New insights into the pathogenesis of dilated cardiomyopathy: possible underlying autoimmune mechanisms and therapy

In the present study, autoimmune processes involved in the pathogenesis of dilated cardiomyopathy (DCM) are discussed. Genetic predisposition, persistent viral infection, and molecular mimicry have previously been described as the underlying mechanisms of the disease, and prevalence of autoantibodies (AABs) against several intra- and extracellular cardiotropic targets has been confirmed. These autoantibodies are able to disturb the normal physiological activity of the cardiomyocytes. They also could function as mediators in an activated immune system and direct a great deal of attention to injured tissue via (1) complement activation and (2) genesis of circulatory immunocomplexes (CICs) in association with self-antigens. The number as well as duration of accessible autoantigens or CICs seem to play an important role in activation of the antigen-presenting cells (APCs) and, consequently, promotion of autoimmunity. Since AABs play such a decisive role, their exclusion by immunoadsorption (IA) therapy has been discussed as a new approach in DCM treatment. Hitherto, all performed pilot studies using this approach have shown improvement in cardiac function and quality of life in the vast majority of treated DCM patients. The removal of circulating AABs may downregulate the autoimmune system, moderate the inflammatory signals, and hasten the recovery of the affected tissue.     

Temperature dependence of mutant mevalonate kinase activity as a pathogenic factor in hyper-IgD and periodic fever syndrome

Hyper-IgD and periodic fever syndrome (HIDS) and mevalonic aciduria are autosomal recessive disorders characterized by recurrent episodes of fever and generalized inflammation. Both syndromes are caused by specific mutations in the gene encoding mevalonate kinase (MK), resulting in a depressed enzymatic activity mainly due to reduced protein levels. We studied the effect of temperature on the activity of wild-type and several mutant MKs in fibroblasts. All fibroblast cell lines from HIDS patients and harbouring the common V377I MVK allele displayed substantially higher MK activities at 30 degrees C as compared to 37 degrees C. As shown by temperature inactivation experiments this resulted in a protein nearly as stable as in control cell lines, indicating that primarily the maturation of the protein is affected. Accordingly, when HIDS cell lines were cultured at 39 degrees C, MK activity decreased further. This triggered a compensatory increase in 3-hydroxy-3-methylglutaryl-CoA reductase activity, indicating that MK becomes progressively rate-limiting. A similar phenomenon occurs in vivo. MK activity in peripheral blood mononuclear cells drops 2-8-fold when HIDS patients experience febrile attacks. Our results suggest that minor elevations in temperature can set off a chain of events with MK becoming progressively rate-limiting, leading to a temporary deficiency of isoprenoid end-products, which induces inflammation and fever.     

Comparison of three methods to measure absolute cerebral hemoglobin concentration in neonates by near-infrared spectrophotometry

Three methods by which to determine absolute total cerebral hemoglobin concentration (tHb in micromol/L) by near-infrared spectrophotometry (NIRS) have evolved: (1) tHbo, requiring oxygenation changes and arterial oxygen saturation measurements as a reference using a relative NIRS algorithm, (2) tHbg, using a geometrical multidistance principle and (3) tHbgo, a combination of both. The aim of this study was to compare the three methods quantitatively. Sixteen clinically stable preterm infants with a mean gestational age of 29.6 (range of 25.1-36.4) weeks, birthweight of 1386 (680-2820) g and a postnatal age of 2.5 (0.5-6) days, who needed supplemental oxygen, were enrolled. The mean+/-standard deviation tHbg was 150.2+/-41.8 micromol/L (range of 61.6-228.9 micromol/L), the tHbo was 62.1+/-27.2 micromol/L (26.0-110.8 micromol/L) and the tHbgo was 89.3+/-45.6 micromol/L (26.5-195.9 micromol/L). The correlation coefficient among the three methods were tHbg and tHbgo r=0.736; tHbo and tHbgo r=0.938; tHbg and tHbo r=0.598. A multiple regression with variable selection by Mellow's C(p) showed, that tHbg was correlated to the birthweight, the postnatal age, the heart rate and the pCO2 (r(2)=0.588), tHbo and tHbgo were associated with the hemoglobin concentration in the blood, the mean arterial blood pressure and the pCO2 (r(2)=0.493 and 0.406, respectively). The three methods (tHbg, tHbo, and tHbgo) give systematically different tHb readings and large intersubject variability.     

