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Biomedical subjects

Martin Herrmann

Publications and source records attributed to Martin Herrmann.

At least 19 recordsLinked to original sources
PubMed2006

Factors masking HMGB1 in human serum and plasma.

High mobility group box 1 protein (HMGB1) is a ubiquitously expressed architectural chromosomal protein. Recently, it has become obvious that HMGB1 can also act as a proinflammatory mediator when actively secreted during cell activation or passively released from necrotic cells. HMGB1 appears to play an important role in the pathogenesis of diseases, including sepsis and rheumatoid arthritis. However, easy, sensitive, and reliable detection systems are required to investigate the clinical significance of HMGB1 in clinical samples for diagnosis and prognosis of diseases. Here, we describe sensitive ELISAs for the detection of HMGB1 in cell culture medium and cell lysates. However, these assays failed to reliably quantitate HMGB1 in serum and plasma when compared with immunoblot analysis. We found that serum/plasma components bind to HMGB1 and interfere with its detection by ELISA systems. In most serum/plasma samples investigated, including those from healthy individuals, we detected IgG antibodies binding to HMGB1. The titers of these antibodies correlated with the capacity of sera to interfere with the detection of recombinant HMGB1 by ELISA. Furthermore, HMGB1 coimmunoprecipitated with several proteins including IgG1, as identified by mass spectrometry. These HMGB1 interacting proteins are currently characterized and may contribute to complex formation, masking, and possibly, modulation of cytokine activity of HMGB1.

Biomarkers↗
PubMed2006

The influence on the immunomodulatory effects of dying and dead cells of Annexin V.

Apoptotic and necrotic cells expose phosphatidylserine (PS). This membrane modification ensures a swift recognition and uptake by phagocytes of the dying and dead cells. Annexin V (AxV) preferentially binds to anionic phospholipids and thereby, modulates the clearance process. First, we analyzed the influence of AxV on the immunogenicity of apoptotic cells. The addition to apoptotic cells of AxV prior to their injection into mice increased their immunogenicity significantly. Next, we studied the influence of endogenous AxV on the allogeneic reaction against apoptotic and necrotic cells. To preserve heat-labile, short-lived "danger signals," we induced necrosis by mechanical stress. Wild-type mice showed a strong, allogeneic delayed-type hypersensitivity (DTH) reaction. In contrast, AxV-deficient animals showed almost no allogeneic DTH reaction, indicating that endogenous AxV increases the immune response against dead cells. Furthermore, AxV-deficient macrophages had a higher immunosuppressive potential in vitro. Next, we analyzed the influence of AxV on chronic macrophage infection with HIV-1, known to expose PS on its surface. The infectivity in human macrophages of HIV-1 was reduced significantly in the presence of AxV. Finally, we show that AxV also blocked the in vitro uptake by macrophages of primary necrotic cells. Similar to apoptotic cells, necrotic cells generated by heat treatment displayed an anti-inflammatory activity. In contrast, mechanical stress-induced necrotic cells led to a decreased secretion of IL-10, indicating a more inflammatory potential. From the experiments presented above, we conclude that AxV influences the clearance of several PS-exposing particles such as viruses, dying, and dead cells.

Animals↗
PubMed2006

Leishmania disease development depends on the presence of apoptotic promastigotes in the virulent inoculum.

The obligate intracellular pathogen Leishmania major survives and multiplies in professional phagocytes. The evasion strategy to circumvent killing by host phagocytes and establish a productive infection is poorly understood. Here we report that the virulent inoculum of Leishmania promastigotes contains a high ratio of annexin A5-binding apoptotic parasites. This subpopulation of parasites is characterized by a round body shape, a swollen kinetoplast, nuclear condensation, and a lack of multiplication and represents dying or already dead parasites. After depleting the apoptotic parasites from a virulent population, Leishmania do not survive in phagocytes in vitro and lose their disease-inducing ability in vivo. TGF-beta induced by apoptotic parasites is likely to mediate the silencing of phagocytes and lead to survival of infectious Leishmania populations. The data demonstrate that apoptotic promastigotes, in an altruistic way, enable the intracellular survival of the viable parasites.

