concentration in the alveolar fluid showed no artificial elution of the proteasomes by the pre-smoking lavage. Tobacco smoke inhalation reduces the concentration of extracellular proteasome in the bronchial space and suggests less bronchiolar protein degradation in the acute phase after smoke exposure. References: * 1. Sixt, S.U. et al. Am J Physiol Lung Cell Mol Physiol 2007; 292:L1280-8. * 2. Sixt, S.U. et al. Am J Respir Crit Care Med 2009; 179:1098-106.

BACKGROUNDRecent data suggest a role of the ubiquitin-proteasome system in various malignancies. In patients with neoplasms, increased extracellular concentrations of circulating 20S proteasome (c-proteasome) have been detected in blood plasma. We tested the hypothesis that the plasma c-proteasome concentration is a biomarker associated with tumor stage and nodal status in patients with the primary diagnosis of non-metastatic breast cancer.PATIENTS AND METHODSVenous plasma concentration of 20S proteasome was measured by ELISA technique in 224 non-metastatic breast cancer patients and in 50 healthy volunteers. To assess the relation of proteasome expression to c-proteasome concentration, tumor specimens from 32 patients were immunohistochemically stained for 20S proteasome using an antibody directed against the core subunits of the catalytic domain of the 20S proteasome.RESULTSThe median c-proteasome concentration was higher (p<0.0001) in breast cancer patients (397.5 ng/ml, range: 200-50,000 ng/ml) than in healthy controls (305 ng/ml, range: 140-425 ng/ml). There was no significant correlation between c-proteasome concentration and strength of proteasomal staining in tumor specimens. Neither tumor size, nor nodal status, nor any other prognostically important clinical parameter, including the presence of disseminated tumor cells in the bone marrow, correlated with high c-proteasome concentrations.CONCLUSIONCirculating proteasome concentrations appear to be higher in patients presenting with primary breast cancer than in healthy controls. Thus, the ubiquitin-proteasome system might represent a potential target in breast cancer treatment.

A higher daily intake of fruits and vegetables in healthy elderly is associated with an improved antioxidant status in comparison to subjects consuming diets poor in fruits and vegetables, but the impact on cognitive performance is unclear. Healthy community dwellers (45 to 102 years old, n=193) underwent cognitive testing and blood withdrawal for the measurement of antioxidant micronutrients and biomarkers of oxidative stress as well as administration of a food frequency questionnaire to assess the daily intake of fruits and vegetables (high intake HI, low intake LI). Ninety-four subjects of the HI group had significantly higher cognitive test scores, higher levels of carotenoids, alpha- and gamma-tocopherol as well as lower levels of F2 alpha isoprostanes than the 99 subjects of the LI group. Cognitive scores were directly correlated with blood levels of alpha-tocopherol and lycopene and negatively correlated with F2 alpha isoprostanes and protein carbonyls. The results were independent of age, gender, body mass index, education, total cholesterol, LDL- and HDL-cholesterol, triglycerides, and albumin. Healthy subjects of any age with a high daily intake of fruits and vegetables have higher antioxidant levels, lower levels of biomarkers of oxidative stress, and better cognitive performance than healthy subjects of any age consuming low amounts of fruits and vegetables. Modification of nutritional habits aimed at increasing intake of fruits and vegetables should be encouraged to lower prevalence of cognitive impairment in later life.

Previous studies indicate that regular consumption of a diet rich in fruits and vegetables is associated with a lower risk for age-related diseases. The aim of the present study was to evaluate whether the often-reported age-related decrease of plasma antioxidants in man depends on differences in dietary intake or on other age- and gender-related factors. In this observational case-control study, thirty-nine community-dwelling healthy subjects aged 65 years and older consuming high intakes of fruits and vegetables daily (HI) and forty-eight healthy subjects aged 65 and older consuming low intakes of fruit and vegetables daily (LI) were enrolled. Plasma levels of retinol, tocopherols, carotenoids and malondialdehyde (MDA) as well as content of protein carbonyls in Ig G were measured. Plasma levels of retinol, tocopherols and carotenoids were significantly higher in group HI than in group LI subjects independent of age and gender. MDA levels were inversely correlated with vitamin A and alpha-carotene. Protein carbonyls were inversely correlated with gamma-tocopherol. In the elderly, a higher daily intake of fruits and vegetables is associated with an improved antioxidant status in comparison to subjects consuming diets poor in fruits and vegetables. Modification of nutritional habits among other lifestyle changes should be encouraged to lower prevalence of disease risk factors in later life.