Mechanical characterization of oligo(ethylene glycol)-based hydrogels by dynamic nanoindentation experiments.

Oligo(ethylene glycol)-based (OEG) hydrogel samples of varying cross-link densities and degrees of swelling were characterized through dynamic nanoindentation testing. Experiments were performed using a non-standard nanoindentation method, which was validated on a standard polystyrene sample. This method maximizes the capability of the instrument to measure the stiffness and damping of highly compliant, viscoelastic materials. Experiments were performed over the frequency range of 1 to 50 Hz, using a 1mm diameter flat punch indenter. A hydration method was adopted to avoid sample dehydration during testing. Values of storage modulus (E') ranged from 3.5 to 8.9 MPa for the different OEG-hydrogel samples investigated. Samples with higher OEG concentrations showed greater scatter in the modulus measurements and it is attributed to inhomogeneities in these materials. The (E') values did not show a strong variation over frequency for any of the samples. Values of loss modulus (E") were two orders of magnitude lower than the storage modulus, resulting in very low values of loss factor (E"/E'<0.1). These are characteristics of strong gels, which present negligible viscous properties.

[Modern diagnostics of cystic liver lesions and hemangiomas]. / Moderne Diagnostik zystischer Leberläsionen und Hämangiome.

CLINICAL ISSUE: Cystic liver lesions incorporate a broad heterogeneous group of mostly benign but also malignant abnormalities. The radiological aim is the non-invasive diagnosis with the use of different imaging modalities to determine the type of lesion. STANDARD RADIOLOGICAL METHODS: The common generally asymptomatic incidental findings of cystic lesions on ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI) must be classified on the basis of specific imaging features. Such a differentiation is essential because the clinical consequences and the appropriate therapy can vary depending on the underlying pathology. Due to the morphological overlap of many cystic lesions, conventional radiological methods are often insufficient. METHODICAL INNOVATIONS: The huge advances in cross-sectional imaging (multidetector CT, MRI with special sequences and different contrast agents and MR cholangiopancreatography) in combination with the clinical history usually enable a non-invasive diagnosis. Pathognomonic morphological and hemodynamic lesion features, as well as a knowledge of the pathomechanisms, help to differentiate this broad spectrum of entities. ACHIEVEMENTS: In this article the different entities of cystic liver lesions, together with the appropriate diagnostic method for detection and distinction and including their strengths and limitations, are demonstrated. PRACTICAL RECOMMENDATIONS: A well-founded knowledge about the development of various cystic liver lesions and the suitable choice of imaging method facilitate a non-invasive diagnosis.

[Focal nodular hyperplasia and hepatocellular adenoma]. / Fokale noduläre Hyperplasie und hepatozelluläres Adenom.

CLINICAL ISSUE: Focal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA) are liver lesions of hepatocellular origin. The FNH is a commonly occurring hepatic lesion whereas HCA is very rare. Non-invasive differentiation between HCA subtypes and atypical FNH may pose a diagnostic challenge as both entities predominantly occur in middle-aged female patients. STANDARD RADIOLOGICAL METHODS: The conventional imaging modalities include ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI). Distinguishing FNH from HCA is of great importance clinically as FNH is considered to be a benign lesion and needs no further management. In contrast HCA is considered to be a borderline tumor due to the risk of hemorrhage, growth and even malignant transformation and requires individualized management. METHODOLOGICAL INNOVATIONS: The above mentioned radiological procedures usually enable an accurate and certain diagnosis of a typical FNH to be achieved. In cases of atypical FNH, particularly in patients with a clinical history of malignancy, these imaging modalities are insufficient to establish a clear diagnosis. In this scenario, the use of modern hepatobiliary contrast-enhanced MRI will enable a differentiation between FNH and metastasis with a high sensitivity and specificity. Furthermore, it allows a differentiation of FNH from 90 % of adenoma subtypes. ACHIEVEMENTS: This article describes the histopathological and radiological features of these lesions and explains the advantages and limitations of various imaging modalities used for the diagnosis and differentiation of these entities. PRACTICAL RECOMMENDATIONS: The new classification of HCAs according to phenotype and genotype and their imaging features, as well as different enhancement patterns, are described. The correlation between HCA subtypes and their individual management are also discussed.

Damage modeling of small-scale experiments on dental enamel with hierarchical microstructure.