Animals↗
PubMed2006

Accumulation of apoptotic cells in the epidermis of patients with cutaneous lupus erythematosus after ultraviolet irradiation.

OBJECTIVE: To examine whether apoptosis contributes to the pathogenesis of skin lesions in patients with cutaneous lupus erythematosus (CLE) after ultraviolet (UV) irradiation. METHODS: In situ nick translation and TUNEL were performed to detect apoptosis in 85 skin biopsy specimens from patients with various subtypes of CLE. Specimens from normal healthy donors and patients with polymorphous light eruption were used as controls. In addition to assessment of primary lesions, provocative phototesting was carried out to investigate events occurring secondary to UV irradiation during a very early stage of lesion formation. RESULTS: A significant increase in apoptotic nuclei was found in the upper epidermal layer of primary and UV light-induced skin lesions of CLE patients compared with controls. In tissue sections obtained from control subjects at 24 hours after a single exposure to UV light, a slight increase in the count of epidermal apoptotic nuclei was present as compared with skin tissue from CLE patients obtained under the same conditions before lesion formation. In sections obtained from controls at 72 hours after irradiation, a significant decrease in the apoptotic nuclei count was observed, consistent with a proper clearance of apoptotic cells in the period between 24 and 72 hours after irradiation. In striking contrast, the number of apoptotic nuclei increased significantly within this period in tissue sections from patients with CLE. CONCLUSION: These data support the hypothesis that apoptotic cells accumulate in the skin of patients with CLE after UV irradiation, as a result of impaired or delayed clearance. The nonengulfed cells may undergo secondary necrosis and release proinflammatory compounds and potential autoantigens, which may contribute to the inflammatory micromilieu that leads to formation of skin lesions in this disease.

Adult↗
PubMed2006

Involvement of phosphatidylserine, alphavbeta3, CD14, CD36, and complement C1q in the phagocytosis of primary necrotic lymphocytes by macrophages.

OBJECTIVE: Uningested dead cells may be an important source of autoantigens and may trigger autoimmune diseases such as systemic lupus erythematosus (SLE). Multiple receptors involved in the clearance of apoptotic cells have been described; however, little is known about the receptors and ligands involved in uptake of necrotic cells that release autoantigens as well. METHODS: The uptake of autologous necrotic peripheral blood lymphocytes into human monocyte-derived macrophages was qualitatively and quantitatively monitored by confocal microscopy and 2-color flow cytometry, respectively. Blocking experiments were performed to examine the receptors and molecules involved in the phagocytosis of necrotic cells. Cytokine secretion by lipopolysaccharide-activated monocytes and macrophages was determined by enzyme-linked immunosorbent assay. RESULTS: Phosphatidylserine, which was exposed on necrotic as well as apoptotic cells, promoted the recognition and removal of primary necrotic lymphocytes. Several macrophage receptor systems, including the thrombospondin-CD36-alphavbeta3 complex, CD14, and the complement component C1q, contributed to the engulfment of necrotic cells. Necrotic peripheral blood lymphocytes slightly increased the lipopolysaccharide-induced secretion of interleukin-10 and reduced the secretion of tumor necrosis factor alpha in monocytes and macrophages. CONCLUSION: Our results indicate that at least some of the receptors and adaptors mediating the uptake of apoptotic cells are also involved in the clearance of necrotic cells. Hence, necrotic cells engage phagocyte receptors such as CD36, which mediate antiinflammatory signals from apoptotic cells. Necrotic cells consequently also have the potency to provide antiinflammatory signals to phagocytes; however, these signals may be overridden by proinflammatory factors released during necrosis. These findings have implications regarding the etiopathogenesis of autoimmune diseases such as SLE, in which impaired clearance of dead cells may foster autoimmunity by the release of potential autoantigens.

CD36 Antigens↗
PubMed2006

Lectins detect changes of the glycosylation status of plasma membrane constituents during late apoptosis.

