Anti-actin specificity of human smooth muscle antibodies in chronic active hepatitis.

Thirty sera reacting by IFL technique in titres greater than or equal to 100 with smooth muscle fibres of rat stomach, rat renal glomeruli, and with the membrane region of thyroid cells were randomly chosen among sera sent in for routine testing of tissue antibodies. All sera but one were found to be derived from patients with chronic active hepatitis. The smooth muscle and other relevant cell staining were abolished after absorption of sera with actin, prepared from rabbit skeletal muscle and found to be homogeneous by SDS gel-electrophoresis and by electron microscopy. The actin anti-bodies were purified by precipitation of sera with F-actin and elution of the precipitates at acid pH. The purified antibodies stained all tissues in the same way as the original sera. In double immunodiffusion tests all thirty sera gave precipitation with actin. Thus, it was concluded that these broad-reacting SMA are directed against actin. The finding of high-titred SMA is of diagnostic value and supports the clinical diagnosis of active chronic hepatitis. In addition, anti-actin antibodies eluted from human sera are a suitable tool for studying actin-containing cellular structures.     

Three-dimensional tissue engineering of hyaline cartilage: comparison of adult nasal and articular chondrocytes

Adult chondrocytes are less chondrogenic than immature cells, yet it is likely that autologous cells from adult patients will be used clinically for cartilage engineering. The aim of this study was to compare the postexpansion chondrogenic potential of adult nasal and articular chondrocytes. Bovine or human chondrocytes were expanded in monolayer culture, seeded onto polyglycolic acid (PGA) scaffolds, and cultured for 40 days. Engineered cartilage constructs were processed for histological and quantitative analysis of the extracellular matrix and mRNA. Some engineered constructs were implanted in athymic mice for up to six additional weeks before analysis. Using adult bovine tissues as a cell source, nasal chondrocytes generated a matrix with significantly higher fractions of collagen type II and glycosaminoglycans as compared with articular chondrocytes. Human adult nasal chondrocytes proliferated approximately four times faster than human articular chondrocytes in monolayer culture, and had a markedly higher chondrogenic capacity, as assessed by the mRNA and protein analysis of in vitro-engineered constructs. Cartilage engineered from human nasal cells survived and grew during 6 weeks of implantation in vivo whereas articular cartilage constructs failed to survive. In conclusion, for adult patients nasal septum chondrocytes are a better cell source than articular chondrocytes for the in vitro engineering of autologous cartilage grafts. It remains to be established whether cartilage engineered from nasal cells can function effectively when implanted at an articular site.     

SKALP/elafin gene polymorphisms are not associated with pustular forms of psoriasis

Psoriasis is a multifactorial skin disease characterised by epidermal abnormalities and infiltration by lymphocytes and polymorphonuclear leukocytes (PMN). Skin-derived antileukoproteinase (SKALP), also known as elafin, is a potent inhibitor of human leukocyte elastase and proteinase 3, two PMN-derived proteinases implicated in tissue destruction and leukocyte migration. We have shown that, at least at the protein level, SKALP is significantly decreased in lesional skin of patients with pustular psoriasis compared with plaque-type psoriasis. This finding raised the possibility that SKALP could be one of the candidate genes for pustular forms of psoriasis. We therefore performed single strand conformation polymorphism (SSCP) analysis on the SKALP gene to screen for mutations/polymorphisms in the exons of 30 patients with plaque-type psoriasis, 15 patients with pustular psoriasis and 48 healthy controls. In exon 1 a polymorphism was detected at position + 43 relative to the translation start site, resulting in a substitution of threonine for alanine in the signal peptide. In the promoter region a dinucleotide repeat polymorphism was identified. Both polymorphisms were not associated with pustular psoriasis, or psoriasis in general. Our data indicate that the decrease in SKALP activity in pustular psoriasis is not caused by mutations in the coding region of the gene, and that there is no allelic association between pustular psoriasis and SKALP gene polymorphisms.     

Interinstitutional variations of sensitometric curves of radiographic dosimetric films

Depth and field size dependence of the sensitometric curves of radiographic films have been studied by various groups. Limited information is, however, available on the magnitude of the variations in sensitometric curves applied in clinical practice in different institutions. In this study we assessed in a systematic way the effect of the various parameters influencing the shape of the sensitometric curve: batch composition, irradiation conditions, film processing, and film scanning. Two types of film, Kodak X-Omat V and CEA TVS, were irradiated, processed, and analyzed in three different institutions. The interinstitutional variation of the sensitometric curves, expressed as the OD variation at 50 cGy, can be up to 32% and is mainly caused by differences in film processing and to a lesser degree to differences in batch composition, film scanning, and irradiation conditions. For the Kodak films, the average OD difference at 50 cGy between the three institutions is 17% as a result of differences in batch composition and 25% due to differences in processing conditions. For the CEA films these data are 6% and 24%, respectively. The long-term variation of the sensitometric curves of KODAK films in one institution was smaller than the differences in batch composition between the three institutions. The sensitometric curves of CEA films showed in one institution a large variation with time; the shape gradually varied from sigmoidal to quasilinear. By using relative OD values rather than absolute OD values, variations in sensitometric curves of KODAK films can be reduced to 2%. Consequently, one sensitometric curve is sufficient to derive relative dose values. If processing conditions are well controlled, it might therefore be advantageous to determine the absolute OD only at one or two dose values, in combination with a "universal" relative sensitometric curve.     

