Analog validation of German-language symptom validity tests and the influence of coaching.

Although symptom validity testing is an integral part of the repertory of neuropsychologists in a number of countries, this is not yet true for Germany. The German adaptations of two effort tests, the Medical Symptom Validity Test (MSVT) by Green and the Amsterdam Short-Term Memory Test (ASTM) by Schmand et al., were investigated with a German-language sample. An analog study was performed with 18 healthy experimental malingerers and 18 controls with a mean age of 25.4 years. The scenario contained detailed information about mild post-traumatic cognitive impairment, as well as an explicit warning against symptom exaggeration. In addition to MSVT and ASTM, the Trail Making Test (TMT), the Complex Figure Test (CFT), and Digit Span were performed. Half of the sample were also given Rey's 15-Item-Test (FIT). Both groups were significantly different in all effort and performance measures, with the exception of the ratio TMT-B:TMT-A. With MSVT and ASTM, correct classification of group membership was between 97 and 100%. For the ratio TMT-B:TMT-A, there was a considerable overlap in the test scores for the two groups and the sensitivity of the FIT was too low. Although the ASTM and the MSVT were identified by a number of subjects as possible effort measures, both tests obtained very good results within this analog design.     

Up-regulation of cathepsin X in Helicobacter pylori gastritis and gastric cancer

Recently, we identified increased cathepsin X expression in H. pylori-infected gastric mucosa. Here, we describe further up-regulation in gastric cancer and report on the role of inflammatory cytokines required for cathepsin X up-regulation in H. pylori-infected gastric mucosa, as well as on consequences for cellular invasion. Biopsy specimens were taken from the antrum, corpus and cardia of H. pylori-infected and non-infected patients. Gastric cancer samples were obtained from patients undergoing gastric surgery. Cathepsin X was detected in gastric mucosa by quantitative real-time RT-PCR, western blotting and immunohistochemistry. Induction of cathepsin X expression in epithelial and inflammatory cells caused by H. pylori infection was tested in in vitro contact and non-contact co-cultures of AGS cells and monocytic cells. Patients with H. pylori gastritis showed significantly higher cathepsin X mRNA (2.5-fold) and protein (1.6-fold) expression than H. pylori-negative patients. Cathepsin X was also up-regulated in gastric cancer (3-12-fold) compared to non-neoplastic mucosa. Cathepsin X was predominantly expressed by macrophages in the mucosal stroma and in glands of the antral mucosa. In addition, tumour cells stained for cathepsin X in 26 (68%) patients with gastric carcinoma. In general, staining was significantly more common (20 vs. 6 patients) and more intense (3.55 vs. 0.83) in intestinal type gastric cancer than in the diffuse type. In vitro cell culture experiments revealed that intercellular signalling between pathogenicity island (PAI)-positive H. pylori-infected epithelial cells and macrophages via soluble factors in the culture medium seems to be responsible for increased expression of cathepsin X in monocytes. Using antisense oligonucleotides, cathepsin X up-regulation was directly associated with higher invasiveness in vitro. Although no correlation of cathepsin X expression and TNM stage was found, our study demonstrates that cathepsin X plays a role not only in the chronic inflammation of gastric mucosa but also in the tumourigenesis of gastric cancer.     

Morphological detection and quantification of lipoprotein(a) deposition in atheromatous lesions of human aorta and coronary arteries

Summary Lipoprotein(a), as an atherogenic particle, represents an independent risk factor for coronary heart disease. In the present study the morphological distribution of apoprotein (a) and apoprotein B within the arterial wall is described. Apoprotein B, a constituent of very low-density lipoprotein, low-density lipoprotein and lipoprotein(a) has previously been demonstrated in atheromatous lesions. Lipoprotein(a) possesses an additional protein, designated apoprotein (a). Autopsy material (n=74) from the left coronary artery and from the thoracic aorta has been examined by means of immunohistochemistry and both apoprotein (a) and apoprotein B were detected, primarily associated with the extracellular matrix and accumulating in lesions in the arterial wall. The staining pattern for both antigens was almost always found to be congruent, suggesting that the detection of (a)-antigen has to be attributed at least in part to the presence of lipoprotein(a). It is concluded that both low-density lipoprotein and lipoprotein(a) have an important role in the pathogenesis of atherosclerosis.     