BACKGROUND: Mechanisms governing the normal resolution processes of inflammation are poorly understood, yet their elucidation may lead to a greater understanding of the pathogenesis of chronic inflammation. The removal of apoptotic cell material and their potentially histotoxic contents is a prerequisite of resolution. Engulfment by macrophages is an important disposal route, and changes in the apoptotic cells that are associated with their recognition by macrophages are the subject of this report. METHODS: Apoptosis and necrosis in primary cells and cell lines were induced by various stimuli. The binding profile of 23 different lectins for vital, apoptotic, and necrotic cells were analyzed by flow cytometry. RESULTS: We observed that lectins were able to attach to the cell surfaces of vital and dying cells. Some lectins exhibited membrane destructive properties and, consecutively, changed the morphology of the cells as detected by flow cytometry. Other lectins did not show differences in their binding to viable and apoptotic cells. Those lectins were, therefore, not used for analyses of surface changes. The lectins Griffonia simplificolia II (GSL II), Narcissus pseudonarcissus (NPn), and Ulex europaeus I (UEA I) showed no cytotoxic activity and bound preferentially to dying cells. Primary and secondary necrotic cells displayed an equal staining intensity, which was substantially higher than for apoptotic cells. The binding of GSL II, NPn, and UEA to dying cells increased in a time-dependent manner and was delayed to AxV positivity and the decrease in the mitochondrial membrane potential of apoptotic cells. The kinetic of the lectin staining correlated with the increase in subG1-DNA. GSL II, NPn, and UEA are specific for N-acetylglucosamine, mannose, and fucose, respectively. CONCLUSION: According to their binding specificity, we conclude that N-acetylglucosamine-, mannose-, and fucose-containing epitopes are increasingly exposed on cells undergoing apoptosis.

Apoptosis↗
PubMed2006

Microscopy under pressure--an optical chamber system for fluorescence microscopic analysis of living cells under high hydrostatic pressure.

High hydrostatic pressure (HHP) becomes more and more interesting for life science research, since it can be employed to inactivate various cells. To directly monitor "cells under pressure," the development of an optical high-pressure chamber is required. Therefore, an optical pressure chamber that can be used for up to 300 MPa was constructed. This chamber has already been described as a tool for in situ observation of dynamic changes of microscopic structures in bright field as well as phase contrast. In combination with an inverted microscope, we obtained brilliant microscopic color pictures with an optical resolution more than 0.56 microm. Here, we demonstrate the capabilities of the HHP cell, in combination with epifluorescence microscopy. Using a nonadherent human B-cell line (Raji, ATCC CCL 86), stained with the fluorescent dyes propidium iodide, Hoechst 33342, or dihexyloxacarbocyanine iodide, we were able to show that the system is suitable to perform fluorescence microscopic analyses, with pressures up to 300 MPa, with viable mammalian cells.

B-Lymphocytes↗
PubMed2005

Cadaveric study on static medial patellar stabilizers: the dynamizing role of the vastus medialis obliquus on medial patellofemoral ligament.

Aim of this cadaveric biomechanical study was to describe the detailed anatomy of the static medial patellar stabilizers and further determine the role of each of them in preventing lateral patellar dislocation. Eight cadaver knees, after removing the skin and subcutaneous tissues, were used in the study. The medial patellofemoral ligament (MPFL), the medial retinaculum (MR) the medial patellomeniscal ligament (MPML), and the medial patellotibial ligament (MPTL) were dissected. Their origins, insertions, orientations and sizes were recorded. To the medial stabilizers, a tension of 10 pounds was applied, using a tensiometer held in a semicircular device while the knees were kept in 30 degrees of flexion. Then, the previously described ligaments were dissected and the resultant displacement recorded. The most anatomically distinct structure is the MPFL, whose length varies from 45-50 mm, and its width from 10-20 mm at its origin (medial femoral epicondyle) to 20-30 mm at its insertion to the patella. The "meshing" of the MPFL fibers to the fibers of the vastus medialis obliquus (VMO) close to its patellar insertion was the most interesting and very important finding. The contribution of MPFL to medial stability was more than 50%. Of the remaining ligaments, MPML contributes 24% and the MPTL and MR contribute only 13% respectively. The MPFL is the strongest medial static patellar stabilizer. Its contribution to patellar stability against lateral dislocation is far more than 50%, since its meshing with the VMO, shortens its fibers which thus pulls the patella to the medial part of the femoral groove and keeps it in the trochlea during the initial 20 degrees -30 degrees of flexion.

