[Viability of autologous fat grafts harvested with the Coleman technique and the tissu trans system (shippert method): a comparative study]. / Vitalitätsvergleich von Fetttransplantaten welche mit der Coleman Technik und dem Tissu Trans System (Shippert Methode) gewonnen wurden.

Various methods for harvesting and refining autologous fat grafts have been described. One of the standard procedures, the Coleman technique, is based on manual aspiration to reduce the negative presssure and the centrifugation of the grafts. The Shippert technique uses automatic liposuction with reduced negative pressure and abstains from centifugation in order not to reduce viability of the graft by exposing it to centrifugal forces. This study intends to compare the viability of fat grafts processed with the above-mentioned methods.Fat grafts were obtained in 9 patients by using both the Tissu Trans system (Shippert technique) and the Coleman technique. To evaluate the impact of centrifugation forces, the grafts harvested with the Coleman technique were treated with standard adjustment of the centrifuge and also with doubled g-force. Viability of fat grafts was analysed with the WST-8 test and with annexin V/PI assay FACS analysis.The viability of fat grafts processed by the Coleman technique was significantly higher compared to the Shippert technique on applying the WST-8 test. Applying the annexin V/PI analysis, the viability of fat grafts was almost equal with both techniques. Whereas the fat grafts processed with the Tissu Trans system are injected without condensation, the grafts refined with the Coleman technique were concentrated 3 times by centrifugation compared to the primary liposuctioned graft volumes.The Coleman technique allows the preparation of a fat graft containing more viable cells than the Shippert technique. This is in part due to the condensation of the graft by centrifugation using the Coleman technique. The factor of condensation of the grafts harvested and refined with the Coleman technique exceeds the factor of increased fat graft viability in comparison to the Shippert technique. The Tissu Trans system is more than twice as fast and easier to use with a preferential use for large volume grafts like in breast augmentation, whereas the Coleman technique produces a more condensed graft, favouring it for fat grafting to the face where less volume is needed.

Suppression of autoimmunity via microbial mimics of altered peptide ligands.

Molecular mimics of self-antigens can behave as altered peptide ligands and serve to ameliorate autoimmune disease. Analysis of experimental autoimmune encephalomyelitis with proteomic autoantibody microarrays reveals that there might exist a wide variety of microbes with features that mimic self-epitopes. Autoimmunity could therefore be modulated via microbial immunity, which may account for relapse and remission of ongoing disease.

Detection of apoptosis-specific autoantibodies directed against granzyme B-induced cleavage fragments of the SS-B (La) autoantigen in sera from patients with primary Sjögren's syndrome.

The objective of this study was to detect autoantibodies against granzyme B cleavage products in sera from patients with primary Sjögren's syndrome (SS). Cell lysates derived from human salivary gland (HSG) cell lines were incubated with granzyme B. The susceptibility to the generation of cleavage fragments of SS autoantigens was assayed by immunoblotting using sera from 57 primary SS patients, 17 primary SS patients with malignant lymphoma (ML), 28 systemic lupus erythematosus (SLE) patients, and 20 healthy controls. A 27 kD protein was recognized by serum autoantibodies in 8 (14.0%) of 57 primary SS patients, 5 (29.4%) of 17 SS patients with ML, 2 (7.1%) of 28 SLE patients, but not in 20 normal subjects. This protein was recognized by anti-SSB (La) monoclonal antibodies. Granzyme B-treated recombinant La protein was also shown to migrate as a discrete 27 kD protein by SDS PAGE. Blocking studies demonstrated the existence of an apoptosis-specific B cell epitope present in sera from 2 of 8 primary SS patients and in 2 of 5 primary SS patients with ML which recognized the 27 kD protein. Granzyme B-induced La fragments are generated during cytotoxicity in vitro. This is the first report describing autoantibodies in sera from primary SS patients that specifically recognize fragments of the La protein that are produced by the granzyme B protease.

The fate of U1 snRNP during anti-Fas induced apoptosis: specific cleavage of the U1 snRNA molecule.

During apoptosis, the U1-70K protein, a component of the spliceosomal U1 snRNP complex, is specifically cleaved by the enzyme caspase-3, converting it into a C-terminally truncated 40-kDa fragment. In this study, we show that the 40-kDa U1-70K fragment is still associated with the complete U1 snRNP complex, and that no obvious modifications occur with the U1 snRNP associated proteins U1A, U1C and Sm-B/B'. Furthermore, it is described for the first time that the U1 snRNA molecule, which is the backbone of the U1 snRNP complex, is modified during apoptosis by the specific removal of the first 5 - 6 nucleotides including the 2,2, 7-trimethylguanosine (TMG) cap. The observations that U1 snRNA cleavage is very specific (no such modifications were detected for the other U snRNAs tested) and that U1 snRNA cleavage is markedly inhibited in the presence of caspase inhibitors, indicate that an apoptotically activated ribonuclease is responsible for the specific modification of U1 snRNA during apoptosis.

Rapid nucleolytic degradation of the small cytoplasmic Y RNAs during apoptosis.

