Biomedical subjects

Gerold Schuler

Publications and source records attributed to Gerold Schuler.

52 records · Page 3Linked to original sources

PubMed2002

Large-scale generation of mature monocyte-derived dendritic cells for clinical application in cell factories.

Dendritic cells (DC) are increasingly used for the immunotherapy of cancer. Both the induction of tumor-specific T cells and some clinical regressions have been observed in early phase I/II trials by using either DC isolated from blood, DC generated from CD34+ precursors ex vivo, and most frequently, by employing monocyte-derived DC. As DC vaccination is now awaiting phase II/III trials with larger patient collectives, it becomes increasingly important to overcome prior limitations such as the repetitive, labor-intensive generation of DC in a large number of open culture vessels. We describe here as a result of several years of optimization, in detail, a procedure that uses the so-called Nunc cell factories to process a whole apheresis product, labor- and cost-effectively in a quasi-closed system to reproducibly generate (by using GM-CSF+IL-4 followed by a maturation cocktail composed of IL-1beta+IL-6+TNF-alpha +PGE(2)) large numbers (8.32+/-3.8% of input peripheral blood mononuclear cells (PBMC)) of mature (>85% CD83+), monocyte-derived DC that can be successfully cryopreserved. Our report is based on the processing of >100 aphereses including 52 unselected aphereses in advanced melanoma patients. This allows us also to suggest meaningful quality and validation criteria. The DC generation method appears particularly promising as respective DC vaccination proved to be immunogenic in cancer patients and cell factories can readily be converted to a fully closed system by using appropriate valves, tubings, and bags.

Blood Component Removal↗

PubMed2002

Differential functions of IL-4 receptor types I and II for dendritic cell maturation and IL-12 production and their dependency on GM-CSF.

Little is known about the distinct roles of the two types of IL-4R on DC. Here we report that IL-4 and IL-13 are able to promote DC maturation, as evaluated by up-regulation of MHC class II and costimulatory molecules, when the concentration of GM-CSF is relatively lower than the dose of IL-4 or IL-13. In addition, under these conditions both cytokines enable DC to respond to maturation stimuli such as bacterial products or proinflammatory cytokines. Both IL-4 and IL-13 act synergistically with weak maturation stimuli such as TNF-alpha or CD40. The IL-4R signaling for DC maturation requires the IL-4R alpha-chain and STAT6, but not Janus kinase 3, indicating that IL-4R type II signaling is preferentially responsible for these effects. In contrast, the production of IL-12 p70, but not IL-10 and TNF, induced by microbial products was enhanced only by IL-4, not by IL-13 or Y119D, a selective type II IL-4R agonist, in vitro and in vivo. This enhancement was dependent on the presence of Janus kinase 3, indicating that this function is exclusively mediated by the type I IL-4R. In short, we discerned the individual roles of the two IL-4R types on DC function, showing that IL-4R type I promotes IL-12 secretion independently of GM-CSF concentration, while IL-4R type II promotes the up-regulation of MHC class II and costimulatory surface markers in a GM-CSF concentration-dependent manner.

Animals↗

PubMed2002

Human CD4(+)CD25(+) regulatory, contact-dependent T cells induce interleukin 10-producing, contact-independent type 1-like regulatory T cells [corrected].

It has been recently demonstrated that regulatory CD4(+)CD25(+) CD45RO(+) T cells are present in the peripheral blood of healthy adults and exert regulatory function similar to their rodent counterparts. It remains difficult to understand how the small fraction of these T cells that regulate via direct cell-to-cell contact and not via secretion of immunosuppressive cytokines could mediate strong immune suppression. Here we show that human CD4(+)CD25(+) T cells induce long-lasting anergy and production of interleukin (IL)-10 in CD4(+)CD25(-) T cells. These anergized CD4(+)CD25(-) T cells then suppress proliferation of syngenic CD4(+) T cells via IL-10 but independent of direct cell contact, similar to the so-called type 1 regulatory T (Tr1) cells. This 'catalytic' function of CD4(+)CD25(+) T cells to induce Tr1-like cells helps to explain their central role for the maintenance of immune homeostasis.

