An Innovative, Centrifugation-free Method to Prepare Human Platelet Mediator Concentrates Showing Activities Comparable to Platelet-rich Plasma .

UNLABELLED: Slow-healing wounds benefit considerably from treatment with platelet-rich plasma (PrP). The drawback of using PrP is its laborious preparation, which requires expensive technical equipment (centrifuges) and well-trained personnel. METHODS: The authors' new method overcomes these issues and provides the practitioner with an innovative tool to freshly prepare a platelet mediator mix with PrP's known biological activities, but is much simpler to obtain. This is achieved by employing the sedimentation of a blood sample at regular gravity (no centrifuge necessary) in the presence of an anti-coagulant and a sedimentation accelerator. Thereafter, the supernatant containing the platelets is concentrated on a unique filter, which causes these platelets to release their mediators (different biologically active molecules resembling the substance mix that is released by the platelets upon degranulation). This solution is eluted from the filter, providing a sterile-filtered, enriched fraction of biologically active mediators (TGF-ß, PDGF, IGF-1, etc.), most of which are active in wound healing disorders. RESULTS: This preparation triggers in-vitro proliferation of fibroblasts and osteoblasts, the secretion of IL-6 by osteoblasts, and differentiation of fibroblasts into cells with an endothelial morphology resembling cells during angiogenesis. CONCLUSION: By providing the practioner a sterile concentrate of a whole range of autologous platelet mediators within 1 hour, this new method has the potency to become a substitute of PrP in wound-healing therapy. PMC (platelet mediator concentrate) eases the manufacturing of such preparations, thereby making them not only more widely applicable, but also reducing treatment costs.

Autoantibodies to muscarinic acetylcholine receptors found in patients with primary biliary cirrhosis.

BACKGROUND: Autoantibodies to the human muscarinic acetylcholine receptor of the M3 type (hmAchR M3) have been suggested to play an etiopathogenic role in Sjögren's syndrome. Primary biliary cirrhosis (PBC) often is associated with this syndrome. Therefore, we studied the co-presence of hmAchR M3 autoantibodies in patients with PBC. METHODS: Frequency of hmAchR M3 autoantibodies was assessed by Western blotting analysis as well as by an ELISA using a 25-mer peptide of the 2nd extracellular loop of hmAchR M3. Co-localization of hmAchR M3/PBC-specific autoantibodies was studied by confocal laser scanning microscopy. Finally, sera from patients with PBC as well as from healthy controls were tested. RESULTS: Western blotting analysis as well as results from ELISA testing revealed a significantly enhanced IgG reactivity in PBC patients in contrast to healthy controls. Co-localization of autoantibodies with the hmAchR M3 receptor-specific autoantibodies was observed in 10 out of 12 PBC-patients but none of the 5 healthy controls. Antibodies of the IgM type were not found to be affected. CONCLUSIONS: For the first time, our data demonstrate the presence of autoantibodies to the hmAchR M3 in PBC patients. These findings might contribute to the understanding of the pathogenesis of this disease. Further studies have to focus on the functionality of hmAchR M3 autoantibodies in PBC patients.

Hepatitis C virus core protein induces apoptosis-like caspase independent cell death.

BACKGROUND: Hepatitis C virus (HCV) associated liver diseases may be related to apoptotic processes. Thus, we investigated the role of different HCV proteins in apoptosis induction as well as their potency to interact with different apoptosis inducing agents. METHODS AND RESULTS: The use of a tightly adjustable tetracycline (Tet)-dependent HCV protein expression cell system with the founder osteosarcoma cell line U-2 OS allowed switch-off and on of the endogenous production of HCV proteins. Analyzed were cell lines expressing the HCV polyprotein, the core protein, protein complexes of the core, envelope proteins E1, E2 and p7, and non-structural proteins NS3 and NS4A, NS4B or NS5A and NS5B. Apoptosis was measured mainly by the detection of hypodiploid apoptotic nuclei in the absence or presence of mitomycin C, etoposide, TRAIL and an agonistic anti-CD95 antibody. To further characterize cell death induction, a variety of different methods like fluorescence microscopy, TUNEL (terminal deoxynucleotidyl transferase (TdT)-catalyzed deoxyuridinephosphate (dUTP)-nick end labeling) assay, Annexin V staining, Western blot and caspase activation assays were included into our analysis.Two cell lines expressing the core protein but not the total polyprotein exerted a strong apoptotic effect, while the other cell lines did not induce any or only a slight effect by measuring the hypodiploid nuclei. Cell death induction was caspase-independent since it could not be blocked by zVAD-fmk. Moreover, caspase activity was absent in Western blot analysis and fluorometric assays while typical apoptosis-associated morphological features like the membrane blebbing and nuclei condensation and fragmentation could be clearly observed by microscopy. None of the HCV proteins influenced the apoptotic effect mediated via the mitochondrial apoptosis pathway while only the core protein enhanced death-receptor-mediated apoptosis. CONCLUSION: Our data showed a caspase-independent apoptosis-like effect of the core protein, which seems to be inhibited in the presence of further HCV proteins like the non structural (NS) proteins. This observation could be of relevance for the viral spread since induction of an apoptosis-like cell death by the core protein may have some impact on the release of the HCV particles from the host cell.

