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Bernd Bonnekoh

Publications and source records attributed to Bernd Bonnekoh.

16 recordsLinked to original sources
PubMed2006

Analyzing proteome topology and function by automated multidimensional fluorescence microscopy.

Temporal and spatial regulation of proteins contributes to function. We describe a multidimensional microscopic robot technology for high-throughput protein colocalization studies that runs cycles of fluorescence tagging, imaging and bleaching in situ. This technology combines three advances: a fluorescence technique capable of mapping hundreds of different proteins in one tissue section or cell sample; a method selecting the most prominent combinatorial molecular patterns by representing the data as binary vectors; and a system for imaging the distribution of these protein clusters in a so-called toponome map. By analyzing many cell and tissue types, we show that this approach reveals rules of hierarchical protein network organization, in which the frequency distribution of different protein clusters obeys Zipf's law, and state-specific lead proteins appear to control protein network topology and function. The technology may facilitate the development of diagnostics and targeted therapies.

Dermatitis, Atopic↗
PubMed2006

UVA1 radiation (340-400 nm) interferes with the perforin-granule system of CD8hi+ cytotoxic T lymphocytes in vitro.

UVA1-irradiation was introduced as an innovative and effective phototherapy of atopic dermatitis (AD) and other skin diseases. In AD, a defect of a central apoptosis inducing effector system involved in immunoregulation and immune defense, i.e., the system of perforin-granules in cytotoxic T lymphocytes (CTL), was recently reported: perforin-reduction and perforin-hyperreleasability. We now investigated UVA1-effects on the perforin-granule system in vitro. Peripheral blood CTLs were exposed in vitro to 10-100 J/cm2 UVA1 (340-400 nm), and to 30-150 mJ/cm2 UVB (280-315 nm) as a control. A time-dependent perforin-granule release was induced by phorbol 12-myristate 13-acetate (PMA) and ionomycin. This release was inhibited dose-dependently by UVA1, but not by UVB. An UVA1-dose dependent pattern of sensitive (80%) and insensitive (20%) individuals was found. The kinetics of perforin release in AD-CTLs, i.e. hyperreleasability, was normalized by 50 J/cm2 UVA1 in vitro. Sodium azide as a quencher of reactive oxygen species prevented the UVA1-mediated inhibition of perforin-granule release. Our data demonstrate for the first time a dose- and wavelength-dependent UVA1-effect in vitro on a major effector system of cytotoxic lymphocytes, the system of perforin-granules. This might contribute to the further understanding of immunomodulatory UVA1-effects in vivo.

CD8-Positive T-Lymphocytes↗
PubMed2005

Treatment of scalp angiosarcoma by controlled perfusion of A. carotis externa with pegylated liposomal doxorubicin and intralesional application of pegylated interferon alfa.

BACKGROUND: Angiosarcoma of the scalp is a rare but highly aggressive malignant tumor that differentiates toward vascular endothelial cells and shows a tendency for diffuse, often clinically occult spread. OBSERVATIONS: A 65-year-old Caucasian man presented with multiple erythematous skin lesions at the right scalp hemisphere and a prominent forehead edema that had developed during a period of 2 months. The clinical diagnosis of angiosarcoma was confirmed by histopathology. Because of the advanced local progression of the tumor and the unilateral localization on the right side of the scalp, we initiated an intra-arterial chemotherapy using pegylated liposomal doxorubicin (Caelyx) (8 mg/cycle every 4 weeks by an A. carotis externa port system). In addition, the patient received pegylated interferon alfa (50 microg/wk) by a percutaneous intralesional injection route. After 2 months, the tumor showed a marked regression; after 4 months, only one nodule located at the margins of the area perfused by the A. carotis externa persisted but regressed after focal irradiation. At 30 months after diagnosis, the patient shows no recurrence of tumor growth. CONCLUSIONS: In combination with intralesional interferon alfa, intra-arterial doxorubicin may be a promising innovative therapeutic option for localized scalp angiosarcoma, a hitherto poorly manageable and aggressive malignant tumor.

Aged↗
PubMed2004

A novel flow cytometric assay focusing on perforin release mechanisms of cytotoxic T lymphocytes.

