Grafting of fibroblasts isolated from the synovial membrane of rheumatoid arthritis (RA) patients induces chronic arthritis in SCID mice-A novel model for studying the arthritogenic role of RA fibroblasts in vivo

The objective of this study was to verify whether isolated rheumatoid arthritis (RA) synovial fibroblasts induce chronic arthritis in SCID mice, in analogy to whole tissue pieces. Fibroblasts were isolated from the synovial membrane of four RA patients (or controls) by out-growth and repeated-passage culture. Following flow-cytometry characterization, 2x10(6)cells were transferred into the left knee joint of SCID mice. The development of arthritis was assessed by joint swelling and histological changes. Human and murine cytokines were measured in vitro in co-cultures (or Transwelltrade mark systems) of human and murine cells. Purified RA synovial fibroblasts, but not healthy synovial or skin fibroblasts, induced hu/mu arthritis within 6 weeks. In-vitro secretion of murine and human interleukin(IL)-6, as well as murine tumour necrosis factor (TNF)-alpha, indicated cross-activation between murine macrophages and human RA fibroblasts. Soluble-factor mechanisms proved more effective than cell-contact mechanisms. Purified RA fibroblasts can, alone, induce hu/mu SCID arthritis. The cytokine profile suggests that xenogeneic interaction between human fibroblasts and murine macrophages may determine the sequence of events leading to hu/mu arthritis.     

In vitro models for the assessment of inflammatory and immuno-modulatory effects of the volatile organic compound chlorobenzene

An in vitro cell culture system based on an air/liquid culture technique was developed which allows a direct exposure of cells to volatile chemicals without medium coverage. For the establishment of the experimental system, chlorobenzene was used as a model compound. Chlorobenzene is a volatile organic compound which is mainly used as a solvent. Beside other adverse health effects, chlorobenzene exposure has been shown to be associated with respiratory tract irritations, Th2 differentiation, and allergic sensitizations. Human peripheral blood mononuclear cells (PBMC) and lung epithelial cells (A549) were exposed to chlorobenzene via gas phase for 20 h. Additionally, PBMC were incubated with culture supernatants from exposed lung epithelial cells. High chlorobenzene concentrations (100 g/m(3)) induced IL-8 production in A549 cells, whereby lower concentrations (10 microg/m(3)-1 g/m(3)) stimulated the secretion of the monocyte chemoattractant protein-1 (MCP-1). A direct effect of chlorobenzene on the cytokine secretion of PBMC was not found. However, if PBMC were incubated with culture supernatants of exposed lung cells, an enhanced production of the Th2 cytokine IL-13 was observed. This induction was prevented in the presence of an anti-MCP-1 antibody. Our data suggest that chlorobenzene induces the production of inflammatory mediators in lung cells. The primary chlorobenzene caused release of MCP-1 in lung epithelial cells may secondarily result in a Th2 differentiation in T lymphocytes. These findings may contribute to the understanding of how chlorobenzene mediates the development of inflammatory reactions in the airways and contributes to the development of an allergic reactivity.     

Detection of low-frequency antigen-specific IL-10-producing CD4(+) T cells via ELISPOT in PBMC: cognate vs. nonspecific production of the cytokine

Single-cell resolution cytokine ELISPOT assays are increasingly used to gain insights into clonal sizes of type 1 and type 2 effector T cell populations in vivo. However, ELISPOT assays permitting monitoring of regulatory IL-10-producing T cells have so far not been established. Unlike IFN-gamma, IL-2, IL-4, and IL-5 assays performed on PBMC in which the recall antigen-induced cytokine spots are T cell-derived, we show here that in such assays IL-10 is primarily monocyte-derived. T cell-derived IL-10 spots were 80 x 10(3) microm(2) in size, seven times larger than spots produced by monocytes, and B cells produced even smaller spots. Based on spot size gating and the use of B cells as APC, we have established test conditions that permit measurement of cognate IL-10 production by low-frequency antigen-specific T cells. IL-10-producing PPD-specific CD4(+) T cells were detected in frequencies comparable to IFN-gamma-secreting CD4(+) T cells in tuberculosis patients, but not in uninfected healthy control individuals. In contrast, IL-10-secreting CD4(+) T cells specific for a panel of recall antigens could not be detected in frequencies >1/100,000 in healthy individuals whose CD4(+) cells responded to these antigens with type 1 or type 2 cytokine production in the 1:100,000-1:1000 frequency range. Therefore, the induction of IL-10-producing T cells seems to be under tighter control than that of Th1/Th2 cells, apparently confined to states of chronic immune stimulation. Access to low-frequency immune monitoring of IL-10-producing T cells will provide new insights into the role of regulatory T cells in health and disease.     