Biomechanical Phenomena↗
PubMed2005

The evolution of human anti-double-stranded DNA autoantibodies.

It has been proposed that the anti-double-stranded DNA (dsDNA) response in patients with systemic lupus erythematosus (SLE) is antigen driven and that DNA or nucleosomes select anti-DNA reactive, somatically mutated B cells. We have used site-directed mutagenesis to systematically revert the somatic mutations of two human anti-dsDNA antibodies from SLE patients to analyze the resulting changes in DNA binding as well as binding to other autoantigens. Our data demonstrate that high-affinity binding to dsDNA and nucleosomes is acquired by somatic replacement mutations in a stepwise manner. Reactivity to surface structures of apoptotic cells is acquired by the same somatic mutations that generate high-affinity dsDNA binding. Importantly, revertant antibodies with germ-line V regions did not show any measurable DNA reactivity. We propose that anti-DNA autoantibodies are generated from nonautoreactive B cells during a normal immune response. B cells may acquire autoreactivity de novo during the process of somatic hypermutation. Nucleosomes, if available in lupus patients because of defects in clearing of apoptotic debris, might subsequently positively select high affinity anti-DNA B cells.

Amino Acid Sequence↗
PubMed2005

Release of high mobility group box 1 by dendritic cells controls T cell activation via the receptor for advanced glycation end products.

High mobility group box 1 (HMGB1) is an abundant and conserved nuclear protein that is released by necrotic cells and acts in the extracellular environment as a primary proinflammatory signal. In this study we show that human dendritic cells, which are specialized in Ag presentation to T cells, actively release their own HMGB1 into the extracellular milieu upon activation. This secreted HMGB1 is necessary for the up-regulation of CD80, CD83, and CD86 surface markers of human dendritic cells and for IL-12 production. The HMGB1 secreted by dendritic cells is also required for the clonal expansion, survival, and functional polarization of naive T cells. Using neutralizing Abs and receptor for advanced glycation end product-deficient (RAGE(-/-)) cells, we demonstrate that RAGE is required for the effect of HMGB1 on dendritic cells. HMGB1/RAGE interaction results in downstream activation of MAPKs and NF-kappaB. The use of an ancient signal of necrosis, HMGB1, by dendritic cells to sustain their own maturation and for activation of T lymphocytes represents a profitable evolutionary mechanism.

Active Transport, Cell Nucleus↗
PubMed2005

Survivin as a radioresistance factor, and prognostic and therapeutic target for radiotherapy in rectal cancer.

Apoptosis levels have been shown to predict tumor response to preoperative radiochemotherapy in rectal cancer. Recently, the prominent role of survivin, a structurally unique member of the inhibitor of apoptosis protein family, has been shown in colorectal cancer tumorigenesis and prognosis. In this study, we investigated whether survivin plays a direct role in mediating radiation resistance. We used short interfering RNA molecules to decrease survivin in radioresistant SW480 and intermediately radioresistant HCT-15 colorectal cancer cells. This resulted in a significant decrease of survivin mRNA and protein expression with a maximum at 24 to 48 hours after transfection. If irradiated during this sensitive period, an increased percentage of apoptotic cells and an increased caspase 3/7 activity in parallel with a decreased cell viability and a reduced clonogenic survival was shown. These effects were more pronounced in the radioresistant SW480 cell line with a radiation-induced cytotoxicity enhancement factor at 10% and 50% survival of 1.8 to 2.2 for SW480 and 1.5 to 1.7 for HCT-15, respectively. Furthermore, transfection with survivin short interfering RNA increased levels of G2-M arrest and levels of DNA double-strand breaks in irradiated cells. These observations indicate that cell cycle and DNA repair mechanisms may be associated with apoptosis induction in tumor cells that are otherwise resistant to killing by radiation. In a translational study of 59 patients with rectal cancer treated with a combination of radiotherapy and chemotherapy, increased survivin expression was inversely related to the levels of apoptosis, and was also associated with a significantly higher risk of a local tumor recurrence.