Nutrient signalling in the regulation of human muscle protein synthesis

The mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) are important nutrient- and energy-sensing and signalling proteins in skeletal muscle. AMPK activation decreases muscle protein synthesis by inhibiting mTOR signalling to regulatory proteins associated with translation initiation and elongation. On the other hand, essential amino acids (leucine in particular) and insulin stimulate mTOR signalling and protein synthesis. We hypothesized that anabolic nutrients would be sensed by both AMPK and mTOR, resulting in an acute and potent stimulation of human skeletal muscle protein synthesis via enhanced translation initiation and elongation.
We measured muscle protein synthesis and mTOR-associated upstream and downstream signalling proteins in young male subjects ( n = 14) using stable isotopic and immunoblotting techniques. Following a first muscle biopsy, subjects in the 'Nutrition' group ingested a leucine-enriched essential amino acid–carbohydrate mixture (EAC). Subjects in the Control group did not consume nutrients. A second biopsy was obtained 1 h later. Ingestion of EAC significantly increased muscle protein synthesis, modestly reduced AMPK phosphorylation, and increased Akt/PKB (protein kinase B) and mTOR phosphorylation ( P < 0.05). mTOR signalling to its downstream effectors (S6 kinase 1 (S6K1) and 4E-binding protein 1 (4E-BP1) phosphorylation status) was also increased ( P < 0.05). In addition, eukaryotic elongation factor 2 (eEF2) phosphorylation was significantly reduced ( P < 0.05). Protein synthesis and cell signalling (phosphorylation status) was unchanged in the control group ( P > 0.05).
We conclude that anabolic nutrients alter the phosphorylation status of both AMPK- and mTOR-associated signalling proteins in human muscle, in association with an increase in protein synthesis not only via enhanced translation initiation but also through signalling promoting translation elongation.     

Neuronal Cell Death in Scrapie-Infected Mice Is Due to Apoptosis

Neuronal loss is a salient yet poorly understood feature in the pathology of transmissible spongiform encephalopathies (prion diseases). Cell culture experiments with neurotoxic prion protein fragments suggest that neuronal cell death in these diseases may be due to apoptosis. To test this hypothesis in vivo we used the in situ end-labeling (ISEL) technique and electron microscopy to study cell death in an experimental scrapie system in the mouse. ISEL, which relies on the incorporation of labeled nucleotides in fragmented DNA by terminal transferase, showed labeled nuclei in the brains and retinae of mice infected with the 79A strain of scrapie, whereas no labeling was observed in control animals. In the retina the highest numbers of labeled nuclei were found in the outer nuclear layer 120 days post infection followed by massive cell loss in this layer. In the brain, labeled nuclei were mainly found in the granular layer of the cerebellum of terminally ill mice. This corresponded to the presence of small dark nuclei with condensed and occasionally fragmented chromatin at the light and electron microscopical levels. Our results support the hypothesis that neuronal loss in spongiform encephalopathies is due to apoptosis. This may explain the almost complete absence of inflammatory response in prion diseases in the face of widespread neuronal cell death, and may also have therapeutic implications in the future.     

Fluorescence in situ hybridization reveals closely correlated results in cytological and histological specimens of hematological neoplasias compared to conventional cytogenetics.

OBJECTIVES: Fluorescence in situ hybridization (FISH) has become a useful tool to identify chromosomal aberrations in non-dividing cells. Numerous studies have compared chromosomal banding analysis (CBA) and FISH on fixed cultured bone marrow cells. However, up to now, there has been no study comparing two main sources of diagnostic material, i.e. bone marrow aspirates and trephine biopsies. We therefore analyzed these materials by FISH in comparison with CBA. METHODS: CBA revealed chromosomal aberrations in 18 patients suffering from myelodysplastic syndrome (n = 13), acute myeloid leukemia (n = 3), or chronic myeloproliferative syndrome (n = 2). FISH was performed on fixed cultured bone marrow cells, aspirates and trephine biopsies from each patient. RESULTS: Percentages of aberrant cells in the different materials correlated highly with Pearson values of 0.909 for biopsy/fixed cultured cells (p < 0.001), 0.830 for biopsy/aspirate (p < 0.001) and 0.768 for aspirate/fixed cultured cells (p < 0.001). Moreover, in bone marrow biopsies peritrabecular and central intertrabecular areas yielded very similar FISH results with a high correlation (r = 0.968, p < 0.001). FISH revealed a lower proportion of aberrant cells than CBA in 90% of the specimens. CONCLUSIONS: In summary, the different materials available for the FISH examination are comparable in sensitivity and show similar quantitative results. Therefore, the use of biopsy sections for the routine FISH examination of chromosomal abnormalities is a valid method.     