Antisense oligonucleotides and short interfering RNAs silencing the cyclin-dependent kinase inhibitor p21 improve proliferation of Duchenne muscular dystrophy patients’ primary skeletal myoblasts

Increased levels of the cyclin-dependent kinase inhibitor p21 associated with decreased myoblast proliferation may be involved in the dystrophic process in Duchenne muscular dystrophy (DMD). Therefore we are interested to improve the proliferation of primary myoblasts of DMD patients by a reduction in p21 using either antisense oligonucleotides (ASO) or short interfering RNAs (siRNA). After transient transfection of myoblasts in cell culture proliferation was analyzed using a 5-bromo-2-deoxyuridine assay comparing specific transfected cells with untransfected cells and cells transfected with scrambled ASO and luciferase siRNA, respectively. Four of five Dystrophin-deficient (Dys^–) cell culture samples revealed an increase in proliferation between 7% and 18% compared to untransfected cells and between 8% and 36% compared to cells transfected with scrambled ASO. Transfection with siRNA was performed for selected samples to determine whether siRNA is more effective in gene silencing than ASO. The increase in proliferation using luciferase siRNA as reference was comparable to or less than ASO data using scrambled ASO as reference. Using untransfected cells as reference, the increase in proliferation was higher for siRNA than ASO (20–47% vs. 7–18%), but the data must be carefully interpreted with respect to nonspecific effects on gene expression by siRNA. Our findings of transient p21 gene silencing represent a basis for viral vector-mediated drug-inducible p21 shRNA expression in Dys^– myoblasts which might enhance, prolong and regulate the proliferation effect.S. Endesfelder and A. Kliche contributed equally to this work     

Optimisation of the CT h4S bioassay for detection of human interleukin-4 secreted by mononuclear cells stimulated by phytohaemaglutinin or by human leukocyte antigen mismatched mixed lymphocyte culture

Limiting dilution analysis has been used in the context of allogeneic bone marrow transplantation to determine anti-recipient interleukin-2 (IL-2) producing helper T lymphocyte precursor (HTLp) frequencies, which in several studies have been predictive of graft-versus-host disease (GVHD). Recently high anti-recipient IL-4 producing HTLp frequencies have been reported and associated with a decreased risk of GVHD. The aim of the present study was to define the optimal conditions for combined determination of IL-2 and IL-4 producing anti-recipient HTLp frequencies. We have optimised the CT.h4S bioassay with regards to specificity, sensitivity, detection limit, and reproducibility. We have found the optimal assay conditions to be 1 x 10 (4) CT.h4S cells/well deprived of IL-4 for 24 h and preincubated for 7 h followed by 18 h of incubation with tritiated methyl-thymidine. In this setting the CT.h4S bioassay detects 5 pg/ml of human recombinant IL-4 with no detection of IL-2 in concentrations below 500 pg/ml. We have found 72 h of culture optimal for detection of IL-2 and IL-4 produced by human mononuclear cells (MNC) in response to stimulation with phytohaemaglutinin and for detection of IL-2 in human leukocyte antigen (HLA)-mismatched mixed leukocyte culture (MLC). An interindividual variation in cytokine accumulation was demonstrated for IL-4 but not for IL-2. With the use of 5x10(4) responder cells/well no IL-4 could be detected in HLA-mismatched MLC between days 1 and 16. The lack of IL-4 detection was not due to high amounts of soluble IL-4 receptor. With the use of 1x10(6) responder cells/well in HLA-mismatched MLC, we found limited IL-4 accumulation still increasing at day 12. We conclude that the CT.h4S bioassay is a reliable and specific method for quantification of IL-4 accumulation in cultures of human MNC. The difference in optimal timing for IL-2 (day 3) and IL-4 (>/=day 12) detection and evidence of very low IL-4 producing HTLp frequencies makes the relevance of a combined IL-2/IL-4 HTLp assay questionable.     

Blood Polymorphonuclear Leukocyte Activation in Atherosclerosis: Effects of Aspirin

The aim of the study was to demonstrate an activation of polymorpho-nuclear leukocytes (PMNs) in chronic progressive atherosclerosis (ATH). A group of patients with ATH, and a group of ATH patients under aspirin (ASA) therapy were compared with control persons without atherosclerotic alterations (healthy controls). Each group comprised 15 male age-matched subjects. The following inflammatory parameters related to PMN activities were measured: the polymorphonuclear leukocyte (PMN) blood count; blood PMN migration and reactive oxygen species release in vitro; the blood levels of PMN elastase, malondialdehyde, antibodies to oxidized LDL and soluble ICAM-1. In ATH patients, the PMN blood counts and the share of blood PMNs migrating upon platelet activating factor and leukotriene B4 stimulation were significnatly above the values of the healthy controls, while the other parameters were not significantly altered. ASA treatment attenuated the inflammatory response and reduced the differences between ATH and the healthy controls. It can be concluded that, in patients with chronic progressive atherosclerosis, PMNs are involved in the inflammatory process underlying the disease.     

Characterization of anti-acetylcholine receptor antibody activity in patients with anti-mitochondrial antibodies

Antibodies binding to the acetylcholine receptor of human skeletal muscle were found in patients with different kinds of anti-mitochondrial antibodies. In patients with primary biliary cirrhosis (PBC), the antibody activity was found in monomeric and pentameric IgM as well as in IgG, whereas patients with other kinds of anti-mitochondrial antibodies had anti-receptor antibodies of predominantly IgG class. Mice transfused with immunoglobulins from patients with PBC showed a reduction of skeletal muscle receptors comparable to that found in mice who had received immunoglobulins from myasthenia gravis (MG) patients. The antibody affinity was of the same order of magnitude in MG and PBC but antibodies with multiple affinities were more common in PBC. In PBC, the anti-receptor antibody associated idiotype repertoire was markedly different from that found in MG.