Mycoplasma antigens as a possible trigger for the induction of antimitochondrial antibodies in primary biliary cirrhosis.

BACKGROUND: In primary biliary cirrhosis (PBC), autoreactivity mainly targets members of the pyruvate dehydrogenase complex (PDC). Because PDC subunits are expressed on the surface of mycoplasma and molecular mimicry may be one aetiological factor, we analysed the presence of mammalian and mycoplasma PDC-specific antibodies in PBC patients. METHODS: Antibodies to porcine PDC and Mycoplasma pneumoniae (mp) antigens mpPDH-C (to be designated mpPDC-E2 chain), mpPDH-B (to be designated mpPDC-E1beta chain), mpCARDS TX and mpP1 were investigated in sera from 43 PBC patients, 19 patients with autoimmune hepatitis and 11 healthy controls by an enzyme-linked immunosorbent assay and Western blotting. To study the rate of acute mycoplasma infection, an adhesin P1-specific polymerase chain reaction (PCR) was performed. RESULTS: Immune reactivity to the mpPDC-E2 antigen was significantly enhanced in PBC patients (83.7%) as compared with controls (overall frequency of 36.7%), while antibodies to the porcine PDC-E2 chain were found only in PBC patients (88%) excluding a simple cross-reactivity of PDC-related antibodies. This observation was confirmed by inhibition studies demonstrating that porcine PDC did not inhibit mycoplasma PDC-specific antibodies and vice versa. The occurrence of antibodies to mpPDC seems to precede the occurrence of antibodies to porcine PDC. Infection with mycoplasma was equally distributed in the groups as evidenced by an antibody frequency comparable to CARDS TX and P1 and PCR reactivity. CONCLUSION: Because PBC patients show a significantly enhanced frequency of mpPDC-E2-related antibodies, besides other factors, molecular mimicry between surface molecules of mycoplasma and epitopes of the autoantigen may play a central role in the aetiopathology of PBC.

Platelets recruit human dendritic cells via Mac-1/JAM-C interaction and modulate dendritic cell function in vitro.

OBJECTIVE: Thrombotic events and immunoinflammatory processes take place next to each other during vascular remodeling in atherosclerotic lesions. In this study we investigated the interaction of platelets with dendritic cells (DCs). METHODS AND RESULTS: The rolling of DCs on platelets was mediated by PSGL-1. Firm adhesion of DCs was mediated through integrin alphaMbeta2 (Mac-1). In vivo, adhesion of DCs to injured carotid arteries in mice was mediated by platelets. Pretreatment with soluble GPVI, which inhibits platelet adhesion to collagen, substantially reduced recruitment of DCs to the injured vessel wall. In addition, preincubation of DCs with sJAM-C significantly reduced their adhesion to platelets. Coincubation of DCs with platelets induced maturation of DCs, as shown by enhanced expression of CD83. In the presence of platelets, DC-induced lymphocyte proliferation was significantly enhanced. Moreover, coincubation of DCs with platelets resulted in platelet phagocytosis by DCs, as verified by different cell phagocytosis assays. Finally, platelet/DC interaction resulted in apoptosis of DCs mediated by a JAM-C-dependent mechanism. CONCLUSIONS: Recruitment of DCs by platelets, which is mediated via CD11b/CD18 (Mac-1) and platelet JAM-C, leads to DC activation and platelet phagocytosis. This process may be of importance for progression of atherosclerotic lesions.

