Giessen international workshop on interactions of exocrine and endocrine pancreatic diseases. Castle of Rauischholzhausen of the Justus-Liebig-University, Giessen, Germany. March 18-19, 2005

The 'Giessen International Workshop on Interactions of Exocrine and Endocrine Pancreatic Diseases' was held on March 18-19, 2005 at the Castle of Rauischolzhausen, Giessen University, Germany. About 50 international clinicians and researchers attended the workshop. It was structured into three sessions: A: Pancreatic Autoimmunity - Interaction Between Exocrine and Endocrine Tissue; B: Diabetes Mellitus - Possible Implications of Exocrine Pancreatic Insufficiency; C: Chronic Pancreatitis - Update on Prevalence, Understanding and Pathophysiological Concepts. Several new aspects of pancreatic diseases were discussed, including new classifications of pancreatitis, new insights into prevalence, pathophysiology and new therapeutical considerations. The meeting resulted in more cooperation and a number of new concepts for clinical study which will provide data for future developments.     

Immuno-SLM-a combined sample handling and analytical technique

Immuno-supported liquid membrane (immuno-SLM) extraction is a new technique that makes use of antibody (Ab)-antigen interactions as the "extraction force" to drive the mass transfer in a selective way. In immuno-SLM, anti-analyte (Ag) Abs are introduced into the acceptor phase of the SLM unit to trap the Ag that passes from the flowing donor through the SLM into the stagnant acceptor. The amount of immuno-extracted analyte (AbAg) is quantified by connecting the immuno-SLM unit on-line with a non-competitive heterogeneous fluorescence flow immunoassay (FFIA) that makes use of a fluorescein-labeled analyte tracer that titrates the residual excess of Ab present in the acceptor. A restricted access (RA) column is used for the separation of the two tracer fractions (Ag* and AbAg*) formed, and the eluted AbAg* fraction is measured downstream by a fluorescence detector. Factors influencing the optimum immuno-SLM extraction parameters, i.e., donor flow rate, extraction time and type of Ab, were investigated for immuno extraction of the model analyte atrazine. Immuno-SLM coupled to FFIA (immuno-SLM-FFIA) and FFIA alone were compared in terms of the assay sensitivities obtained and the sample matrix influence. The concentration at the mid-point of the calibration curve (IC(50)) was 16.0+/-1.4 and 36+/-16 microg/l, the limit of detection (LOD) was 2.0+/-1.1 and 20+/-10 microg/l, and the dynamic range was 2-100 and 20-500 microg/l atrazine for immuno-SLM-FFIA and FFIA, respectively. The matrix influence on the FFIA was significant in orange juice and surface water, whereas the influence was minor for immuno-SLM-FFIA with recoveries between 104% and 115% for 5 microg/l atrazine in tap water, orange juice and river water.     

Regional ventilation and distribution of emphysema - a quantitative comparison

The distribution of ventilation and emphysema throughout the lung was compared in 20 emphysematous patients who were candidates for lung volume reduction surgery. Ventilation distribution among 1.6-cm-high stripe regions was obtained in supine position by planar gamma camera scintigraphy after inhalation of technegas. Results from nine healthy subjects provided normal stripe count rates. Patient count rates were expressed as the difference from predicted normal stripe count rate (DeltaSVI). Thus, DeltaSVI is a relative index of abnormal ventilation. Emphysema was assessed quantitatively by a density mask method on corresponding slices of high resolution computed tomography (HRCT). The results show that in the majority of patients, there is a highly significant relationship between the degree of stripe hypoventilation and the amount of distribution of emphysema, but in some patients no such relationship is found. We speculate that the distribution of airway obstruction not necessarily follows the distribution of emphysema, which could explain the lack of concordance between ventilation and emphysema distribution in some patients. We conclude that ventilation scintigraphy contains complementary information to lung HRCT.     

Catastrophic failure of an uncemented acetabular component due to high wear and osteolysis: an analysis of 154 omnifit prostheses with mean 6-year follow-up

BACKGROUND: The purposes of this study were (1) to evaluate the wear pattern of the hydroxyapatite-coated "Dual Radius" Omnifit cup, (2) to investigate whether wear is correlated to any demographic or prosthesis-related factors, and (3) to describe micromotion of both the cup and the stem. PATIENTS AND METHODS: 154 hips were implanted between 1990 and 1996 and followed for an average of 6 years. Wear was measured according to the "Charnley-duo" method and, in 79 hips, with radiostereometry IRSA). RSA was also used to evaluate micromotion. We analyzed the femoral heads using scanning electron microscopy, energy dispersive X-ray spectroscopy and an atomic force microscope. RESULT: 66 cups were revised and had a mean annual wear of 0.32 mm compared to 0.12 mm in hips not revised. Osteolytic processes were observed in 35 hips but at revision osteolysis was present in 51 cases. 43/66 sockets were loose. Micromotion evaluated by RSA, weight, age, side, size of cup, screws, polyethylene thickness or shelf-life of the polyethylene did not correlate to wear, whereas male gender did. INTERPRETATION: It is still unclear why about half of our cases had an abnormal wear rate. Annual wear exceeding 0.2 mm is prognostic of late failure and should be considered a warning sign.     