The effect of dietary supplementation with n-3 polyunsaturated fatty acids on the synthesis of interleukin-1 and tumor necrosis factor by mononuclear cells

We examined whether the synthesis of interleukin-1 or tumor necrosis factor, two cytokines with potent inflammatory activities, is influenced by dietary supplementation with n-3 fatty acids. Nine healthy volunteers added 18 g of fish-oil concentrate per day to their normal Western diet for six weeks. We used a radioimmunoassay to measure interleukin-1 (IL-1 beta and IL-1 alpha) and tumor necrosis factor produced in vitro by stimulated peripheral-blood mononuclear cells. With endotoxin as a stimulus, the synthesis of IL-1 beta was suppressed from 7.4 +/- 0.9 ng per milliliter at base line to 4.2 +/- 0.5 ng per milliliter after six weeks of supplementation (43 percent decrease; P = 0.048). Ten weeks after the end of n-3 supplementation, we observed a further decrease to 2.9 +/- 0.5 ng per milliliter (61 percent decrease; P = 0.005). The production of IL-1 alpha and tumor necrosis factor responded in a similar manner. Twenty weeks after the end of supplementation, the production of IL-1 beta, IL-1 alpha, and tumor necrosis factor had returned to the presupplement level. The decreased production of interleukin-1 and tumor necrosis factor was accompanied by a decreased ratio of arachidonic acid to eicosapentaenoic acid in the membrane phospholipids of mononuclear cells. We conclude that the synthesis of IL-1 beta, IL-1 alpha, and tumor necrosis factor can be suppressed by dietary supplementation with long-chain n-3 fatty acids. The reported antiinflammatory effect of these n-3 fatty acids may be mediated in part by their inhibitory effect on the production of interleukin-1 and tumor necrosis factor.     

External quality control assessment in PCR diagnostics of dengue virus infections

BACKGROUND: Increased travelling to countries endemic for dengue fever (DF) demands efficient laboratory diagnostics. Nucleic acid amplification techniques (NAT) are now frequently used for rapid diagnosis of imported viral diseases. Different PCR systems are available. OBJECTIVES: In order to assess the quality of molecular diagnostics of dengue virus infections, an external quality assurance (EQA) in PCR diagnostics was conducted. Study design: A panel of 10 human plasma samples was prepared and spiked with dengue virus types DEN-1 to DEN-4. In addition, a 10-fold dilution series (1:10-1:10(4) ) of DEN-3 virus was included. The panel was pre-tested by nested RT-PCR, in-house real-time PCR, and a commercial real-time PCR kit. The samples were inactivated by gamma irradiation and shipped in freeze dried state. Thirteen laboratories, within the European network for the diagnostics of imported viral diseases (ENIVD) took part using either single-round, nested, or real-time RT-PCR methods. Two laboratories used two methods in parallel, summarising up to 15 comparable results. RESULTS: 33-100% correct results were achieved. All laboratories detected DEN-2 correctly, followed by DEN-1 (14 positive results of 15), DEN-3 (12/15) and DEN-4 (11/15). Testing of the serial dilution revealed low sensitivity in many labs, with results ranging from 33 to 80% of correctly tested samples. CONCLUSION: The EQA gives a feedback of the quality of the RT-PCR system used by each respective laboratory. The different test systems and amplification conditions demonstrate the importance of external quality control measures.     

A modified chorioallantoic membrane (CAM) assay for qualitative and quantitative study of growth factors

Summary The application of Thermanox tissue culture coverslips to the day 9 CAM of the chick causes constant effects beneath the carrier after 3 days, and these are associated with a change in the blood vessel pattern. Histological sections show enormous thickening of the CAM in the reactive areas. The stroma of the CAM shows fibrocyte proliferation, leucocyte infiltration, and clusters of dispersed ectodermal epithelial cells exhibiting signs of necrosis. The latter obviously cause a strong vascular response. The same effects are seen when the Thermanox discs are applied at day 11. Following application on day 12 a positive or negative response to the carrier is observed, whereas on day 13 no such carrier effects are seen. The only remaining effect is compression of the intra-ectodermal capillary plexus of the CAM. This can macroscopically be seen after peroxidase staining of the blood vessels. The effect of 5 l PBS dried on the Thermanox disc and applied to the day 13 CAM is to cause, after 3 days, hyperosmotic damage to the ectodermal epithelium, which becomes overgrown by fibrocytes. We found dose-dependent effects of salt-free human bFGF applied to the day 13 CAM. The first and main effect is fibrocyte proliferation (0.5 g). New capillaries appear with higher doses, but are not as frequent as would be expected for an angiogenic substance (1.25–2.5 g). Also with higher doses additional hyperplasia of the endodermal (3.75 g) and ectodermal (5 g) epithelium can be seen. The latter might be a non-specific hyperosmotic effect. Leucocytes are regularly present within the reactive areas. When salt-free angiogenin is applied to the day 13 CAM, some effects appear with doses of 4.6 g and more. The ectodermal epithelium of the reactive areas is discontinuous, exhibiting signs of necrosis. It is overgrown by parallel fibrocytes. Whether this is a non-specific hyperosmotic effect, or indicates enhancement of invasive growth, calls for further investigation.     