We have investigated the fate of the RNA components of small ribonucleoprotein particles in apoptotic cells. We show that the cytoplasmic Ro ribonucleoprotein-associated Y RNAs are specifically and rapidly degraded during apoptosis via a caspase-dependent mechanism. This is the first study describing the selective degradation of a specific class of small structural RNA molecules in apoptotic cells. Cleavage and subsequent truncation of Y RNAs was observed upon exposure of cells to a variety of apoptotic stimuli and were found to be inhibited by Bcl-2, zinc, and several caspase inhibitors. These results indicate that apoptotic degradation of Y RNAs is dependent on caspase activation, which suggests that the nucleolytic activity responsible for hY RNA degradation is activated downstream of the caspase cascade. The Y RNA degradation products remain bound by the Ro60 protein and in part also by the La protein, the only two proteins known to be stably associated with intact Ro ribonucleoprotein particles. The size of the Y RNA degradation products is consistent with the protection from degradation of the most highly conserved region of the Y RNAs by the bound Ro60 and La proteins. Our results indicate that the rapid abrogation of the yet unknown function of Y RNAs might be an early step in the systemic deactivation of the dying cell.

The 72-kDa component of signal recognition particle is cleaved during apoptosis.

Proteins cleaved by apoptotic caspases are commonly recognized by autoantibodies found in the serum of patients with rheumatic disease. We report that the 72-kDa signal recognition particle (SRP) protein, a rare target of autoantibodies found in the serum of patients with dermatomyositis and systemic lupus erythematosus, is rapidly cleaved in Jurkat T cells treated with apoptotic (i.e. Fas ligation, treatment with gamma or ultraviolet radiation, or co-culture with anisomycin or staurosporine) but not proliferative (CD3 cross-linking) stimuli. Cleavage of SRP 72 produces a 66-kDa amino-terminal fragment and a 6-kDa carboxyl-terminal fragment that is selectively phosphorylated on serine residues. Cleavage of SRP 72 is prevented by chemical and peptide caspase inhibitors, and by overexpression of bcl-2, an inhibitor of apoptotic cell death. Analysis of the carboxyl terminus of SRP 72 has identified a putative cleavage site (SELD/A) for group III caspases, and carboxyl-terminal serine residues that are highly conserved in phylogeny. Both serine phosphorylation and caspase cleavage of SRP 72 are observed in cells derived from human, dog, rat, and mouse. Canine SRP 72 is cleaved in vitro by recombinant caspase 3 but retains the ability to mediate transport of a signal peptide-containing protein into the endoplasmic reticulum lumen. The 72-kDa component of the SRP joins a growing list of autoantigens that undergo post-translational modifications during programmed cell death.

Association of phosphorylated serine/arginine (SR) splicing factors with the U1-small ribonucleoprotein (snRNP) autoantigen complex accompanies apoptotic cell death.

Proteins subject to proteolysis or phosphorylation during apoptosis are commonly precipitated by autoantibodies found in the serum of patients with systemic lupus erythematosus (SLE). We screened a panel of murine monoclonal and human monospecific sera reactive with known autoantigens for their ability to selectively precipitate phosphoproteins from apoptotic Jurkat T cell lysates. Sera known to recognize the U1-small nuclear ribonucleoprotein (snRNP) complex (confirmed by their ability to precipitate U1-snRNA) selectively precipitated a phosphoprotein complex (pp54, pp42, pp34, and pp23) from apoptotic lysates. Monoclonal antibodies reactive with U1-snRNP proteins precipitated the same phosphoprotein complex from apoptotic lysates. The phosphorylation and/or recruitment of these proteins to the U1-snRNP complex is induced by multiple apoptotic stimuli (e.g., Fas ligation, gamma irradiation, or UV irradiation), and is blocked by overexpression of bcl-2. The U1-snRNP-associated phosphoprotein complex is immunoprecipitated by monoclonal antibodies reactive with serine/arginine (SR) proteins that comprise a structurally related family of splicing factors. The association of phosphorylated SR proteins with the U1-snRNP complex in cells undergoing apoptosis suggests a mechanism for regulation of alternative splicing of apoptotic effector molecules.

Proteins phosphorylated during stress-induced apoptosis are common targets for autoantibody production in patients with systemic lupus erythematosus.

Proteins cleaved by interleukin-1 beta converting enzyme family proteases during apoptosis are common targets for autoantibody production in patients with systemic lupus erythematosus (SLE). We have tested the possibility that proteins phosphorylated in cells undergoing apoptosis are also targets for autoantibody production in patients with autoimmune disease. Sera from 9/12 patients containing antinuclear antibodies (10/12 meeting diagnostic criteria for SLE or a lupus overlap syndrome), precipitated new phosphoproteins from lysates derived from Jurkat T cells treated with apoptotic stimuli (i.e., Fas-ligation, gamma irradiation, ultraviolet irradiation), but not with an activation (i.e., CD3-ligation) stimulus. Sera derived from individual patients precipitated different combinations of seven distinct serine-phosphorylated proteins. None of these phosphoproteins were included in precipitates prepared using sera from patients with diseases that are not associated with autoantibody production or using serum from rheumatoid arthritis patients. Protein phosphorylation precedes, or is coincident with, the induction of DNA fragmentation, and is not observed when apoptosis is inhibited by overexpression of bcl-2. Serum from four patients precipitated a serine/threonine kinase from apoptotic cell lysates that phosphorylates proteins of 23-, 34-, and 46-kD in in vitro kinase assays. Our results suggest that proteins phosphorylated during apoptosis may be preferred targets for autoantibody production in patients with SLE.