Role of actin filaments in targeting of Crimean Congo hemorrhagic fever virus nucleocapsid protein to perinuclear regions of mammalian cells

Crimean-Congo hemorrhagic fever virus is the causative agent of a severe disease throughout Africa, Europe, and Asia. Like other members of the genus Nairovirus, the Crimean-Congo hemorrhagic fever virus contains three genomic RNA segments, the small (S), medium (M), and large (L) segments. The S segment encodes the viral nucleocapsid protein (NP), while the M and L segments encode the glycoproteins and the RNA-dependent RNA polymerase, respectively. In this study, the site of expression and assembly of Crimean-Congo hemorrhagic fever virus NP in mammalian cells have been investigated. It was found that the NP is localized in the perinuclear region of infected cells. By using the Semliki forest virus expression system, it was shown that the Crimean-Congo hemorrhagic fever virus NP is targeted to the perinuclear region of cells in the absence of native RNA segments and virally encoded glycoproteins. It was also demonstrated that the Crimean-Congo hemorrhagic fever virus NP was not expressed as a Golgi-membrane associated protein. By using Cytochalasin D, an agent that disrupts actin filaments, it was found that actin filaments are involved in targeting the viral NP to perinuclear regions. We also demonstrated that disruption of actin filaments reduced the assembly of infectious Crimean-Congo hemorrhagic fever virus up to 97%. Furthermore, we showed that the NP of Crimean-Congo hemorrhagic fever virus NP interacts with actin.     

Splicing profile based protein categorization between human and mouse genomes by use of the DDBJ Web services

In one scenario of gene evolution, exon shuffling plays a fundamental role in increasing gene diversity. This paper is an appraisal of the biological relevance of categorising proteins by their splicing profiles (exon-intron structures). The central question is whether protein function is more correlated with splicing profiles than sequence similarity, or not. To approach this question, a splicing profile similarity (SPS) index, which measures relative exon length discrepancy, was devised. Arbitrary human proteins were compared, in terms of SPS and amino acid sequence similarity, to their 1) mouse orthologues and 2) human paralogues, which epitomise functional equivalence and non-equivalence, respectively, to methodically elucidate the global relationship between a) biological function, b) splicing profile similarity, and c) sequence similarity. Protein function is more correlated with splicing profile similarity than sequence similarity as demonstrated by the fact that human-mouse orthologues (HMOs) display significantly higher splicing profile similarity than do human-human paralogues (HHPs), despite the mutual sequence similarity between these two categories. This finding indicates that splicing profile-based protein categorisation is biologically meaningful.     

Developmental Exposure to 4-hydroxy-2,3,3',4',5-pentachlorobiphenyl (4-OH-CB107): Long-Term Effects on Brain Development, Behavior, and Brain Stem Auditory Evoked Potentials in Rats

In the present study the developmental neurotoxic effects ofthe PCB metabolite 4-OH-2,3,3',4',5-pentachlorobiphenyl (4-OH-CB107)were compared with effects caused by a mixture of parent polychlorinatedbiphenyl (PCB) congeners (Aroclor 1254). Pregnant female Wistarrats were exposed to 0.5 or 5 mg 4-OH-CB107, or 25 mg Aroclor1254 per kg body weight from gestation days 10 to 16. Plasmathyroid hormone levels were significantly decreased in the offspringof all treatment groups at postnatal day 4 (PND 4). Behavioralexperiments using an open field paradigm revealed an impairedhabituation in male offspring of all treatment groups at PND130. Passive avoidance experiments indicated significant influenceson the time course of step-down latencies across trials in exposedmale rats. Catalepsy induced by haloperidol showed increasesin latencies to movement onset in female offspring exposed to0.5 mg 4-OH-CB107 compared to Aroclor 1254 treated offspringat PND 168–175. Male offspring exposed to 4-OH-CB107 orAroclor 1254 showed decreases in latencies compared to controlanimals. Brain stem auditory evoked potentials (BAEPs) measuredat PND 300–310 showed significant increases in auditorythresholds in the low frequency range between Aroclor 1254 and4-OH-CB107 (5 mg/kg bw) treated animals. Measurements of neurotransmitterlevels revealed effects of Aroclor 154 exposure on both thedopaminergic and the serotonergic systems, whereas 4-OH-CB107exposure affected dopaminergic and noradrenergic systems, withslight but not significant effects on the serotonergic system.These results indicate that 4-OH-CB107 is able to induce long-termeffects on behavior and neurodevelopment. The observed effectsfor 4-OH-CB107 are similar to, but in some aspects differentfrom, the effects observed after Aroclor 1254 exposure.     

