Quantitative analysis of receptor-mediated uptake and pro-apoptotic activity of mistletoe lectin-1 by high content imaging.

Ribosome inactivating proteins (RIPs) are highly potent cytotoxins that have potential as anticancer therapeutics. Mistletoe lectin 1 (ML1) is a heterodimeric cytotoxic protein isolated from European Mistletoe and belongs to RIP class II. The aim of this project was to systematically study ML1 cell binding, endocytosis pathway(s), subcellular processing and apoptosis activation. For this purpose, state of the art cell imaging equipment and automated image analysis algorithms were used. ML1 displayed very fast binding to sugar residues on the membrane and energy-dependent uptake in CT26 cells. The co-staining with specific antibodies and uptake blocking experiments revealed involvement of both clathrin-dependent and -independent pathways in ML1 endocytosis. Co-localization studies demonstrated the toxin transport from early endocytic vesicles to Golgi network; a retrograde road to the endoplasmic reticulum. The pro-apoptotic and antiproliferative activity of ML1 were shown in time lapse movies and subsequently quantified. ML1 cytotoxicity was less affected in multidrug resistant tumor cell line 4T1 in contrast to commonly used chemotherapeutic drug (ML1 resistance index 6.9 vs 13.4 for doxorubicin; IC50: ML1 1.4 ng/ml vs doxorubicin 24000 ng/ml). This opens new opportunities for the use of ML1 as an alternative treatment in multidrug resistant cancers.

[Multidisciplinary health care needs of psychologically distressed cancer patients in a Comprehensive Cancer Center]. / Multidisziplinärer Versorgungsbedarf psychisch belasteter Tumorpatienten an einem Comprehensive Cancer Center.

BACKGROUND AND OBJECTIVE: Considering the prolonged life-expectancies and the resulting demands that are placed on cancer patients and their relatives, the importance of specific counseling and support services including psycho-oncology, social services, nutritional, and exercise counseling has profoundly increased. The main focus of the current study was to evaluate the multidisciplinary health care needs of emotionally distressed cancer patients whoe were treated in a Comprehensive Cancer Center. METHODS AND STUDYGROUP: 831 out-patients were evaluated with regard to their psychological distress level and their multidisciplinary health care needs for specialist services of psycho-oncology, social services, nutritional, and exercise counseling using a tablet-PC assisted screening questionnaire. Separate analyses were completed for patients with and without psychological distress. RESULTS: One third of the screened patients showed clinically relevant psychological distress. Health care needs for all specialist services were significantly greater among these patients compared to patients without psychological distress (all p-values < 0.005). The higher needs were foremost presented by the number of needed specialist services (p < 0.001): two thirds of the psychologically distressed patients demonstrated, besides the need for a psycho-oncological service, a need for two or three further specialist services, whereas among patients without psychological distress more than 70% showed a need for at most one specialist service. CONCLUSION: Multidisciplinary health care needs of psychologically distressed cancer patients should be systematically addressed in a Comprehensive Cancer Center, and patients should be offered a coordinated and integrated health care program.

[Smoking cessation in patients with COPD]. / Tabakentwöhnung bei COPD.

Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide. Cigarette smoking is the main cause of COPD. Quitting smoking is thus the most effective treatment strategy and central in COPD prevention. A number of guidelines on prevention, diagnosis, therapy and rehabilitation of COPD have been published. To help implementing and standardizing smoking cessation in COPD a guideline was published 2008 in Germany focusing on this complex issue. The present guideline is an update of the 2008 guideline and has a high grade of evidence (S3 according to the AWMF; Arbeitsgemeinschaft wissenschaftlicher medizinischer Fachgesellschaften). The guideline gives comprehensive and practical information on how to integrate smoking cessation as an central part of COPD therapy.

[Photodynamic therapy of bladder cancer. A new option]. / Die photodynamische Therapie des Harnblasenkarzinoms. Eine neue Option.

BACKGROUND: Bladder cancer (BCa) is the third most common tumor in Germany. Currently, resection therapy for superficial BCa (Ta, CIS) includes photodynamic diagnostics (PDD) using HEXVIX® for improved assessment of tumor spread. Trials using these photosensitizers for photodynamic therapy (PDT) showed only limited success. Especially low tissue penetration due to short-wave excitation was a limiting factor. METHODS: This study which was funded by the German Research Foundation (DFG) examined the feasibility of the novel photosensitizer tetrahydroporphyrin-tetratosylate (THPTS) for PDT in a rat bladder cancer model. RESULTS: As THPTS is very effectively excitable at a near infrared wavelength of 760 nm it is within the so-called phototherapeutic window and allows tissue penetration of up to 15 mm. Thus THPTS can also be used for PDT of larger, solid tumors as was previously demonstrated for other tumor entities. Therefore, effective treatment of even muscle-invasive bladder cancer (≥T2) may become an option using THPTS. In this current study the effectiveness and safety of THPTS-PDT was examined in an orthotopic bladder cancer rat model.