Tributyltin (TBT) induces ultra-rapid caspase activation independent of apoptosome formation in human platelets.

Activation of caspases has been demonstrated to be involved in thrombocytopenia and prolonged storage of platelet concentrates. Platelets represent enucleate cells that comprise all elements of the mitochondrial apoptosis pathway. However, no apoptotic stimuli capable of activating the endogenous caspase cascade have been identified so far. Using tributyltin (TBT) we could identify a compound that is capable of activating caspase-9 and -3 in platelets. Recent studies implicate that TBT induces apoptosis via the mitochondrial signaling pathway that is characterized by the formation of a high-molecular-weight complex (apoptosome) containing the adapter protein Apaf-1 and active caspase-9. Interestingly, addition of TBT induced the activation of caspase-9 in an ultra-rapid kinetic within the first 2 min. In addition, size exclusion chromatography revealed that TBT-mediated processing of caspase-9 occurs in the absence of the apoptosome. Thus, these data implicate that TBT induces the activation of caspase-9 by a mechanism not involving the formation of the apoptosome.

Prevention of surgery-induced suppression of granulocyte function by intravenous application of a fermented extract from Viscum album L. in breast cancer patients.

Surgical stress and anaesthetics are able to suppress the immune system. This may accelerate the growth and metastasis of residual cancer cells. As Viscum album L. extracts (VA-E) are known to exert both effects, immunomodulating and apoptosis-inducing properties, a Good-Clinical-Practice-guided, prospective bi-centric phase II study was conducted to measure the influence of a perioperative intravenous application of a VA-E on granulocyte function. In 98 patients with breast cancer, it was shown that a single intravenous application of the standardized VA-E "Iscador M special" in a final concentration of 1 mg/individual prior to surgery prevented the surgery-associated inhibition of the oxidative burst. As no VA-E-related side-effects were observed, this distinct route of application may be a rationale to restrict immunosuppression by surgical stress and anaesthesia.

Stimulation of the maturation of dendritic cells in vitro by a fermented mistletoe extract.

BACKGROUND: Dendritic cells (DC) play a key role during the initiation of specific immune responses. In cancer patients, however, an alteration of their function was observed. In our investigation we analysed the influence of a fermented mistletoe extract often used for adjuvant treatment of cancer patients on the generation and maturation of DC. MATERIALS AND METHODS: Monocytes from healthy individuals were incubated with a fermented mistletoe extract in the presence or absence of GM-CSF/IL-4. Surface marker expression was measured by flow cytometry. RESULTS: While there was no relevant effect on the generation of DC in the absence or presence of GM-CSF/IL-4 in 5-day cultures, the mistletoe extract significantly stimulated the maturation of pre-generated immature DC, as evidenced by a heightened expression of CD83. Like the positive control TNF-alpha, the mistletoe extract significantly activated CD80 and CD86 as well as HLA class I and II molecules on these cells. CONCLUSION: Our data clearly demonstrate an influence of the mistletoe extract on the maturation of DC, but it remains to be elucidated whether the function of DC is also activated and, especially, whether this effect can be observed in tumour patients as well.

Activation of dendritic cells by an aqueous mistletoe extract and mistletoe lectin-3 in vitro.

BACKGROUND: The function of dendritic cells (DC), the most important cell population for the induction of specific immune responses, is often altered in cancer patients. Since mistletoe extracts are applied to tumour patients, their influence on the activation/generation of DC was investigated. MATERIALS AND METHODS: Monocytes from healthy individuals were stimulated with the mistletoe extract (HM) or the mistletoe lectin 3 (ML-3) in the presence or absence of GM-CSF/IL-4. RESULTS: HM slightly but significantly stimulated the expression of CD83 but not CD1a in the presence of GM-CSF/IL-4, while in their absence only CD86 was activated. In pre-generated immature DC, HM and ML-3 enhanced the expression of CD86 but did not significantly activate CD83/CD1a. There was no relevant influence on the expression of HLA-class-I or class-II molecules. CONCLUSION: For the first time, our data demonstrate an influence of a mistletoe extract on the activation/generation of DC, but it remains to be elucidated whether the function of DC is also activated and especially whether this rather slight effect is of importance for tumour patients.

Characterization of immunologically active drugs in a novel organotypic co-culture model of the human gut and whole blood.