Knowledge about asthma and COPD: associations with sick leave, health complaints, functional limitations, adaptation, and perceived control

We sought to investigate associations between knowledge about the disease and sick leave, health complaints, functional limitations, adaptation and perceived control. Patients with asthma (n = 101) and COPD (n = 64) underwent lung function tests and completed questionnaires. In addition, all were asked the question: ‘what is the diagnosis of your disease?’, with the response categories: ‘asthma’ and ‘COPD (chronic bronchitis or emphysema)’. Thirty-five percent of the asthma patients and 30% of the COPD patients did not know their correct diagnosis. Sick leave was not associated with knowledge about the disease in asthma and COPD. In asthma, much knowledge about management of the disease was associated with better adaptation (P = 0.01) and less perceived control over health by external factors (P = 0.02). Knowing the correct diagnosis was associated with less control over health by powerful others (P = 0.02). For COPD, more knowledge about management of the disease was associated with better adaptation (P = 0.02) and less control over health by internal factors (P = 0.01). Knowing the correct diagnosis was associated with less control over dyspnea at work (P = 0.01).     

Advanced 1H Solid‐State NMR Spectroscopy on Hydrogels, 1

Summary: The nature of the pH dependent collapse of poly(methacrylic acid) (PMAA) hydrogels is investigated using recent ¹H solid‐state NMR methods. In aqueous solution, PMAA changes from an expanded conformation at high pHs to a compact contracted form at low pHs, where hydrogen bonds play a central role. In solid‐state ¹H NMR spectra, recorded under fast magic angle spinning (MAS), dried PMAA samples previously collapsed at low pHs show characteristic signals in the spectral region of the carboxylic acid protons. With the aid of 2D ¹H‐¹H double‐quantum (DQ) MAS NMR spectra, three signals can be distinguished at 8, 10.5 and 12.5 ppm, which are attributed to free carboxylic groups and two different types of hydrogen bonded forms, respectively. The 12.5 ppm signal arises from the hydrogen bond with the shortest H? H distance, corresponding to the form that is most stable with respect to increasing temperature and pH. The weaker hydrogen‐bonded form (with a signal at 10.5 ppm) requires a slightly lower pH, while the free acid signal (at 8 ppm) emerges under the most acidic medium. Moreover, the stabilities of the hydrogen‐bonded carboxylic acid dimers can be inferred from the proton‐proton distances within the dimers, i.e. (275 ± 5) pm and (295 ± 15) pm for the protons at 12.5 and 10.5 ppm, respectively, which are determined by means of DQ MAS sideband patterns. Both the stability of the hydrogen bonds and the acidity of the protons may be related to the stereochemistry and the conformation of the PMAA chains.

     

Isolation of human immunodeficiency virus (HIV) from plasma during primary HIV infection

Human immunodeficiency virus (HIV) has been isolated from plasma in 6 of 7 patients showing clinical symptoms of a primary HIV infection. Parallel cultures from peripheral blood mononuclear cells (PBMC) yielded virus in 5 patients. In one case, virus could only be isolated from the cerebrospinal fluid but not from peripheral blood. Detectable viremia was transient and preceded the appearance of HIV specific antibodies. After cessation of acute symptoms, the frequency of HIV isolations was similar to that of asymptomatic carriers (23 and 26%, respectively). The role of the immune response in terminating detectable viremia remains to be established.     

Lymphocytic choriomeningitis virus

Lymphocytic choriomeningitis (LCM) virus-specific complement-fixing (CF) antigen (ECFA) has been solubilized, concentrated, and partially purified. When inoculated together with Freund's adjuvant, ECFA induced CF antibody but not neutralizing antibody or protective immunity. By itself it boosted pre-existing CF antibody but not neutralizing antibody. In double diffusion tests one line developed between ECFA and its antiserum, and a corresponding line became visible when ECFA interacted with an antiserum directed against all LCM virus-specific antigens. Absorption of either serum with ECFA abolished all ECFA-precipitating qualities. Ouchterlony tests also revealed that ECFA prepared from cells and tissues of various species is immunologically identical. By a variety of procedures ECFA was not found to be represented on the surface of either the virion or the infected cell. When purified infectious LCM virus was disrupted, a CF antigen corresponding immunologically to ECFA was set free. In double diffusion tests this antigen gave a line of identity with ECFA. Thus, ECFA appears to be an internal component of the infectious LCM virus.Part of the work reported here has been published in preliminary communications [8, 10, 24, 25].