[Paraganglioma--a rare cause of mediastinal lesions]. / Das Paragangliom--seltene Ursache einer mediastinalen Raumforderung.

A 73-year-old female patient was admitted to our clinic for further clarification of a suspicious mediastinal lesion. Endosonographically, we performed a fine-needle aspiration biopsy. Clinical and radiological findings as well as cytology hardened the suspicion of a paraganglioma as being the cause of the mediastinal lesion. Finally, this diagnosis was confirmed histologically after complete surgical resection of the lesion. This case report on a mediastinal paraganglioma includes a concise summary of diagnostic procedures and therapeutic options for this rare disease.

The use of citrullinated peptides and proteins for the diagnosis of rheumatoid arthritis.

The presence or absence of antibodies to citrullinated peptides/proteins (ACPA) is an important parameter that helps a clinician set a diagnosis of early rheumatoid arthritis and, hence, initiate treatment. There are several commercial tests available to measure ACPA levels, although it can be difficult to decide what the best test for a given clinical question is. We analyzed literature data in which the diagnostic and other properties of various ACPA tests are compared. The results show that for diagnostic purposes the CCP2 test has the highest specificity, the highest sensitivity in stratified studies and the highest positive predictive value. For the prediction of future joint destruction the CCP2, MCV, and CCP3 tests may be used. The ability to predict the likelihood of not achieving sustained disease-modifying antirheumatic drug-free remission was highest for the CCP2 test. Finally, the levels of anti-CCP2 and anti-CCP3 (and possibly anti-mutated citrullinated vimentin) in rheumatoid arthritis patients are not significantly influenced by TNFalpha blocking agents.

DTI of commissural fibers in patients with Chiari II-malformation.

Chiari II-malformation is a complex congenital deformity of the brain which is frequently associated with hydrocephalus. Abnormalities of the corpus callosum are known to occur in the majority of patients. The objective of the present study was to study the microstructure of the corpus callosum (CC) and the anterior commissure (AC) to differentiate between different mechanisms of damage to these structures. We investigated 6 patients with Chiari II-malformation and 6 well-matched healthy volunteers employing T1-weighted 3D imaging and diffusion tensor imaging (DTI) to determine the fractional anisotropy (FA) and cross-sectional area of the CC and AC, as well as with neuropsychological testing. Four patients showed hydrocephalus, two patients had callosal dysplasia and four had a hypoplastic CC. The callosal FA in the patients was significantly reduced which was less pronounced for the genu alone. The area of CC was also reduced in Chiari II-patients. There was a strong correlation between the size and FA of the CC in the patients. In contrast, the thickness of the AC was significantly increased and was associated with higher FA in the patients. In psychological tests all patients showed reduced verbal memory; all but one patient showed reduced IQ as well as impaired visuo-spatial performance, indicating deficits in tasks requiring parieto-occipital integration. The existence of callosal dysplasia in two patients, the diminished FA reduction in the genu and the correlation of the cross-sectional area and FA in the patients point to a developmental white matter damage beside that exerted by hydrocephalus alone.

Cartilage-hair hypoplasia-associated mutations in the RNase MRP P3 domain affect RNA folding and ribonucleoprotein assembly.

Cartilage-hair hypoplasia (CHH) is caused by mutations in the gene encoding the RNA component of RNase MRP. Currently it is unknown how these mutations affect the function of this endoribonuclease. In this study we investigated the effect of mutations in the P3 domain on protein binding and RNA folding. Our data demonstrate that a number of P3 nucleotide substitutions reduced the efficiency of its interaction with Rpp25 and Rpp20, two protein subunits binding as a heterodimer to this domain. The CHH-associated 40G>A substitution, as well as the replacement of residue 47, almost completely abrogated Rpp25 and Rpp20 binding in different assays. Also other CHH-associated P3 mutations reduced the efficiency by which the RNase MRP RNA is bound by Rpp25-Rpp20. These data demonstrate that the most important residues for binding of the Rpp25-Rpp20 dimer reside in the apical stem-loop of the P3 domain. Structural analyses by NMR not only showed that this loop may adopt a pseudo-triloop structure, but also demonstrated that the 40G>A substitution alters the folding of this part of the P3 domain. Our data are the first to provide insight into the molecular mechanism by which CHH-associated mutations affect the function of RNase MRP.

[Sleep apnoea in women?--The forgotten gender]. / Schlafapnoe bei Frauen?--Das vergessene Geschlecht.