Selective induction of metabolic activation programs in peritoneal macrophages by lipopolysaccharide substructures.

The structural elements of Salmonella typhimurium lipopolysaccharides (LPS) that are able to stimulate peritoneal macrophages to produce increased amounts of prostaglandin E2, ornithine, and citrulline, agents known to modulate immune responses, are described. Two different incomplete lipid A structures which lack the carbohydrate portion, the nonhydroxylated fatty acids lauric acid and myristic acid (lipid A precursor IB), and additional palmitic acid (lipid A precursor IA) stimulated increased prostaglandin E2 synthesis but were unable to augment ornithine and citrulline production at concentrations of up to 0.5 microgram/ml. Acyl-deficient smooth LPS containing lipid A precursors IA and IB substituted by the complete carbohydrate region were able to augment prostaglandin E2 and ornithine production but failed, even at a high concentration (0.5 microgram/ml), to stimulate citrulline production. Moreover, Re glycolipids and smooth intact LPS containing the lipid A region with 3-acyloxyacyl residues possessed all of the structural requirements to induce increased prostaglandin E2, ornithine, and citrulline synthesis. Finally, all of the LPS structures, including lipid A precursors IA and IB stimulated, in combination with gamma interferon, production of citrulline with similar efficiencies. These results demonstrate that LPS contains various substructures including regions of the carbohydrate and lipid A structure that can deliver signals for the activation of peritoneal macrophages. Signals for partial activation of macrophages to produce prostaglandins and ornithine can be delivered by acyl-deficient LPS structures. In contrast, full activation of macrophages to produce citrulline requires an additional signal that is delivered by 3-acyloxyacyl residues of the lipid A region or gamma interferon.     

Improvements in Gene Therapy

Gene therapy is an interesting approach for the correction of defective genes, the treatment of cancer and the introduction of immunomodulatory genes. Various techniques for gene transfer into cells or tissues have been developed within the last decade; these can be divided generally into viral and nonviral gene transfer systems. Nonviral techniques include the liposome- or gene gun-mediated introduction of therapeutic genes; however, the efficiency of gene transfer by these applications is still very low. In contrast, viruses have optimised their strategies for efficient infection of virtually any cell type in a mammalian organism. The genetic modification of genomes from different virus families (Adenoviridae, Retroviridae, Herpesviridae) led to the development of gene therapy vectors with a similar capacity to infect cells or tissues as that of wild type viruses. In contrast to wild type viruses, gene therapy vectors are engineered to transfer therapeutic genes into the target cells or tissues. In addition, they have lost their capacity for replication in target cells, because of the removal of essential genes, which allows replication only in specialised packaging cell lines engineered for the production of recombinant viruses. Despite considerable progress over the past decade in the generation of gene transfer systems with reduced immunogenic properties, the remaining immunogenicity of many gene therapy vectors is still the major hurdle, preventing their frequent application in clinical trials. Recombinant adenoviruses have been shown to be promising vectors for gene therapy, since they are able to transduce both quiescent and proliferating cells very efficiently. However, a major disadvantage of adenoviral vectors lies in the activation of both the innate and adaptive parts of the recipient's immune system when applied in vivo. The inflammatory responses induced by adenovirus particles can be very strong and can be fatal in patients treated with these adenoviral constructs. Therefore, many experiments have been performed in the effort to prevent these inflammatory responses mediated by adenoviral particles. The depletion of cell populations responsible for these inflammatory responses as well as the application of immunosuppressive drugs have been investigated. Moreover, the generation of less immunogenic adenoviral vectors by further genetic modification within the adenoviral genome has led to vectors with reduced immunogenic properties. Both strategies to reduce inflammatory responses against adenoviral particles are discussed in this review.     