CD8(hi+) cytotoxic T lymphocytes (CTL) are major players in immune defense. In addition, they contribute to the maintenance of immune homeostasis. We now describe a hitherto unavailable, but simple assay to determine ex vivo lytic granule-based cytotoxic functions of human CD8(hi+) CTL subgroups in a clinical setting, under target cell free conditions. Ficoll-isolated peripheral blood lymphocytes from 17 healthy volunteers were stimulated either by phorbol 12-myristate 13-acetate (PMA) in combination with ionomycin or by antibody mediated crosslinking of the CD3 molecule on the T cell surface. Using perforin as a marker for lytic granules, the reduction of CTL granules over time intervals up to 120 min was quantified by FACScan flow cytometry. The kinetics of perforin reduction were compared to the kinetics of NA-CBZ-L-lysine-thiobenzyl ester hydrochloride (BLT)-esterase release and of CD63 upregulation. The reduction in the perforin(+) portion of CD8(hi+) CTLs was correlated inversely with BLT-esterase release and CD63 upregulation. At 30 and 120 min after PMA/ionomycin stimulation, 55 +/- 14% and 42 +/- 14%, respectively, of CD8(hi+) CTLs still stained perforin(+) (time point 0 min = 100%). Perforin-granule release induced by CD3-crosslinking occurred as fast within 30 min (55 +/- 17%), but over the 120 min time interval it was not as complete when compared to PMA/ionomycin-stimulated perforin-reduction. Thus, the combination of an established degranulation assay with the power of immuno flow cytometry allows one to investigate the cytotoxic capability of CTL-subtypes and the kinetics of perforin-granule release. In addition, the assay may prove useful in the elucidation of intracellular signaling cascades governing the perforin-granule release process.

Biological Assay↗
PubMed2004

Imiquimod, a Toll-like receptor-7 agonist, induces perforin in cytotoxic T lymphocytes in vitro.

Imiquimod (IMQ), an activator of Toll-like receptor-7 (TLR-7), induces by several routes a profound anti-viral and anti-tumor effect in vivo. Physiologically, the immune system is using perforin-containing granules of cytotoxic T lymphocytes (CTL) towards the same biological purpose. This functional synergism prompted our current experiments addressing the question whether IMQ may influence perforin-release and/or induce perforin in CTLs in vitro. In peripheral lymphocytes of healthy and diseased subjects, IMQ induced a significant increase of perforin(+) CTLs within 12h in all experiments performed. This effect was most pronounced in CTLs of patients suffering from atopic dermatitis, a model disorder for subnormal perforin expression: as compared to perforin(+) CTLs detected at time point zero (100%), up to 270% of perforin(+) CTLs were induced by 2.5 microg/ml [corrected] IMQ. Perforin release from peripheral blood CTLs after PMA/ionomycin-stimulation was not influenced significantly by IMQ. Thus, the biological activity of IMQ appears to exceed its previously known functions, inasmuch as it boosts up significantly the perforin-granule system.

Adjuvants, Immunologic↗
PubMed2004

[Photodynamic therapy with methylaminooxopentanoate (Metvix) and a broad band light source (PhotoDyn 501): practical experiences in problem-patients with actinic keratoses and basal cell carcinomas].

BACKGROUND: Actinic keratoses (AK) and basal cell carcinomas (BCC) may represent a therapeutic challenge because of special subtypes, location, previous therapy or accompanying diseases. Photodynamic therapy (PDT) offers a semi-conservative treatment option for selected indications. PATIENTS AND METHODS: 28 outpatients who had been admitted as complicated dermato-oncologic cases because of AK (n = 22) and BCC (n = 6) were treated with PDT, using methylaminooxopentanoate (MAOP, Methyl-Ala, Metvix) and a broad band light source (PhotoDyn 501). The treatment was evaluated for efficacy and subjective tolerance (local discomfort and pain). RESULTS: A complete remission (CR) was achieved in 11/22 AK (50%) and 4/6 BCC (67%) cases. All three cases of a superficial BCC subtype underwent a CR. Among responders, tolerance was good in 12/15 cases (80%), as compared to 4/13 cases (31%) in non-responders. Focusing on 16/28 patients with good tolerance (57%), there was a CR in 12 cases (75% rate), whereas for the 12/28 patients with moderate to poor tolerance a CR was achieved in only 3 cases (25% rate). In a subgroup of 8 patients who, partly due to secondary diseases, were taking systemic retinoids or immunosuppressive-cytostatic medications, a CR was achieved in 3/8 cases (38%) with a good tolerance in only 1/8 cases (13%). CONCLUSION: These observations confirm a good efficacy and tolerance of PDT in > or = 50% of a AK/BCC problem patient cohort. We found indications for 1) a positive correlation between efficacy and subjective tolerance as well as 2) the presumptive existence of a retinoid-dependent cutaneous PDT hyperalgesia. Effective pain control seems to be an essential cofactor for the success of PDT.