Dental enamel is a highly anisotropic and heterogeneous material, which exhibits an optimal reliability with respect to the various loads occurring over years. In this work, enamel's microstructure of parallel aligned rods of mineral fibers is modeled and mechanical properties are evaluated in terms of strength and toughness with the help of a multiscale modeling method. The established model is validated by comparing it with the stress-strain curves identified by microcantilever beam experiments extracted from these rods. Moreover, in order to gain further insight in the damage-tolerant behavior of enamel, the size of crystallites below which the structure becomes insensitive to flaws is studied by a microstructural finite element model. The assumption regarding the fiber strength is verified by a numerical study leading to accordance of fiber size and flaw tolerance size, and the debonding strength is estimated by optimizing the failure behavior of the microstructure on the hierarchical level above the individual fibers. Based on these well-grounded properties, the material behavior is predicted well by homogenization of a representative unit cell including damage, taking imperfections (like microcracks in the present case) into account.

[New developments in MRI of the liver]. / Neuere Entwicklungen der Leber-MRT.

Radiology has gained an exceptional position in medicine because a correct diagnosis is the most crucial issue in determining an accurate and personalized therapeutic strategy. This has a direct influence not only on the individual patient but also on the socio-economic aspects of healthcare services in terms of shortening the time interval to establish a diagnosis and to avoid risk-associated invasive diagnostic methods or long-term, cost-intensive follow-up. Magnetic resonance imaging (MRI) is an excellent example of this which due to continuous technological developments and emerging techniques allows a non-invasive diagnosis of the different hepatic diseases. In this article, we illustrate the direct correlation between the recent technical advances in MRI, such as 3.0 T, diffusion-weighted imaging, perfusion imaging, spectroscopy, texture analysis and MR elastography and obtaining a confident non-invasive diagnosis of focal and diffuse liver diseases.

Dominant GDAP1 mutations cause predominantly mild CMT phenotypes.

OBJECTIVE: Ganglioside-induced differentiation associated-protein 1 (GDAP1) mutations are commonly associated with autosomal recessive Charcot-Marie-Tooth (ARCMT) neuropathy; however, in rare instances, they also lead to autosomal dominant Charcot-Marie-Tooth (ADCMT). We aimed to investigate the frequency of disease-causing heterozygous GDAP1 mutations in ADCMT and their associated phenotype. METHODS: We performed mutation analysis in a large cohort of ADCMT patients by means of bidirectional sequencing of coding regions and exon-intron boundaries of GDAP1. Intragenic GDAP1 deletions were excluded using an allele quantification assay. We confirmed the pathogenic character of one sequence variant by in vitro experiments assaying mitochondrial morphology and function. RESULTS: In 8 Charcot-Marie-Tooth disease (CMT) families we identified 4 pathogenic heterozygous GDAP1 mutations, 3 of which are novel. Three of the mutations displayed reduced disease penetrance. Disease onset in the affected individuals was variable, ranging from early childhood to adulthood. Disease progression was slow in most patients and overall severity milder than typically seen in autosomal recessive GDAP1 mutations. Electrophysiologic changes are heterogeneous but compatible with axonal neuropathy in the majority of patients. CONCLUSIONS: With this study, we broaden the phenotypic and genetic spectrum of autosomal dominant GDAP1-associated neuropathies. We show that patients with dominant GDAP1 mutations may display clear axonal CMT, but may also have only minimal clinical and electrophysiologic abnormalities. We demonstrate that cell-based functional assays can be reliably used to test the pathogenicity of unknown variants. We discuss the implications of phenotypic variability and the reduced penetrance of autosomal dominant GDAP1 mutations for CMT diagnostic testing and counseling.

LA-ICP-MS analysis of waste polymer materials.

Waste polymer materials were analyzed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The concentrations of 35 elements were determined by using different types of external standards, namely glass and polyethylene (PE) based. Prior to the LA-ICP-MS analysis of determined elements, Na and/or Zn were used as internal standards. The investigations concentrated mainly on the detection of Cr, As, Cd, Sn, Sb, Hg, and Pb. Using PE-based calibration standards, the measured concentrations in the waste polymers were within 49% of the wet chemical data. The determined deviation was up to 102% when using the glass standards. Trace concentration of As and Hg (and also of S) could be determined with a concentration below 1 mg/kg. However, Hg provided very low intensity with a high relative standard deviation (RSD) and was therefore not further evaluated. Cryomilling of polymers was applied to reduce the particle size of the material and improved the precision and accuracy of LA-ICP-MS analysis. On average, the LA-ICP-MS results showed a deviation from the wet chemical reference analysis of 38% and an RSD of 56% for pressed polymer powder samples prepared by cryomilling. In general, waste pellets without sample preparation (i.e., use of pellets as delivered) are too heterogeneous, not suitable for micro-beam techniques, and showed a strong matrix dependence. With homogeneous pellets that appear similar to each other agreement in the determined concentrations was found for some elements.