SUP-HD1: a new Hodgkin's disease-derived cell line with lymphoid features produces interferon-gamma.

A new cell line, SUP-HD1, was established from the pleural effusion of a patient with nodular sclerosing Hodgkin's disease (NSHD). The SUP-HD1 cells had the characteristic morphology of Reed-Sternberg cells and contained acid phosphatase and nonspecific esterase. The cells lacked the Epstein-Barr virus (EBV) genome and reacted with monoclonal antibodies (MoAbs) against CD15 (Leu-M1), CD25 (Tac), CD71 (OKT9), Ki67, and HLA-Dr. However, the SUP-HD1 cells were nonreactive with MoAbs that specifically identify T lymphocytes, B lymphocytes, and macrophage/myeloid cells. Karyotype analysis of the cell line showed clonal abnormalities involving 1p13, 7p15, 8q22, and 11q23, chromosomal locations, at which breakpoints have been reported in HD. Southern blot analysis demonstrated rearrangement of the immunoglobulin heavy chain and kappa light chain genes as well as the gene for the beta chain of the T-cell receptor (TCR). Transcriptional analysis showed expression of RNAs for kappa light chain, interferon-gamma (IFN-gamma), and interleukin-2 receptor (IL-2R) but not IL-2. The SUP-HD1 cells lacked cytoplasmic and surface immunoglobulin heavy chain, but a small amount of cytoplasmic kappa light chain was detected. The presence of nuclear factor kappa B (NF kappa B), a B-lymphocyte-associated transcription factor, was demonstrated in stimulated and unstimulated cells. In addition, the SUP-HD1 cell line, produced IFN-gamma, a T-lymphocyte-associated lymphokine. Based on these data, the SUP-HD1 cells appear to be aberrant lymphocytes with characteristics of both activated B and T lymphocytes. Elaboration of lymphokines such as IFN-gamma by the malignant cells may represent one explanation for the unique clinical and pathologic features of HD.

Effects of the thiazolidinedione derivative CGP 19984 on growth and endocrine function of the MtT-W10 transplantable mammosomatotropic pituitary tumor in female rats.

The thiazolidinedione derivative CGP 19984 has previously been shown to suppress the growth of hormone-dependent mammary and prostatic tumors, primarily by reducing gonadotropin and subsequently gonadal steroid secretion. The present study examines the effects of CGP 19984 on the growth and hormone secretion of the autonomous, but estrogen-responsive, MtT-W10 mammosomatotropic transplantable rat pituitary tumor. Intact tumor-bearing Wistar/Furth female rats were administered vehicle or 25, 100, or 250 mg/kg CGP 19984 p.o., 5 x week for 4 weeks. CGP 19984 was found to significantly reduce MtT-W10 tumor growth and weight and reduce prolactin and growth hormone (GH) secretion in a dose-responsive manner. A similar study in ovariectomized rats also showed that CGP 19984 treatment suppressed MtT-W10 pituitary tumor growth, weight and hormone secretion in a dose-responsive manner, suggesting a direct inhibitory action of this drug on the tumor. In a third study, bromocryptine (CB-154; 5 mg/kg) and CGP 19984 (50 mg/kg) were both found to be effective in suppressing growth of the MtT-W10 tumor in intact female rats. However, rats treated with CGP 19984 alone had reduced serum and tumor GH and prolactin concentrations, while rats treated with CB-154 alone had reduced serum and tumor prolactin, but no change in GH concentrations. These results suggest that CGP 19984 effectively inhibits growth and hormone secretion of the autonomous MtT-W10 pituitary tumor by apparently suppressing both somatotropic and lactotropic cell populations within the tumor. Furthermore, these findings indicate that CGP 19984 may be an effective alternative to CB-154 in the clinical treatment of prolactin-producing adenomas, as well as other types of pituitary adenomas.

Identification of a putative regulator of early T cell activation genes.

Molecules involved in the antigen receptor-dependent regulation of early T cell activation genes were investigated with the use of functional sequences of the T cell activation-specific enhancer of interleukin-2 (IL-2). One of these sequences forms a protein complex, NFAT-1, specifically with nuclear extracts of activated T cells. This complex appeared 10 to 25 minutes before the activation of the IL-2 gene. Studies with inhibitors of protein synthesis indicated that the time of synthesis of the activator of the IL-2 gene in Jurkat T cells corresponds to the time of appearance of NFAT-1. NFAT-1, or a very similar protein, bound functional sequences of the long terminal repeat (LTR) of the human immunodeficiency virus type 1; the LTR of this virus is known to be stimulated during early T cell activation. The binding site for this complex activated a linked promoter after transfection into antigen receptor-activated T cells but not other cell types. These characteristics suggest that NFAT-1 transmits signals initiated at the T cell antigen receptor.