Adult↗

PubMed2002

TCR-like human antibodies expressed on human CTLs mediate antibody affinity-dependent cytolytic activity.

The permanent genetic programming via gene transfer of autologous T cells with cell surface receptors directed toward tumor-related Ags holds great promise for the development of more-specific tumor therapies. In this study we have explored the use of Abs directed to MHC-peptide complexes (or TCR-like Abs) to engraft CTLs with exquisite specificity for cancer cells. First, we affinity matured in vitro a previously selected TCR-like Ab, Fab-G8, which is highly specific for the peptide melanoma-associated Ag-A1 presented by the HLA-A1 molecule. A combination of L chain shuffling, H chain-targeted mutagenesis, and in vitro selection of phage display libraries yielded a Fab-G8 Ab derivative, Fab-Hyb3, with an 18-fold improved affinity yet identical peptide fine specificity. Fab-G8 and Fab-Hyb3 were expressed on primary human T lymphocytes as cell surface-anchored Fab, demonstrating that T cells expressing the high-affinity Fab-Hyb3 molecule eradicate tumor cells much more effectively. Furthermore, the gain in ligand-binding affinity resulted in a 2-log improvement in the detection of peptide/MHC complexes on melanoma-associated Ag-A1 peptide-loaded cells. In summary, an affinity-matured Ab specifically recognizing a cancer-related peptide/MHC complex was generated and used to improve the tumor cell killing capacity of human T cells. This strategy, based on engraftment of T cells with in vitro engineered Abs, is an attractive alternative to the laborious, and in many cases unsuccessful, generation of highly potent tumor-specific T lymphocytes.

Antibody Affinity↗

PubMed2002

Rapid induction of tumor-specific type 1 T helper cells in metastatic melanoma patients by vaccination with mature, cryopreserved, peptide-loaded monocyte-derived dendritic cells.

There is consensus that an optimized cancer vaccine will have to induce not only CD8+ cytotoxic but also CD4+ T helper (Th) cells, particularly interferon (IFN)-gamma-producing, type 1 Th cells. The induction of strong, ex vivo detectable type 1 Th cell responses has not been reported to date. We demonstrate now that the subcutaneous injection of cryopreserved, mature, antigen-loaded, monocyte-derived dendritic cells (DCs) rapidly induces unequivocal Th1 responses (ex vivo detectable IFN-gamma-producing effectors as well as proliferating precursors) both to the control antigen KLH and to major histocompatibility complex (MHC) class II-restricted tumor peptides (melanoma-antigen [Mage]-3.DP4 and Mage-3.DR13) in the majority of 16 evaluable patients with metastatic melanoma. These Th1 cells recognized not only peptides, but also DCs loaded with Mage-3 protein, and in case of Mage-3DP4-specific Th1 cells IFN-gamma was released even after direct recognition of viable, Mage-3-expressing HLA-DP4+ melanoma cells. The capacity of DCs to rapidly induce Th1 cells should be valuable to evaluate whether Th1 cells are instrumental in targeting human cancer and chronic infections.

Adult↗

PubMed2002

Matrix metalloproteinases 9 and 2 are necessary for the migration of Langerhans cells and dermal dendritic cells from human and murine skin.