Aged↗
PubMed2004

[Sarcoidosis, myasthenia gravis and anterior ischaemic optic neuropathy: severe side effects of adjuvant interferon-alpha-therapy in malignant melanoma?].

In dermato-oncology, interferon-alpha is widely used as treatment of cutaneous lymphoma and as adjuvant therapy in high risk malignant melanoma. With increasingly wide administration of interferon-alpha (IFN-alpha), not only early-onset side effects, such as influenza-like symptoms, but also uncommon late-onset side effects are being recognized. We present three patients with melanoma who developed rare side effects in the course of adjuvant IFN-alpha-therapy: sarcoidosis, myasthenia gravis and anterior ischaemic optic neuropathy. Based on current knowledge a causal relationship between these diseases and the administration of IFN-alpha can neither be affirmed or absolutely ruled out. Therefore special attention should be paid to such coincidences.

Aged↗
PubMed2003

Lung carcinoma with congenital plantar keratoderma as a variant of Clarke-Howel-Evans syndrome.

A 32-year-old man was admitted to the Magdeburg University Hospital with icterus and for further diagnosis of suspected hepatitis. He also complained of generalized pruritus, abdominal pain, nausea, and diarrhea. The patient's history revealed the excision of a lymph node metastasis of the left groin showing pleomorphic macrocellular infiltrates, 2 months previously. The patient presented to our department with prominent hyperkeratosis of both feet, which had been present since early youth. The family history was negative. Both soles showed very thick, white and blackish hyperkeratosis with predominance of the heels and the forefeet (Fig. 1). The naturally occurring wrinkles of the skin of the toes were flattened. The palms were not affected, and neither was the oral mucosa. Further investigations revealed icterus of the sclera and multiple, firm tumors, which were located in the deep subcutaneous tissue, on the left hip, thigh, and buttock. From thorough clinical, laboratory and staging investigations, a non-small-cell bronchogenic carcinoma, with metastases of the liver, kidneys, adrenal glands, and several skin sites, was diagnosed. A skin biopsy specimen of the foot showed substantial acanthosis of the epidermis with hypergranulosis and excessive orthohyperkeratosis. The corneocytes were enlarged and arranged in a tile-like pattern (Fig. 2). The dermis was free of inflammatory infiltrates and human papillomavirus infection was ruled out by immunohistochemistry. Polychemotherapy was immediately started with 5-fluorouracil, mitomycin, and cisplatin, which was well tolerated. When the patient was admitted for the second cycle, however, his general health had worsened markedly. He complained of abdominal pain, severe weight loss, and nausea. Generalized metastases showed substantial progression. Chemotherapy could not be continued because of a Karnowsky index below 20%. The patient died 2 weeks later.

Adult↗
PubMed2003

The defect of the perforin granule system in cytotoxic T lymphocytes of atopic patients--are perforin reduction and hyperreleasability of clinical relevance?

Perforin-containing lytic granules are secretory lysosomes of cytotoxic lymphocytes. They act as a negative regulator of activated T cells, control immunoglobulin production, contribute to the regulation of the TH1/TH2 balance, and occupy a central role in anti-viral defense mechanisms. This review focuses on recent evidence for a fundamental defect in the lymphocytic perforin system of atopic patients, namely perforin reduction and the hyperreleasability of perforin granules. These findings are set in relation to the immune imbalance in atopy, which is characterized by a weakly restrained proliferation of allergen-specific T and B cells, a predominance of Ttype-2 cytokines, and an increased susceptibility to cutaneous infections. In the context of the wellknown defect of secretory lysosomes in mast cells and keratinocytes of atopic patients, the possibility of a cell type-independent major pathological factor in atopy is discussed: pan-cellular reduction and hyperreleasability of secretory lysosomes.

Animals↗
PubMed2003

Routine flow cytometric immuno-staining of T-cell perforin is preserved using diethylene glycol for erythrocyte-lysis but lost by the use of ammonium chloride.