Anti-apoptotic and growth-stimulatory functions of CK1 delta and epsilon in ductal adenocarcinoma of the pancreas are inhibited by IC261 in vitro and in vivo.

BACKGROUND: Pancreatic ductal adenocarcinomas (PDACs) are highly resistant to treatment due to changes in various signalling pathways. CK1 isoforms play important regulatory roles in these pathways. AIMS: We analysed the expression levels of CK1 delta and epsilon (CK1delta/in) in pancreatic tumour cells in order to validate the effects of CK1 inhibition by 3-[2,4,6-(trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261) on their proliferation and sensitivity to anti-CD95 and gemcitabine. METHODS: CK1delta/in expression levels were investigated by using western blotting and immunohistochemistry. Cell death was analysed by FACS analysis. Gene expression was assessed by real-time PCR and western blotting. The putative anti-tumoral effects of IC261 were tested in vivo in a subcutaneous mouse xenotransplantation model for pancreatic cancer. RESULTS: We found that CK1delta/in are highly expressed in pancreatic tumour cell lines and in higher graded PDACs. Inhibition of CK1delta/in by IC261 reduced pancreatic tumour cell growth in vitro and in vivo. Moreover, IC261 decreased the expression levels of several anti-apoptotic proteins and sensitised cells to CD95-mediated apoptosis. However, IC261 did not enhance gemcitabine-mediated cell death either in vitro or in vivo. CONCLUSIONS: Targeting CK1 isoforms by IC261 influences both pancreatic tumour cell growth and apoptosis sensitivity in vitro and the growth of induced tumours in vivo, thus providing a promising new strategy for the treatment of pancreatic tumours.

Different effects of antisense RelA p65 and NF-kappaB1 p50 oligonucleotides on the nuclear factor-kappaB mediated expression of ICAM-1 in human coronary endothelial and smooth muscle cells.

BACKGROUND: Activation of nuclear factor-kappaB (NF-kappaB) is one of the key events in early atherosclerosis and restenosis. We hypothesized that tumor necrosis factor-alpha (TNF-alpha) induced and NF-kappaB mediated expression of intercellular adhesion molecule-1 (ICAM-1) can be inhibited by antisense RelA p65 and NF-kappaB1 p50 oligonucleotides (RelA p65 and NF-kappaB1 p50). RESULTS: Smooth muscle cells (SMC) from human coronary plaque material (HCPSMC, plaque material of 52 patients), SMC from the human coronary media (HCMSMC), human endothelial cells (EC) from umbilical veins (HUVEC), and human coronary EC (HCAEC) were successfully isolated (HCPSMC, HUVEC), identified and cultured (HCPSMC, HCMSMC, HUVEC, HCAEC). 12 hrs prior to TNF-alpha stimulus (20 ng/mL, 6 hrs) RelA p65 and NF-kappaB1 p50 (1, 2, 4, 10, 20, and 30 microM) and controls were added for a period of 18 hrs. In HUVEC and HCAEC there was a dose dependent inhibition of ICAM-1 expression after adding of both RelA p65 and NF-kappaB1 p50. No inhibitory effect was seen after incubation of HCMSMC with RelA p65 and NF-kappaB1 p50. A moderate inhibition of ICAM-1 expression was found after simultaneous addition of RelA p65 and NF-kappaB1 p50 to HCPSMC, no inhibitory effect was detected after individual addition of RelA p65 and NF-kappaB1 p50. CONCLUSIONS: The data point out that differences exist in the NF-kappaB mediated expression of ICAM-1 between EC and SMC. Experimental antisense strategies directed against RelA p65 and NF-kappaB1 p50 in early atherosclerosis and restenosis are promising in HCAEC but will be confronted with redundant pathways in HCMSMC and HCPSMC.

Partial sequence of human platelet heparitinase and evidence of its ability to polymerize.