Dendritic cells migrate from the skin to the draining lymph nodes. They transport immunogenic MHC-peptide complexes, present them to Ag-specific T cells in the T areas, and thus generate immunity. Migrating dendritic cells encounter physical obstacles, such as basement membranes and collagen meshwork. Prior work has revealed that matrix metalloproteinase-9 (MMP-9) contributes to mouse Langerhans cell migration. In this study, we use mouse and human skin explant culture models to further study the role of MMPs in the migration and maturation of skin dendritic cells. We found that MMP-2 and MMP-9 are expressed on the surface of dendritic cells from the skin, but not from other sources. They are also expressed in migrating Langerhans cells in situ. The migration of both Langerhans cells and dermal dendritic cells is inhibited by a broad spectrum inhibitor of MMPs (BB-3103), by Abs to MMP-9 and -2, and by the natural tissue inhibitors of metalloproteinases (TIMP), TIMP-1 and TIMP-2. Inhibition by anti-MMP-2 and TIMP-2 define a functional role for MMP-2 in addition to the previously described function of MMP-9. The importance of MMP-9 was emphasized using MMP-9-deficient mice in which Langerhans cell migration from skin explants was strikingly reduced. However, MMP-9 was only required for Langerhans cell migration and not maturation, since nonmigrating Langerhans cells isolated from the epidermis matured normally with regard to morphology, phenotype, and T cell stimulatory function. These data underscore the importance of MMPs, and they may be of relevance for therapeutically regulating dendritic cell migration in clinical vaccination approaches.

Animals↗

PubMed2002

Repetitive injections of dendritic cells matured with tumor necrosis factor alpha induce antigen-specific protection of mice from autoimmunity.

Mature dendritic cells (DCs) are believed to induce T cell immunity, whereas immature DCs induce T cell tolerance. Here we describe that injections of DCs matured with tumor necrosis factor (TNF)-alpha (TNF/DCs) induce antigen-specific protection from experimental autoimmune encephalomyelitis (EAE) in mice. Maturation by TNF-alpha induced high levels of major histocompatibility complex class II and costimulatory molecules on DCs, but they remained weak producers of proinflammatory cytokines. One injection of such TNF/DCs pulsed with auto-antigenic peptide ameliorated the disease score of EAE. This could not be observed with immature DCs or DCs matured with lipopolysaccharide (LPS) plus anti-CD40. Three consecutive injections of peptide-pulsed TNF/DCs derived from wild-type led to the induction of peptide-specific predominantly interleukin (IL)-10-producing CD4(+) T cells and complete protection from EAE. Blocking of IL-10 in vivo could only partially restore the susceptibility to EAE, suggesting an important but not exclusive role of IL-10 for EAE prevention. Notably, the protection was peptide specific, as TNF/DCs pulsed with unrelated peptide could not prevent EAE. In conclusion, this study describes that stimulation by TNF-alpha results in incompletely matured DCs (semi-mature DCs) which induce peptide-specific IL-10-producing T cells in vivo and prevent EAE.

Animals↗

PubMed2002

Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity?

Dendritic cells (DCs) are currently divided into tolerogenic immature and immunogenic mature differentiation stages. However, recent findings challenge this model by reporting mature DCs as inducers of regulatory CD4+ T cells in vivo. This implies that decisive tolerogenic and immunogenic maturation signals for DCs might exist. Closer inspection reveals that tolerance is observed when partial- or semi-maturation of DCs occurs, whereas only full DC maturation is immunogenic. The decisive immunogenic signal seems to be the release of proinflammatory cytokines from the DCs. Moreover, the semi-mature DC phenotype is comparable to steady-state migratory veiled DCs within the lymphatics, which seem to continuously tolerize lymph node T cells against tissue-derived self-antigens or apoptotic cells.

Animals↗

PubMed2002

Severity and phenotype of bullous pemphigoid relate to autoantibody profile against the NH2- and COOH-terminal regions of the BP180 ectodomain.

