Treatment of renal amyloidosis with etanercept in tumour necrosis factor receptor-associated periodic syndrome.

OBJECTIVE: To describe the effect of Etanercept treatment in systemic AA amyloidosis in tumour necrosis factor receptor-associated periodic syndrome (TRAPS). METHODS: Etanercept therapy was given to a 27 year old woman, with systemic amyloidosis and nephrotic syndrome, and to her 51 year old father, also affected by TRAPS, who had previously undergone renal transplant for amyloidosis. Serum SAA levels, plasma cytokines, glomerular filtration rate and serum amyloid P scanning were monitored. RESULTS: Etanercept treatment resulted in initial clinical resolution of nephrotic syndrome in the 27 year old female. Both subjects demonstrated improvements in GFR and initial reduction or stabilisation of amyloid deposits on SAP scanning. CONCLUSION: Etanercept may reverse or slow the progression of systemic AA amyloidosis in subjects with C33Y TNFRSF1A mutation. Treatment may however need to be continuous and life-long to prevent progression to end stage disease.

Assessment of Be1 and Be2 cells in systemic lupus erythematosus indicates elevated interleukin-10 producing CD5+ B cells.

Recent studies indicate that normal B cells can be primed to differentiate into two distinct cytokine-secreting effector subsets, Be1 and Be2. The aim of this study was to analyse, for the first time, Be1 and Be2 cells at the single cell level in SLE patients using the recently developed technique of flow cytometry for intracellular cytokines. Peripheral blood mononuclear cells (PBMC) from SLE patients and age- and sex-matched normal controls were cultured for 24 h in the presence or absence of phorbal myristate acetate and ionomycin (PMA/I) or lipopolysaccharide (LPS). The production of type I (IFN-gamma, IL-2) and type 2 (IL-4, IL-5, IL-6, IL-10, IL-13) cytokines by B cells (and IL-10 production by fractionated CD5+ and CD5- B cells) was investigated using an intracellular cytokine staining technique and flow cytometry. In the absence of PMA/I stimulation, the percentage of B cells from SLE patients was significantly lower than those of normal subjects and significantly more SLE B cells spontaneously produced IL-10 than controls. Moreover, CD5+ B cells from SLE patients were enriched for cells with signs of previous in vivo activation and for high levels of IL-10 production. A significant positive correlation was observed between the percentage of IL-10- and IL-6-producing PMA/I-stimulated B cells in SLE patients, but not in controls. There were no significant differences in the production of other cytokines by B cells of SLE patients and normal subjects. In conclusion, a general alteration of type 1 and type 2 cytokine production by B cells is not observed in SLE patients. The role of B cell cytokines in the pathogenesis of SLE appears to be exerted by elevated secretion of in vivo IL-10, which may play an important role in the immune dysregulation observed in SLE patients. Moreover, the cross regulation of IL-10 and IL-6 is disrupted in SLE patients.

Retroviral activity in Behçet's syndrome and systemic lupus erythematosus detected by a PCR-based reverse transcriptase assay.

The objective of this study was to assess retroviral activity in Behçet's syndrome (BS) and systemic lupus erythematosus (SLE) patients. Serum and peripheral blood mononuclear cells (PBMC) were obtained from patients and normal volunteers and PBMC cultured with and without phytohaemagglutinin stimulation. Reverse transcriptase (RT) activity in serum and culture supernatants was measured using a sensitive polymerase chain reaction-based assay. An RT activity above the levels in normal controls was detected in a minority of patients with BS (2/15) and SLE (1/13) and was typically present in all three types of sample. Elevated levels of RT activity were not detected in follow-up samples from the two BS patients. Our findings indicate that elevated RT activity is present in only a minority of patients with BS and SLE. Simultaneously elevated activity in all three sample types implicates PBMC as the source of this retroviral activity.

Electrical stimulation of transforming growth factor-beta 1 secretion by human dermal fibroblasts and the U937 human monocytic cell line.