Adenocarcinoma↗
PubMed2005

SLE--a disease of clearance deficiency?

Systemic lupus erythematosus (SLE) is a multifactorial disease and its pathogenesis and precise aetiology remain unknown. Under physiological conditions, neither apoptotic nor necrotic cell material is easily found in tissues because of its quick removal by a highly efficient scavenger system. Autoantigens are found in apoptotic and necrotic material and they are recognized by autoimmune sera from SLE patients. The clearance of dying cells is finely regulated by a highly redundant system of receptors on phagocytic cells and bridging molecules, which detect molecules specific for dying cells. Changes on apoptotic and necrotic cell surfaces are extremely important for their recognition and further disposal. Some SLE patients seem to have an impaired ability to clear such apoptotic material from tissues, and this could cause the breakdown of central and peripheral mechanisms of tolerance against self-antigens. In this article, we address the cells, receptors and molecules involved in the clearance process and show how deficiencies in this process may contribute to the aetiopathogenesis of SLE.

Apoptosis↗
PubMed2005

An optimized conditional suicide switch using doxycycline-dependent expression of human tBid.

We have examined in a HeLa model system whether tight doxycycline-dependent expression of human tBid can be used as a fast and efficient suicide switch. A stably transfected cell line expressing human tBid in a strictly dox-dependent manner showed fast and efficient killing with up to 98 % specific cell death 24 h after induction. The survival rate was only 0.6 % after eleven days of dox treatment. Very low amounts of tBid were sufficient to induce apoptosis indicating that stringent control of the dox-dependent suicide gene is essential for cell survival in the absence of inducer. Using human tBid, an endogenous effector protein with low immunogenic potential, and doxycycline, an inducer with low cytotoxicity and a long record of safe use in humans, this conditional suicide switch provides a tool for various applications, e.g., adoptive immune transfer.

Apoptosis↗
PubMed2005

Beneficial therapeutic effects with different particulate structures of murine polyomavirus VP1-coat protein carrying self or non-self CD8 T cell epitopes against murine melanoma.

Polyomavirus-like-particles (PLPs) are empty, non-replicative, non-infectious particles that represent a potent antigen-delivery system against malignant disease. Protective anti-tumour immunity can be induced under therapy conditions by subcutaneous (s.c.) treatment with particulate antigenic structures like chimerical polyomavirus-pentamers (PPs). These PPs displaying an immunodominant H-2Kb-restricted ovalbumin (OVA)257-264 epitope evoked nearly complete tumour remission in MO5 (B16-OVA) melanoma-bearing C57BL/6 mice by two s.c. applications in a weekly interval. The immunotherapeutic intervention started at day 4 after melanoma implant. Furthermore, 40% of melanoma-bearing mice vaccinated with heterologous PPs carrying a H-2Kb-restricted cytotoxic T lymphocyte (CTL) epitope derived from of tyrosinase-related protein 2 (TRP2) survived similar treatment conditions. However, a late immunotherapeutic onset at day 10 post melanoma inoculation revealed no significant differences between the therapeutic values (40-60% survival) of VP1-OVA252-270 and VP1-TRP2180-192 PPs, respectively. These experiments underlined the capacity of PPs to break T cell tolerance against a differentially expressed self-antigen. As a correlate for preventive and therapeutic immunity against MO5 melanoma the number of OVA257-264- or TRP2180-188-specific CD8 T cells were significantly increased within the splenocyte population of treated mice as measured by H-2Kb-OVA257-264-PE tetramer staining or appropriate ELISPOT assays, respectively. These results reveal that heterologous PLPs and even chimerical PPs represent highly efficient antigen carriers for inducing CTL responses underlining their potential as immunotherapeutics against cancer.

Animals↗
PubMed2005

The CFSE distribution assay is a powerful technique for the analysis of radiation-induced cell death and survival on a single-cell level.