Bullous pemphigoid, the most common autoimmune subepidermal bullous disorder, is associated with autoantibodies targeting antigenic sites clustered within the extracellular domain of BP180. To investigate epitope and subclass specificity of autoantibodies in bullous pemphigoid, we developed an enzyme-linked immunosorbent assay utilizing baculovirus-expressed recombinant forms of the NH2- and COOH-terminal regions of the extracellular domain of BP180 and examined sera obtained from patients with active bullous pemphigoid (n=116) and controls (n=100). Ninety-three (80%) and 54 (47%) of the 116 bullous pemphigoid sera recognized the NH2- and COOH-terminal regions, respectively, of the extracellular domain of BP180. Detailed analysis demonstrates that (i) this novel enzyme-linked immunosorbent assay is highly specific (98%) and sensitive (93%) as 108 of 116 bullous pemphigoid sera reacted with at least one of the baculovirus-derived recombinants, (ii) in active bullous pemphigoid, autoantibodies against the NH2-terminus of the extracellular domain of BP180 were predominantly of the IgG1 class, whereas a dual IgG1 and IgG4 response to this region was related to a more severe skin involvement, (iii) autoreactivity against both the NH2- and COOH-terminal regions was more frequently detected in patients with mucosal lesions, and (iv) levels of IgG (and IgG1) against the NH2-terminal, but not against the COOH-terminal portion of the extracellular domain of BP180, reflected disease severity indicating that autoantibodies against the NH2-terminus are critical in the pathogenesis of bullous pemphigoid. In conclusion, this novel enzyme-linked immunosorbent assay represents a highly sensitive and specific assay for rapid diagnosis of bullous pemphigoid and related disorders and may provide predictive parameters for the management of bullous pemphigoid patients.

Autoantibodies↗

PubMed2002

Cloning and characterization of the promoter region of the human CD83 gene.

Human CD83 (hCD83) is a glycoprotein expressed predominantly on the surface of dendritic cells (DC). To get insight into the regulation of hCD83 expression, we cloned a 3037 bp fragment up-stream of the translation initiation codon. Deletion mutants were constructed revealing highest promoter activity in the -261 fragment containing four SP1 binding sites and one NF-kappaB element. Electrophoretic mobility shift assays demonstrated the specific interaction of NF-kappaB factors with the NF-kappaB element as well as specific binding of SP1 and SP3 to the SP1 binding site. The hCD83 promoter was inducible by TNF-alpha. This inducibility was strictly dependent on the intact NF-kappaB element.

Antigens, CD↗

PubMed2002

Segmental neurofibromatosis.

Segmental neurofibromatosis is characterised by a limited, segmental distribution of cutaneous neurofibromatosis type 1 (NF1) lesions. It has been suggested that segmental NF results from a postzygotic NF1 gene mutation, and, recently, this hypothesis has been proven in a patient with regionally distributed café-au-lait (CAL) spots and freckles by demonstrating an NF1 microdeletion restricted to fibroblasts cultured from CAL spots. We describe here a patient with segmental NF in which we could not demonstrate any NF1 gene mutation in fibroblasts cultured from neurofibromas by use of the protein truncation test, enzymatic mutation detection and fluorescence in situ hybridisation. These data are in line with the concept that NF1 mutations in Schwann cells, but not in fibroblasts, correlate with neurofibroma formation.

Humans↗

PubMedDate not supplied

Dendritic cells matured with TNF can be further activated in vitro and after subcutaneous injection in vivo which converts their tolerogenicity into immunogenicity.

Dendritic cell (DC) maturation can occur by different types of stimuli. Previously, we described that murine DC matured with tumor necrosis factor (TNF) up-regulate surface MHC and costimulatory molecules but lack cytokine release, and therefore termed them semi-mature DC. These TNF/DC-induced tolerance after intravenous (i.v.) injection in a model of experimental autoimmune encephalomyelitis (EAE). Here, we show that TNF/DC are not terminally differentiated but can still respond to the microbial stimulus lipopolysaccharide. Subcutaneously injected TNF/DC induce an unpolarized T(H)1/T(H)2 response and are not protective in the experimental autoimmune encephalomyelitis model. Although TNF/DC home to the draining lymph node, they remain negative for intracellular cytokine stainings. However, the nonmigrating, endogenous DC started to produce interleukin (IL)-12p40, TNF and little IL-6, IL-10, and MCP-1 in a bystander fashion. Together, DC matured with the inflammatory stimulus TNF remains responsive to further signals in vitro and in vivo. These signals can be provided by pathogens or the subcutaneous injection route, which can convert them from tolerogenic to immunogenic DC. These findings are important for selecting the appropriate injection route of human DC for tumor immunotherapy.