Regulation of matrix metalloproteinase 13 expression by androgen in prostate cancer

The matrix metalloproteinases (MMPs) are members of a family of endopeptidases that are able to degrade extra-cellular matrix. MMPs and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), play a key role in the migration of normal and malignant cell. Interaction of MMPs and TIMPs has been involved in the process of tumor invasion and metastasis. Using cDNA microarray as a screening tool to find androgen regulated gene in prostate cancer, we have found that expression of MMP-13 is regulated by androgen in prostate cancer derived cell line LNCaP. This regulation was further confirmed and quantified by real-time RT-PCR. In addition, the upregulation of MMP-13 mRNA by androgen could be abolished by the androgen antagonist Casodex but not the protein inhibitor cycloheximide. Western blot and immunohistochemistry of MMP-13 confirmed the androgen regulation at the protein level. We have furthermore shown that MMP-13 expression is presented in human prostate cancer obtained from aspiration biopsy. In summary, MMP-13 is androgen regulated and detectable in prostate cancer. Further study of MMP-13 in prostate cancer may help us to understand the progression of the cancer and can lead to new therapeutic options.     

BRCA1 and BRCA2 mutation analysis in breast-ovarian cancer families from northeastern Poland

Sixty high-risk breast and/or ovarian cancer families from North-Eastern Poland were screened for germline mutations in BRCA1 (MIM# 113705) and BRCA2 (MIM# 600185), using a combination of protein truncation test, denaturing high-performance liquid chromatography and direct sequencing. Sixteen (27%) of the families were found to carry nine different BRCA mutations, including 14 families with BRCA1 mutation and two families with BRCA2 mutation. The results suggest the presence of two strong BRCA1 founder mutations in the Polish population - 5382insC (6 families) and 300T>G (Cys61Gly; 3 families). The remaining seven mutations were found in single families and included three previously reported BRCA1 mutations (185delAG, 2682C>T [Gln855Ter] and 3819del5), a novel BRCA1 mutation (IVS14+1G>A), as well as two BRCA2 mutations (4088delA and 7985G>A [Trp2586Ter]) not previously observed in Polish families. We confirm the strong influence of two Central-Eastern European BRCA1 founder mutations in familial breast and/or ovarian cancer in Poland. We also conclude that the Polish population has a more dispersed BRCA mutation spectrum than had been earlier thought. This warrants further careful BRCA mutation screening in order to optimise genetic counselling and disease prevention in affected families.     

Genetic characterization of new Dobrava hantavirus isolate from Greece

The first complete genome sequence of Dobrava hantavirus isolated from yellow-necked mouse Apodemus flavicollis trapped in the northeastern Greece is described. The S, M, and L segments of the Greek isolate of Dobrava virus are 1673, 3635, and 6532 nucleotides (nt) long, respectively, and encode the nucleocapsid (N) protein of 429 amino acids (aa), glycoprotein precursor of 1135 aa, and the L protein of 2151 aa. N protein contains three cysteine residues conserved in all known hantaviruses, as well as structural domains responsible for the RNA binding and presumable interaction with the apoptosis enhancer Daxx. All cysteine residues and glycosylation sites that are conserved among G1G2 sequences of all hantaviruses species were also found in the Greek isolate. The L protein contains all the polymerase motifs and structural domains found in other hantavirus polymerases. Comparison of the Greek isolate of Dobrava virus with other hantaviruses showed the highest level of sequence homology with Dobrava virus isolate from Slovenia. Other hantaviruses carried by Murinae rodents (Saaremaa, Hantaan, Seoul, and Thailand viruses) were more divergent and hantaviruses carried by Arvicolinae or Sigmodontinae rodents showed the highest genetic diversity with the Greek isolate of Dobrava. The results of phylogenetic analyses confirmed these observations and showed a monophily of all the Dobrava virus strains that, in turn, shared more ancient ancestors first with Saaremaa virus and then with other Murinae-borne hantaviruses.