The system of perforin-containing lytic granules of cytotoxic lymphocytes plays an important role in the immune defense machinery. Investigating the capacity and efficacy of this system in and ex vivo is helpful to understand immune responses and their modulation by therapeutic interventions. With regard to its pathophysiological function, we recently demonstrated a substantial increase of perforin-positive CD8+ T cells in the peripheral blood of patients with acute exacerbated psoriasis and severe generalized drug reactions, and, in marked contrast, a highly significant perforin-depletion and a perforin-hyperreleasability in atopic dermatitis (AD). To streamline the perforin staining procedure, isolation of peripheral blood mononuclear cells (PBMC) by Ficoll density centrifugation was to be replaced by lysis of erythrocytes. Ammonium chloride lysis, however, reduced the perforin content of CD8+ T cells substantially (up to 75-100%) as compared with Ficoll isolation of PMC. Incubation of cells in concanamycin A, a selective inhibitor of H+-ATPases, resulted in a similar loss of perforin staining pointing to the critical influence of lysosomal pH. Using diethylene glycol-mediated erythrocyte lysis, perforin was well preserved to be readily detectable by immuno flow cytometry. Representative examples of the application of this optimized perforin staining procedure as well as accumulated data are given for various dermatological disorders (psoriasis, atopic dermatitis, cutaneous drug reactions, graft-versus-host disease (GVHD) with strong involvement of the cytotoxic T-cell population. Our findings may help to explain recent conflicting reports about a widely varying range of the portion of perforin-positive cells in healthy individuals as a reflection of such artificial methodological influences.

Adenosine Triphosphatases↗
PubMed2002

Imiquimod, a topical immune response modifier, in the treatment of cutaneous metastases of malignant melanoma.

BACKGROUND: Imiquimod 5% cream (Aldara, a novel topical immune response modifier, has been approved for the topical treatment of anogenital HPV-induced warts. In addition, several studies have demonstrated antitumoral activity in solar keratoses, superficial basal cell carcinomas and Bowen's disease. AIM: Given the convincing therapeutic results of imiquimod when used for treating selected types of epithelial skin cancer, we became interested to study imiquimod as an adjuvant for treating cutaneous metastases of malignant melanoma. METHODS: Three patients with multiple, i.e. more than 15, cutaneous in-transit metastases of malignant melanoma in unilateral localization on the leg were treated topically with imiquimod 5% cream. RESULTS: Twice daily application under occlusive conditions for a period of 21-28 weeks resulted in >90% regression of cutaneous metastases in 2 patients. The third patient showed marked response only when topical imiquimod was intermittently supplemented by intralesional interleukin (IL)-2 for 2 weeks. Unwanted side effects were mild in all patients. CONCLUSION: Overall, imiquimod as a single agent or in combination with intralesional IL-2 may be a promising immunomodulatory compound for the adjuvant topical treatment of patients with multiple cutaneous metastases of malignant melanoma.

Adjuvants, Immunologic↗
PubMedDate not supplied

Profiling lymphocyte subpopulations in peripheral blood under efalizumab treatment of psoriasis by multi epitope ligand cartography (MELC) robot microscopy.

CD11a-blocking efalizumab has recently been approved as a systemic treatment of moderate to severe chronic plaque psoriasis. When treating 6 psoriasis patients with efalizumab over 12 weeks in the present study, we observed an overall good tolerability and 5 treatment responders characterized by a decrease of PASI from 21.3 +/- 5.4 to 3.9 +/- 0.6. The accompanying significant increase of peripheral blood lymphocytes from 1.9 +/- 0.7 to 4.3 +/- 1.0 x 10(9)/L (p < 0.05) was analyzed by multi epitope ligand cartography (MELC) robot microscopy. Thereby a high-dimension simultaneous multiplex immunophenotyping was pursued using 39 fluorophore-labeled antibodies including labeled efalizumab and 3 other affinity reagents such as lectins. Due to efalizumab treatment there was a substantial decrease of the cellular expression of CD11a (detected by mab clone 25.3.1) and efalizumab binding sites (EfaBSs). This was paralleled by an increase of the number of EfaBS- and EfaBS+ lymphocytes by a factor of 2.4x and 2.2x, respectively. The latter effect was mainly derived from a subpopulation showing a low degree of EfaBS expression. Efalizumab treatment led furthermore to an increase of the numbers of CD3+, CD4+, CD8+, CD44+, CD45+, CD45R0+, CD45 RA+, CD52+, CD58+, CD247+, HLA-DR+ and Sambucus nigra lectin-reactive lymphocytes (by factors from 2.0 to 3.3x). In terms of a combinatorial molecular phenotype we identified a CD3+/CD4+/CD44+/CD52+ lymphocyte subpopulation which accumulated most predominantly from 0.824 +/- 0.270 x 10(9)/L up to 1.616 +/- 0.152 x 10(9)/L under efalizumab treatment (p < 0.01). Thus, the current study extends the knowledge of efalizumab-dependent perturbations of recirculating blood lymphocyte subpopulations in psoriasis patients.

Adult↗