The human immune system represents a highly complex multicellular network that protects the organism against the environment and pathogens. Within this system, different immune cells communicate with each other, as well as with adjacent organs and tissues, using an impressive network of regulatory signals. This inherent complexity makes it rather difficult to mimic these processes in vitro. Unpredictable drug-induced side effects can be the consequence when moving from preclinical animal models into clinical phase. Therefore, there is a demand for more elaborate in vivo like human cell culture models. In this study, an in vitro co-culture model consisting of Caco-2 human gut epithelial cells and human whole blood representing the immune system is applied to investigate the intestinal absorption of anti-inflammatory drugs and the subsequent modulation of the immunoregulatory signaling processes. By using blood of different donors, the individuality of the immune system is integrated into the overall analysis. The anti-inflammatory drugs prednisolone and ibuprofen were applied on top of the Caco-2 epithelial cells and alterations in the extracellular communication via cytokines and chemokines were visualized using miniaturized multiplexed sandwich immunoassays. Optionally, pretreatment of the Caco-2 epithelial cells with pro-inflammatory mediators can be used to modulate the epithelial barrier function similar to the situation observed during inflammatory conditions of the gut. The presented translational test system, consisting of differentiated Caco-2 intestinal epithelial cells and whole blood substantially improves preclinical screening of immunologically active drugs with respect to an approximation of the human "in vivo" conditions.

Interferon-gamma and tumor necrosis factor-alpha sensitize primarily resistant human endometrial stromal cells to Fas-mediated apoptosis.

The subtle interaction between the implanting embryo and the maternal endometrium plays a pivotal role during the process of implantation. Human endometrial stromal cells (ESCs) express Fas and the implanting trophoblast cells secrete Fas ligand (FASLG, FasL), suggesting a possible role for Fas-mediated signaling during early implantation. Here we show that ESCs are primarily resistant to Fas-mediated apoptosis independently of their state of hormonal differentiation. Pre-treatment of ESCs with interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha sensitizes them to become apoptotic upon stimulation of Fas by an agonistic anti-Fas antibody. Incubation of ESCs with the early embryonic signal human chorionic gonadotropin (hCG, CGB) does not influence their reaction to Fas stimulation. The sensitizing effect of IFN-gamma and TNF-alpha was accompanied by a significant upregulation of Fas and FLICE-inhibitory protein (FLIP, CFLAR) expression in ESCs. Additionally, we observed an activation of caspase 3, caspase 8 and caspase 9 upon apoptotic Fas triggering. In summary, we demonstrate that IFN-gamma and TNF-alpha sensitize primarily apoptosis-resistant ESCs to Fas-mediated cell death. This might be due to an upregulation of Fas expression, and apoptosis seems to be mediated by active caspase 3, caspase 8 and caspase 9. The observed pro-apoptotic effect of IFN-gamma and TNF-alpha on ESCs could play an important role in the modulation of early implantation.

Demonstration of PDC-E1 subunits as major antigens in the complement-fixing fraction M4 and re-evaluation of PDC-E1-specific antibodies in PBC patients.

BACKGROUND: Primary biliary cirrhosis (PBC) is characterized by the presence of antimitochondrial antibodies (AMA). Autoantibodies specific for the mitochondrial M4 antigen can be detected by a complement fixation test (CFT) but not by immunoblotting. The aim of this study was to elucidate the identity of the M4 antigen. PATIENTS AND METHODS: M4 proteins were purified by affinity chromatography using IgG fractions of PBC marker sera being CFT positive (n=5) or negative (n=5) and identified by Western blotting, silver staining and sequence analysis. Further, a cohort of 57 PBC patients was tested for the reactivity to M4 and pyruvate dehydrogenase complex (PDC). RESULTS: Two AMA patterns of the marker sera were visualized: CFT-positive sera were defined as PDC-E2(+)/E1(+) and the CFT-negative sera as PDC-E2(+)/E1(-). The major proteins in the M4 fraction could be related to the PDC-E1 subunits. A clear-cut association between anti-M4 reactivity in the CFT and the reactivity to both PDC subunits could also be documented in the cohort of 57 PBC patients showing anti-PDC-E1alpha and E1beta antibodies at a frequency of 74% and 67%. CONCLUSIONS: CFT reactivity against M4 antigens could be preferentially identified as a reaction against PDC-E1. As PDC-E1 subunits as compared with PDC-E2 lack lipoyl-binding sites, they probably have to be considered as an independent and important target.