The in vitro effects on human dermal fibroblasts and the U937 human monocytic cell line of three phases of electrical microcurrents generated by the ACE Stimulator were investigated. The growth and viability of growing and confluent dermal fibroblasts were not directly influenced by the separate microcurrent phases. One form of microcurrent (designated phase 1) stimulated both dermal fibroblasts and U937 cells to secrete transforming growth factor-beta 1 (TGF-beta 1), which is an important regulator of cell-mediated inflammation and tissue regeneration, but none of the three phases stimulated secretion of the pro-inflammatory cytokine interleukin-6 by U937 cells. The stimulation of TGF-beta 1 secretion in these experiments was not dramatic (a median increase over control levels of 20-30%), although it could be biologically significant.

Quantitative and qualitative analysis of the balance between type 1 and type 2 cytokine-producing CD8(-) and CD8(+) T cells in systemic lupus erythematosus.

The production of type 1 (IFN-gamma, IL-2) and type 2 (IL-4, IL-5, IL-10, IL-13) cytokines by CD8(-) and CD8(+) T cells from systemic lupus erythematosus (SLE) patients and normal subjects was investigated using an intracellular cytokine-staining technique. This flow cytometric method facilitates analysis of both surface markers and cytoplasmic cytokines, after a short term (6 h) culture with or without phorbol myristate acetate and ionomycin (PMA/I) stimulation. In SLE patients, more unstimulated T cells produced IL-10 in comparison with controls; other cytokines were not detected in unstimulated cells. The percentage of IL-10-secreting T cells did not significantly increase after PMA/I stimulation of cells from SLE patients. The mean intensity of fluorescence (MIF) of intracellular IL-4 staining was significantly higher in CD8(-) T cells of SLE patients than controls. Significantly fewer CD8(-) and CD8(+) T cells from SLE patients secreted IFN-gamma after PMA/I stimulation compared with controls. The MIF and percentage of IL-2, IL-5, and IL-13-secreting cell subsets were not significantly different between SLE patients and controls. These findings indicate that T cells of SLE patients are already stimulated to produce IL-10 in vivo, which may result in downregulation of IFN-gamma secreting CD8(-) and CD8(+) T cells observed following PMA/I stimulation. Thus, the population size of Th1 and Tc1 cells are reduced in SLE patients whereas the effector function of Th2 cells, with respect to IL-4 production, is enhanced in SLE patients. Furthermore, although the balance between Th1/Th2 and between Tc1/Tc2 is disrupted in SLE patients, it is significantly biased in favour of the Th2 subset only.

Antibodies from systemic lupus erythematosus (SLE) sera define differential release of autoantigens from cell lines undergoing apoptosis.

SLE is an autoimmune disease characterized by a wide range of anti-cellular and anti-nuclear autoantibodies. Many of these antigens are exposed or altered during apoptosis when the nucleus is dismantled in a controlled manner by caspases. We used Western blotting techniques to demonstrate that autoantibodies in SLE sera recognize antigens released during apoptosis. Reproducible bands, not seen in the untreated cells, with the characteristics of histones were seen when staining apoptotic cell lysates with SLE sera. Normal sera recognized some of these bands but much less strongly. Different triggers of apoptosis did not produce marked differences in the antigens recognized. We also compared different cell lines (Jurkat and U937) and found that the staining differed for one autoantigen in particular. The differential release of autoantigens by apoptotic cells may have relevance to the variety of autoantibodies seen in SLE.

Modulation of autoimmune disease in the MRL-lpr/lpr mouse by IL-2 and TGF-beta1 gene therapy using attenuated Salmonella typhimurium as gene carrier.