The prevalence of clinically relevant, obstructive sleep apnoea syndrome (OSAS) in the general population is 2% in women and 4% in men. With increasing age and onset of postmenopausal status, the prevalence of OSAS in women becomes comparable to that of males. However, compared to prevalence data, women are under-represented in clinical sleep laboratories. The present overview deals with the potential reasons for clinical under-recognition of OSAS in women. The fact that OSAS frequency is underestimated in women probably derives from the atypical clinical symptoms, dominated by difficulties of initiating and maintaining sleep and by a depressive mood. There are several protecting mechanisms in women that prevent or postpone OSAS development to higher age groups or until the onset of menopause. These factors include craniofacial morphology and function, gender-specific body-fat distribution and hormonal influences on ventilation and dilating muscles in the oropharynx. Physicians should be aware of the presence of sleep-disordered breathing in women and of their special features.

ABAP: antibody-based assay for peptidylarginine deiminase activity.

Members of the family of peptidylarginine deiminases (PADs, EC 3.5.3.15) catalyze the posttranslational modification of peptidylarginine into peptidylcitrulline. Citrulline-containing epitopes have been shown to be major and specific targets of autoantibodies produced by rheumatoid arthritis patients. Recently, the citrullination of histone proteins by PAD enzyme was reported to influence gene expression levels. These findings greatly increase the interest in the PAD enzymes and their activities. A few procedures to monitor PAD activity in biological samples have been described previously. However, these assays either have low sensitivity or are rather laborious. Here we describe a reliable and reproducible method for the determination of PAD activity in both purified and crude samples. The method is based on the quantification of PAD-dependent citrullination of peptides, immobilized in microtiter plates, using antibodies that are exclusively reactive with the reaction product(s). Our results demonstrate that this antibody-based assay for PAD activity, called ABAP, is very sensitive and can be applied to monitor PAD activity in biological samples.

Somatic CTG*CAG repeat instability in a mouse model for myotonic dystrophy type 1 is associated with changes in cell nuclearity and DNA ploidy.

BACKGROUND: Trinucleotide instability is a hallmark of degenerative neurological diseases like Huntington's disease, some forms of spinocerebellar ataxia and myotonic dystrophy type 1 (DM1). To investigate the effect of cell type and cell state on the behavior of the DM1 CTG*CAG repeat, we studied a knock-in mouse model for DM1 at different time points during ageing and followed how repeat fate in cells from liver and pancreas is associated with polyploidization and changes in nuclearity after the onset of terminal differentiation. RESULTS: After separation of liver hepatocytes and pancreatic acinar cells in pools with 2n, 4n or 8n DNA, we analyzed CTG*CAG repeat length variation by resolving PCR products on an automated PAGE system. We observed that somatic CTG*CAG repeat expansion in our DM1 mouse model occurred almost uniquely in the fraction of cells with high cell nuclearity and DNA ploidy and aggravated with aging. CONCLUSION: Our findings suggest that post-replicative and terminal-differentiation events, coupled to changes in cellular DNA content, form a preconditional state that influences the control of DNA repair or recombination events involved in trinucleotide expansion in liver hepatocytes and pancreatic acinar cells.

Proteomic analysis of secreted proteins in early rheumatoid arthritis: anti-citrulline autoreactivity is associated with up regulation of proinflammatory cytokines.

OBJECTIVES: To identify peripheral blood autoantibody and cytokine profiles that characterise clinically relevant subgroups of patients with early rheumatoid arthritis using arthritis antigen microarrays and a multiplex cytokine assay. METHODS: Serum samples from 56 patients with a diagnosis of rheumatoid arthritis of <6 months' duration were tested. Cytokine profiles were also determined in samples from patients with psoriatic arthritis (PsA) and ankylosing spondylitis (n = 21), and from healthy individuals (n = 19). Data were analysed using Kruskal-Wallis test with Dunn's adjustment for multiple comparisons, linear correlation tests, significance analysis of microarrays (SAM) and hierarchical clustering software. RESULTS: Distinct antibody profiles were associated with subgroups of patients who exhibited high serum levels of tumour necrosis factor (TNF)alpha, interleukin (IL)1beta, IL6, IL13, IL15 and granulocyte macrophage colony-stimulating factor. Significantly increased autoantibody reactivity against citrullinated epitopes was observed in patients within the cytokine "high" subgroup. Increased levels of TNFalpha, IL1alpha, IL12p40 and IL13, and the chemokines eotaxin/CCL11, monocyte chemoattractant protein-1 and interferon-inducible protein 10, were present in early rheumatoid arthritis as compared with controls (p<0.001). Chemokines showed some of the most impressive differences. Only IL8/CXCL8 concentrations were higher in patients with PsA/ankylosing spondylitis (p = 0.02). CONCLUSIONS: Increased blood levels of proinflammatory cytokines are associated with autoantibody targeting of citrullinated antigens and surrogate markers of disease activity in patients with early rheumatoid arthritis. Proteomic analysis of serum autoantibodies, cytokines and chemokines enables stratification of patients with early rheumatoid arthritis into molecular subgroups.