Heparitinase cleaves heparan sulfate, a glycosaminoglycan associated with all nucleated mammalian cells and extracellular matrices. Despite the important physiologic role heparitinase is postulated to play in such processes as tumor metastasis and inflammation, the identity of the enzyme remains a matter of controversy and there is a question of whether heparitinase is CTAP III. We report a 900,000-fold purification of heparitinase from human platelets. A multi-step procedure utilizing chromatography on heparin, DEAE, hydroxyapatite and size exclusion matrices was employed and yielded a single protein as judged by Coomassie staining of protein separated by SDS-PAGE. The purified protein had an apparent molecular mass of 35 kDa by size exclusion chromatography and 55 kDa by SDS-PAGE. During purification, heparitinase activity co-eluted from the hydroxyapatite and size exclusion columns with the 35-55 kDa protein, confirming that the purified protein was indeed heparitinase. The 35-55 kDa protein reacted strongly with concanavalin A, a lectin known to bind to heparitinase, further confirming that the protein was heparitinase. Platelet heparitinase formed dimers and tetramers upon storage in a purified form, possibly accounting for the various molecular weights previously reported for the enzyme. A partial amino acid sequence of the protein revealed that heparitinase has not been previously sequenced.

A transcription factor with AP3-like binding specificity mediates gene regulation after an allergic triggering with IgE and Ag in mouse mast cells.

Mast cells are an important source of a number of lymphokines and chemokines primarily those released after challenge with the allergic trigger IgE and Ag. However, the mechanisms of lymphokine and chemokine gene activation in this cell type, as opposed to the mechanisms of activation in T cells, are poorly understood. As a model system, we addressed this issue in mast cells by using the recently cloned chemokine MARC gene, which belongs to the RANTES/sis gene family. After allergic stimulation, MARC induction is pronounced and mast cell specific. Northern blot analysis, in combination with two inhibitors, actinomycin D and cycloheximide, resulted in the formation of our initial hypothesis, which was that both transcriptional and post-transcriptional regulation are involved after stimulation through the Fc epsilon R. We performed a detailed promoter analysis of the cloned MARC gene by using transient assays of transfected reporter gene constructs. Thereby, two potential promoter regions were identified as being crucial for transcriptional stimulation. Additional fine mapping of the proximal element and subsequent electrophoretic mobility shift assays, combined with competitions of known transcription factor binding sites, identified one of the transcription factors in stimulated mast cells as an AP3 or AP3-like binding activity.

Immunoglobulin E plus antigen challenge induces a novel intercrine/chemokine in mouse mast cells.

In an attempt to characterize genes participating in the allergic late phase reaction, we have isolated a novel intercrine/chemokine (called MARC) from a cDNA library of the stimulated mouse mast cell line, CPII. As measured by Northern blotting, it is strongly upregulated at the mRNA level after the physiological challenge of the cells with immunoglobulin (Ig)E plus antigen. Unstimulated cells completely lack significant, stable expression, as do a number of other, different cell lines (uninduced and induced) and mouse tissues. In contrast to the Northern blot analysis, a polymerase chain reaction (PCR) analysis, performed on CPII cells and on Percoll gradient purified mouse peritoneal mast cells, revealed a basal level of transcription in the uninduced stage. After 2 h of IgE plus antigen challenge, a quantitative reverse transcriptase-PCR, using a spiked in MIMIC, showed a level of transcripts more than 100-fold higher in the CPII cells and 5-20-fold higher in purified mouse peritoneal cavity mast cells. This rapid induction after the Fc epsilon RI challenge, the identification of the gene as a member of the chemokine family, and its upregulated expression in peritoneal mast cells, all suggest an involvement in certain acute and chronic pathological mast cell-driven diseases.

A novel transiently expressed, integral membrane protein linked to cell activation. Molecular cloning via the rapid degradation signal AUUUA.

A novel cDNA clone termed E16 which codes for an integral membrane protein of 241 amino acids with six transmembrane domains was isolated from peripheral blood lymphocytes. The cDNA clone is 4000 base pairs in length and exhibits an unusually long 3'-untranslated region of about 3000 nucleotides. Its expression at the mRNA level is closely linked to cellular activation and division. In all myeloid and lymphoid cells, as well as in primary lymphocytes from peripheral blood, E16 transcripts are rapidly induced and rapidly degraded after stimulation. This pattern of expression is unusual for an integral membrane protein and resembles more closely the kinetic seen for protooncogenes and lymphokines in the T cell system. Its isolation was made possible by a novel approach especially designed to selectively clone cDNAs which exhibit such an expression kinetic. It is based on a combination of the differential screening of a subtracted cDNA library and the subsequent hybridization of the resulting phages to a short oligonucleotide (5'-TAAATAAA-TAAATA-3'). This oligonucleotide is complementary to a trimer of the rapid degradation signal (AUUUA) which is present as a single or reiterated motif in the 3'-untranslated region of many short-lived transcripts.

A new superfamily of lymphoid and melanoma cell proteins with extensive homology to Schistosoma mansoni antigen Sm23.