Animals↗

PubMedDate not supplied

Mycosis fungoides palmaris et plantaris--an unusual variant of cutaneous T-cell lymphoma.

Mycosis fungoides (MF) represents a low-risk, cutaneous, non-Hodgkin, T-cell lymphoma with a wide spectrum of clinicopathological manifestations and therefore may mimic a number of other dermatoses. Sometimes the clinical diversity makes the diagnosis of MF, and especially its atypical forms, challenging. We report on an 18-year old male patient, who had been previously diagnosed with palmoplantar eczema. Clinical, histopathological, immunohistochemical and molecular findings revealed an atypical case of MF.

Adolescent↗

PubMedDate not supplied

Delayed response of oral pemphigus vulgaris to rituximab treatment.

Rituximab is a monoclonal antibody directed against the CD20 antigen of B cells, which has been developed for the treatment of lymphomas and has been successfully used in the treatment of recalcitrant pemphigus vulgaris. Here, we report on a 26-year-old patient with an 18 month history of pemphigus vulgaris of the oral mucosa where treatment with rituximab led to a delayed but sustained therapeutic response. This case is of interest since most of the previous reports have described a more rapid clinical improvement of refractory pemphigus by rituximab treatment. The delayed clinical response to rituximab of the present case may be explained by the persistence of long-lived plasma cells that continued to produce pathogenic autoantibodies against desmoglein 3.

Adult↗

PubMedDate not supplied

Topical imiquimod as treatment for different kinds of cutaneous lymphoma.

Imiquimod as a topical immune response modifier leads to a localized production of interferon and other cytokines. Apart from its use for genital warts it has therefore been used as treatment for different cutaneous neoplasms, including a few cases of cutaneous T-cell lymphoma. We treated 8 patients (4 with mycosis fungoides, 1 with CD30+ anaplastic large cell lymphoma and 3 with primary cutaneous B-cell lymphoma) with topical imiquimod. Therapy was started three times per week, in cases without response, the frequency was increased to a daily application. Two patients with mycosis fungoides and the patient with the CD30+ anaplastic large cell lymphoma had a complete clinical remission, the other two patients with mycosis fungoides did not show a response to imiquimod. Of the patients with cutaneous B-cell lymphoma, two reached a partial remission, one did not respond to therapy. Two patients had side effects such as erythema and pruritus which disappeared when the frequency of therapy was reduced. Our preliminary data show that imiquimod might be effective in some cases with therapy resistant lesions of cutaneous T-cell lymphoma as well as of cutaneous B-cell lymphoma, but more controlled studies are needed.

Administration, Topical↗

PubMedDate not supplied

Late syphilis mimicking a pseudolymphoma of the skin.

After a period of declining incidence of syphilis in most of Western Europe until the late 1990s, reports about an increasing trend have been published recently. In contrast to the rising incidence of early syphilis, cases of late (tertiary) syphilis are rarely seen in developed countries. In this report, we describe a 54-year-old patient with infiltrated erythematous plaques on his forehead and neck that histologically revealed a dense lymphocytic cell infiltrate with numerous plasma cells. The serologic examination was characteristic of syphilis and in conjunction with the clinical presentation and the patient's history led to the diagnosis of tertiary syphilis. The diagnosis of this late stage of syphilis can be difficult as clinical pictures can be misleading. Peculiar skin lesions should always remind clinicians of this infectious disease which has still to be considered in differential diagnoses in dermatology.

Diagnosis, Differential↗