Analysis of cell type-specific responses mediated by the type IV secretion system of Helicobacter pylori

Helicobacter pylori persistently infects the human stomach and can cause gastritis, gastric ulceration, and gastric cancer. The type IV secretion system (TFSS) of virulent H. pylori strains translocates the CagA protein, inducing the dephosphorylation of host cell proteins and leading to changes in the morphology or shape of AGS gastric epithelial cells. Furthermore, the TFSS is involved in the induction of proinflammatory cytokines. While the H. pylori genes required for TFSS function have been investigated systematically, little is known about possible host cell factors involved. We infected 19 different mammalian cell lines individually with H. pylori and analyzed CagA translocation, dephosphorylation of host cell proteins, chemokine secretion (interleukin-8 and macrophage inflammatory protein 2), and changes in cellular phenotypes. Our results demonstrate that not only bacterial but also host cell factors determine the cellular response to infection. The identification of such unknown host cell factors will add to our understanding of host-pathogen interactions and might help in the development of new therapeutic strategies.     

Detection of cytomegaloviral DNA in human milk cells and cell free milk whey by nested PCR

Human cytomegalovirus (HCMV) DNA can be detected in different compartments of human milk. A protocol for the preparation of milk whey free of fat and cells for the detection of human cytomegalovirus (HCMV) by nested PCR is presented. This is based upon the experience of the separation of more than 200 milk specimens of healthy seropositive breast feeding mothers. HCMV DNA could be detected in freshly centrifuged and filtrated milk whey specimens without contamination by cellular DNA. In limiting dilution experiments using HCMV plasmid DNA, the effect of different DNA extraction procedures from native milk and milk whey on the detection limit of cytomegaloviral DNA was demonstrated. About 200 viral genome equivalents/ml in milk whey or native milk were detectable by classical organic phenol/chloroform extraction or a spin column method, respectively. The detection of viral DNA in milk cells depended on a minimum number of milk cells (10⁵–2×10⁵) available for DNA extraction. In contrast to the findings of cytomegaloviral DNA in native sera or plasma of immunosuppressed patients we failed to amplify low level viral DNA from native breast milk by nested PCR due to an inhibition of Taq polymerase by lipid components. Finally, the course of cell associated and cell free DNAlactia was monitored. Analyzing sequential milk specimens, in some cases the presence of HCMV DNA in colostrum could be demonstrated. DNAlactia of milk cells and whey was partially discordant. Onset (week 1–4 after delivery) and duration (2 weeks up to more than 3 months) of DNAlactia showed distinct individual patterns. The methods described, allow further analysis of the mechanisms involved in the postnatal HCMV transmission by breast feeding seropositive mothers.     

Autoimmunity and inflammation due to a gain-of-function mutation in phospholipase C gamma 2 that specifically increases external Ca2+ entry

The identification of specific genetic loci that contribute to inflammatory and autoimmune diseases has proved difficult due to the contribution of multiple interacting genes, the inherent genetic heterogeneity present in human populations, and a lack of new mouse mutants. By using N-ethyl-N-nitrosourea (ENU) mutagenesis to discover new immune regulators, we identified a point mutation in the murine phospholipase Cg2 (Plcg2) gene that leads to severe spontaneous inflammation and autoimmunity. The disease is composed of an autoimmune component mediated by autoantibody immune complexes and B and T cell independent inflammation. The underlying mechanism is a gain-of-function mutation in Plcg2, which leads to hyperreactive external calcium entry in B cells and expansion of innate inflammatory cells. This mutant identifies Plcg2 as a key regulator in an autoimmune and inflammatory disease mediated by B cells and non-B, non-T haematopoietic cells and emphasizes that by distinct genetic modulation, a single point mutation can lead to a complex immunological phenotype.