A novel cDNA clone termed R2 was isolated by subtractive hybridization of a cDNA library of phytohemagglutinin (PHA)/phorbol myristate acetate-stimulated Jurkat cells and by rescreening a cDNA library of PHA-stimulated peripheral blood lymphocytes. It hybridizes to a single mRNA species of about 2.2 kb, which is inducible in lymphoid cells and codes for a protein of 267 amino acids which contains four potential transmembrane domains. A computer-aided comparison showed strong homology to four other membrane proteins, the pan B cell marker CD37, the pan leukocyte marker CD53, the melanoma antigen ME491 and, surprisingly, the Schistosoma mansoni antigen Sm23. The four human proteins share a number of additional similarities in their overall structure. These include identical spacing of the transmembrane domains, similar hydrophobicity plots, possible N-linked glycosylation sites of similar number and position as well as similar distribution of the cysteine residues. The majority of these characteristics are still conserved in the evolutionary most distant member of this family, the Schistosoma mansoni antigen Sm23. Here we introduce this new protein superfamily and characterize the inducible, lymphoid-specific member R2.

Animal exposures and antibodies to Toxoplasma gondii in a university population.

To determine the risk of toxoplasma infection to individuals exposed to cats in a research institution, we compared the prevalence of toxoplasma antibodies with exposure to cats in university employees. Of 116 employees tested, 42 (36 percent) had toxoplasma antibodies as determined by the indirect fluorescent antibody test. Women and individuals aged 35 years or more had a greater prevalence of antibodies. The antibody prevalence by occupation was 72.1 percent for physicians and those with doctorates, 45.3 percent for animal and veterinary technicians, 33.3 percent for research technicians, 28.2 percent for administrative staff, 25.0 percent for graduate students and fellows, and 13.4 percent for veterinarians. There was no significant positive association between exposure to cats and the prevalence of toxoplasma antibodies. A follow-up of seronegative employees, 6 and 18 months later, revealed no seroconversions indicative of acute toxoplasma infection. We concluded that there was no significant risk of toxoplasma infection in university employees exposed to cats.

Bovine leukemia virus infection in a large Holstein herd: prospective comparison of production and reproductive performance in antibody-negative and antibody-positive cows.

Serum samples from lactating cows in a purebred Holstein herd were tested annually (from 1977 to 1980) for antibodies to bovine leukemia virus (BLV), using the agargel immunodiffusion test. Production and reproductive variables were obtained from Dairy Herd Improvement Association records. All milk and fat production values were converted to 3.5% fat-corrected milk (FCM). Variables examined included: FCM, 305-day actual; FCM, 305-day mature-equivalent; FCM, total lactation; FCM per day, 305-day actual; total days milked during lactation; days nonlactating; age at calving; calving interval; days open, and number of times bred. Lactations were stratified from 1 to greater than 5 for comparison of variables. A matched case-control analysis was performed to assess the risk of clinical mastitis in BLV-infected cows. The retention of BLV antibody-negative and antibody-positive cows in the herd was compared. There were no significant trends in the means of production and reproductive variables between BLV antibody-negative and antibody-positive cows. The relative risk of clinical mastitis in BLV antibody-positive cows was 1.3, which was not significant (P greater than 0.05). Survivorship analysis over 3 years demonstrated no significant difference in the retention of BLV antibody-negative and antibody-positive cows in the herd. The BLV-infected cows did not have lower milk production, poorer reproductive efficiency, increased prevalence of mastitis, or lesser longevity in the herd than did noninfected cows.

Prevalence of bovine leukemia virus antibody in seven herds of Holstein-Friesian cattle.

Sera from 959 Holstein-Friesian cattle in 7 herds were tested for antibody to bovine leukemia virus, using the agar gel immunodiffusion test with glycoprotein antigen. Seropositive cattle were found in 6 herds; 120 (12.5%) were reactors. In 1 of the herds, the prevalence of seropositive cattle was significantly greater in purebred than in grade cows. Purebred cows raised on the premises and an overall antibody prevalence similar to purchased purebred cows. Purebred cattle more than 2 years old had a significantly greater prevalence of reactors than younger cattle. There was no difference in prevalence by sex.

Use of the MMPI in predicting completion and evaluating changes in a long-term alcoholism treatment program.

Veterans admitted to a 90-day alcoholism treatment program were administered the MMPI, and those who completed the program were retested before discharge. Completers differed significantly from noncompleters on the Pd scale. Although responses on most MMPI scales indicated improvement after treatment, those on the MacAndrew Alcoholism Scale were not affected. Neither the MacAndrew nor the Unitary Alcoholism Factor differentiated between completers and noncompleters.