We have investigated the effects of interleukin-2 (IL-2) and transforming growth factor-beta (TGF-beta) gene therapy on the progress of autoimmune disease in MRL-lpr/lpr mice, a murine model of systemic lupus erythematosus (SLE). These mice have uncontrolled proliferation of T cells, an impaired response to T cell mitogen and produce autoantibodies against nuclear antigens, including DNA. Immune complexes formed by these autoantibodies are believed to cause glomerulonephritis and vasculitis in lupus mice and human SLE. Since there is an imbalance of cytokine production in both SLE patients and lupus mice, we examined the effects of cytokine gene therapy on the progression of autoimmune disease in MRL-lpr/lpr mice. The mice were treated orally with a non-pathogenic strain of Salmonella typhimurium bearing the aroA-aroD- mutations and carrying the murine genes encoding IL-2 and TGF-beta. The bacteria synthesise and slowly release the cytokines in vivo. Our results show that, contrary to expectation, TGF-beta gene therapy produced no improvement in pathology and generally had opposite effects to those of IL-2. IL-2 gene therapy restored the defective T cell proliferative response to mitogen and suppressed the autoantibody response, glomerulonephritis and growth of lymphoid tumours.

Lung eosinophilia in response to intravenously injected Trichinella spiralis.

Mice were injected intravenously with killed muscle-stage larvae of the parasitic nematode Trichinella spiralis. This induced eosinophilic lung inflammation, the degree of eosinophilia was detected and measured by broncho-alveolar lavage. Subsequent in vivo restimulation by Trichinella antigens produced an enhanced lung response, showing that exposure to killed larvae generated an effective immunological memory. The kinetics and characteristics of the eosinophil response suggested that it was locally rather than systemically controlled. This conclusion is based on the fact that, in contrast to the situation seen after oral infection with T. spiralis, there was little change in bone marrow eosinophilopoiesis, blood eosinophil numbers or in serum interleukin-5 levels.

APIS: a software for model identification, simulation and dosage regimen calculations in clinical and experimental pharmacokinetics.

APIS is a software package based on mathematical modelling which provides a reliable approach in optimizing drug therapy. It was designed to assist clinicians in interpreting blood drug levels so that drug therapy may be better and more cost-effective. It is a methodological approach to describe, predict and control the kinetic behaviour of a drug. This software incorporates the principle of Bayesian procedures, i.e. one can use all available patient information (population) to determine patient-specific parameter estimates. These estimates can then be used to design an optimal and individualized drug regimen. APIS is an attractive and useful tool for clinical and experimental pharmacokinetics. APIS may be used on any IBM compatible computer using the Microsoft-Windows environment. The software is menu driven to provide a very user-friendly tool for analysing pharmacokinetic data and for designing dosage regimens.

The structure of alpha-keratin.

Known structural principles (close packing, maximum hydrogen bonding, the tendency of like groups to be surrounded in like manner, and the approximate constancy of interatomic distances and bond angles) are used, with meridional and equatorial x-ray data, to deduce and check the structure pattern for alpha-keratin. Internally hydrogen-bonded polypeptide helices are grouped into "3-stacks", in which each chain is rotated and shifted vertically a distance equal to the helix pitch (5.15 A, average), relative to the other two. This shift accounts simply for the meridional x-ray reflection at this spacing. The 3-stack structure repeats after three turns, except for differences in the R groups and a slight twist, required to give satisfaction of both intrachain and interchain forces. The 3-stacks are grouped into 9-stacks and these into 27-stacks (all twisting), giving a crystallographic unit containing 81 chains, with the chain axes spaced approximately like those of close-packed cylinders. When the twisting reaches the limit of stability for good interchain contacting and cross-linking, the residue/turn ratio in each chain helix shifts to another, with twisting in the opposite direction. The twisting reversal mechanism keeps all the helix axes approximately straight, parallel, and in a close-packed arrangement. Interchain distances and orientations are suitable for cystine cross-linking. The dimensions of the 27-stacks agree well with estimates of the "effective radius" of microfibrils. X-ray measurements of spacing changes during fiber extension are explained as due to alternation of zones with much cross-linking and zones with few cross-links.