Proliferation studies on chromosome preparations of bone marrow in hematological disease.

The growth rate of neoplastic cells has been the subject of numerous scientific and diagnostic approaches. The study presented here analyses the relationship between mitotic activity in standardised cytogenetic bone marrow preparations from three haematological diseases and diagnostic and clinical parameters, most importantly the outcome. The disorders studied were: Acute lymphoblastic leukemia (ALL) (N=107), chronic myeloid leukemia (CML) (N=166) and aplastic anemia in childhood (AA) (N=39). A strict protocol of quantitative standardisation of cytogenetic slides was adhered to ensuring comparability both cross-sectionally and longitudinally. The samples were studied after short-term incubation without mitogenic in vitro stimuli. The most important findings include: i) ALL: Immunological subtypes can be differentiated according to their proliferation profile; there is a striking difference between childhood and adult ALL in proliferation activity; most importantly initial proliferation is much higher in patients who will relapse than in those with stable remission. ii) CML: Philadelphia-positive CML shows proliferation activities quite distinct from Philadelphia-negative CML; however there is only a small change in the proliferative activity from the chronic phase to the accelerated phase or blast crisis. iii) AA: Very low proliferation scores rise quickly to near normal levels during immunosuppressive therapy in most patients. Higher levels at diagnosis are associated with a faster and better response to therapy. In conclusion, assessment of the proliferative activity in cytogenetic preparations made from bone marrow samples of patients with haematological disease may add valuable information as to diagnostic sub-groups and clinical course and may contribute to therapeutic decisions.

Differential association of protein subunits with the human RNase MRP and RNase P complexes.

RNase MRP is a eukaryotic endoribonuclease involved in nucleolar and mitochondrial RNA processing events. RNase MRP is a ribonucleoprotein particle, which is structurally related to RNase P, an endoribonuclease involved in pre-tRNA processing. Most of the protein components of RNase MRP have been reported to be associated with RNase P as well. In this study we determined the association of these protein subunits with the human RNase MRP and RNase P particles by glycerol gradient sedimentation and coimmunoprecipitation. In agreement with previous studies, RNase MRP sedimented at 12S and 60-80S. In contrast, only a single major peak was observed for RNase P at 12S. The analysis of individual protein subunits revealed that hPop4 (also known as Rpp29), Rpp21, Rpp20, and Rpp25 only sedimented in 12S fractions, whereas hPop1, Rpp40, Rpp38, and Rpp30 were also found in 60-80S fractions. In agreement with their cosedimentation with RNase P RNA in the 12S peak, coimmunoprecipitation with VSV-epitope-tagged protein subunits revealed that hPop4, Rpp21, and in addition Rpp14 preferentially associate with RNase P. These data show that hPop4, Rpp21, and Rpp14 may not be associated with RNase MRP. Furthermore, Rpp20 and Rpp25 appear to be associated with only a subset of RNase MRP particles, in contrast to hPop1, Rpp40, Rpp38, and Rpp30 (and possibly also hPop5), which are probably associated with all RNase MRP complexes. Our data are consistent with a transient association of Rpp20 and Rpp25 with RNase MRP, which may be inversely correlated to its involvement in pre-rRNA processing.

PM-Scl-75 is the main autoantigen in patients with the polymyositis/scleroderma overlap syndrome.

OBJECTIVE: To compare the autoantigenicity of the recently described N-terminally elongated PM-Scl-75 protein with that of PM-Scl-100 and the originally defined PM-Scl-75 polypeptide, and to determine its value for analyzing sera from patients with the polymyositis (PM)/scleroderma overlap syndrome. METHODS: Serum samples obtained from patients with the PM/scleroderma overlap syndrome and from patients with several other diseases were analyzed for the presence of autoantibodies reactive with recombinant PM-Scl-100 and PM-Scl-75 (both the original and the longer form) proteins, in an enzyme-linked immunosorbent assay (ELISA). RESULTS: Autoantibodies recognizing the longer PM-Scl-75 protein isoform were detected in 28% of the patients with PM/scleroderma. This percentage is slightly higher than that for PM-Scl-100 (25%) and is significantly higher than that for the previously defined PM-Scl-75 protein (11%). In addition, we identified a significant number of patients who had anti-PM-Scl-75 but not anti-PM-Scl-100 antibodies. This finding contrasts with what has been previously reported for the shorter version of the PM-Scl-75 protein. CONCLUSION: Our data indicate that use of the long PM-Scl-75 isoform in addition to PM-Scl-100 in ELISAs significantly increases the number of patients in whom anti-PM-Scl autoantibodies can be detected.