Application of capillary electrophoresis in clinical chemistry: the clinical value of urinary modified nucleosides

Urinary modified nucleosides were determined by capillary electrophoresis using a 300 mM SDS-25 mM sodium tetraborate-50 mM sodium dihydrogenphosphate buffer. The nucleosides were extracted from urine by phenylboronate affinity gel chromatography. In cancer patients the levels of the modified nucleosides are generally elevated. By an artificial neural network method breast cancer patients were differentiated from normal individuals, which indicates that the modified nucleosides could be of clinical value as tumor markers.     

Linking: a dynamic electrophysiologic phenomenon in macroreentry circuits

The term "linking" has been used specifically to describe the mechanism for perpetuation of functional anterograde bundle branch block: namely, repetitive transseptal retrograde concealed penetration by impulses propagating along the contralateral bundle. We present selected examples that demonstrate tht linking-type phenomena actually have a wide spectrum of expression in human macroreentry circuits, particularly those incorporating either the bundle branches and His bundle or the normal pathway and Kent bundle. The examples presented are as follows: (1) persistent retrograde functional conduction delays in the His-Purkinje system during right ventricular pacing, (2) anterograde Kent bundle condution at rapid rates, dependent on prior block in the normal pathway, (3) persistent anterograde functional infra-His block of atrial impulses during rapid ventricular pacing in the presence of a retrogradely conducting accessory pathway, and (4) transient advancement of His activation with ventricular fusion complexes during overdrive ventricular pacing of bundle branch reentrant tachycardia. Based on these examples, we characterize linking as a generalized electrophysiologic phenomenon in which each successive impulse entering a macroreentry circuit propagates preferentially along one limb because of functional block in the contralateral limb resulting from the effects of the prior impulse. It is proposed that such functional block may be dynamically maintained either by repetitive impulse interference, which perpetuates local refractoriness (examples No. 1 to 3), or by repetitive impulse collision (example No. 4). The general conceptual scheme outlined can be applied to specific electrophysiologic phenomena associated with a wide variety of reentry circuits in man.     

Spironolactone as a source of interference in commercial digoxin immunoassays

Eight commercial digoxin immunoassay methods were tested in 17 subjects taking spironolactone (but not digoxin) to evaluate cross-reactivity from parent drug and/or metabolites. Four of these methods showed significant (up to 1.9 nmol/L) and variable "apparent digoxin" concentrations, despite the absence of digoxin in the drug regimen. The results suggest that clinical laboratories require a knowledge of their method with respect to spironolactone-related cross-reactivity and should exercise caution when interpreting digoxin results where spironolactone is coadministered. Further, the presence of concurrent renal and/or hepatic impairment could delay clearance of spironolactone metabolites (as well as digoxin metabolites and endogenous substances) and further distort a genuine digoxin result.     

Chest wall moulages for radiotherapy of resected breast cancer with fast electrons: comparative tests of different materials

Irradiation of the thoracic wall with high-speed electrons is one of the standard methods of prophylaxis and therapy of local recurrences and cutaneous metastases of an operated mammary carcinoma. The surface dose, however, is only 85% of the maximum dose, due to the depth dose curve of the electron beams with the preponderantly applied energy of 7MeV. This is a poor value, since most of all recurrences appear near to the surface and so the risk of giving an insufficient dose is involved. The dose distribution could be essentially improved by the use of moulages on the chest. These moulages were made of different materials which were tested and compared with respect to their suitability for radiotherapeutic purposes. The best materials proved to be "Urgo-Plastan" (manufacturer: Holphar, Sulzbach) and "Orthoplast" (manufacturer: Johnson & Johnson, Düsseldorf). Both materials are synthetic substances which after heating can easily be adapted to the body shape and which offer a good stability, little inconvenience for the patient and a relative easy handling. With these moulage materials, the surface dose is increased to 98% ("Urgo-Plastan") and 99% ("Orthoplast") of the maximum dose.