BACKGROUND AND PURPOSE: To analyze radiation sensitivity of cells and to monitor cellular responses to irradiation, sensitive test systems for cell death and proliferation on a single-cell level are required. Traditionally, cellular radiation survival is measured using the clonogenic assay as the gold standard. Here it is reported, that labeling of cells with 5-(and 6-)carboxyfluorescein diacetate succinimidyl ester (CFDASE) can be used as a highly sensitive assay to determine cellular response toward irradiation on a single-cell level. MATERIAL AND METHODS: The human malignant cell lines U937 (myelomonocytic, nonadherent), SW48 and SW480 (colorectal, adherent) were labeled with CFDASE, irradiated with either UVB (0-540 mJ/cm(2)), or X-rays (0-16 Gy). Cell death and proliferation were monitored by cytofluorometry and compared to the clonogenic assay for adherent SW48 and SW480 cells. RESULTS: Dividing nonadherent U937 cells displayed a shift in carboxyfluorescein (CF) fluorescence in parallel with an increased cell count indicating cell proliferation. By comparison, UVB-irradiated U937 cells did not show a shift in CF fluorescence and an increase in cell count indicating cell-cycle arrest. In a mixed cell culture, only the nonirradiated cells divided and concomitantly reduced their fluorescence. Calculating the number of cell divisions it was observed that the nonirradiated cells underwent approximately six cell divisions within 7 days, whereas the irradiated cells divided only once on average. The adherent SW480 colorectal cells showed a more pronounced cell-cycle arrest after irradiation with 240 mJ/cm(2) UVB as compared to cells treated with X-ray up to 16 Gy. Furthermore, the CFSE assay also discriminated colorectal cell lines of different intrinsic radiosensitivities and yielded results comparable to the standard clonogenic assay. CONCLUSION: Analysis of CF distribution can be employed as a powerful add-on to the clonogenic assay to simultaneously monitor cellular responses toward irradiation on a single-cell level. It constitutes an add-on to the clonogenic assay, especially for nonadherent cells.

Cell Adhesion↗
PubMed2005

The low-throughput protein A adsorber: an immune modulatory device. Hypothesis for the mechanism of action in the treatment of rheumatoid arthritis.

To achieve specific removal of pathogenic antibodies (Ab) or immune complexes (IC), several adsorbers have been developed. We discuss the mode of action of low-throughput staphylococcal protein A (SPA) immunoadsorption. The SPA-based Prosorba apheresis is likely to modify some of the autoantibodies (autoAb) or IC. The low-throughput adsorber showed very limited adsorption capacity of circulating autoAb and/or circulating IC. Besides changes of humoral diagnostic parameters, cellular changes could be observed in the Prosorba-treated patients. These changes were rather similar to those that have been observed in a patient successfully treated with Ab against tumor necrosis factor alpha. We propose an adsorber-catalyzed conversion of small, tissue-penetrating, scarcely detectable, non-complement-binding, proinflammatory IgG-rheumatoid factor (RF)-based IC into the more readily phagocytosed species of IC: intermediate-sized, partially cryoprecipitable, non-tissue penetrating IC that are opsonized with complement. These IC are rather short-lived and could quickly be cleared by the body's scavenging system.

Journal Article↗
PubMed2005

Removal of dying cells and systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a very heterogeneous systemic autoimmune disease, in which autoantibody synthesis against nuclear constituents is the main immunological characteristic. These autoantibodies underwent affinity maturation and isotype switching. Additionally, T-cell tolerance against nuclear autoantigens should be affected in these autoimmune patients. Nuclear material derived from apoptotic and/or necrotic cells may serve as an important source of autoantigens. However, dead and dying cells as well as cellular debris are rapidly removed from tissues by phagocytes without eliciting inflammation or immune responses under healthy conditions. During apoptosis nuclear components are strongly modified through enzymatic reactions. If these cells are not timely cleared, those autoantigens may be released, taken up, and presented by dendritic cells in tissues or presented by follicular dendritic cells in lymph nodes to T and B cells, respectively. This could be a mechanism for breaking the peripheral self-tolerance. In this article we focus on the deficient clearance of apoptotic cells in SLE patients and its importance in development of this autoimmune disease.

Journal Article↗