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Biomedical subjects

G Köhler

Publications and source records attributed to G Köhler.

At least 37 records · Page 2Linked to original sources

Evolution of microbial pathogens.

Various genetic mechanisms including point mutations, genetic rearrangements and lateral gene transfer processes contribute to the evolution of microbes. Long-term processes leading to the development of new species or subspecies are termed macroevolution, and short-term developments, which occur during days or weeks, are considered as microevolution. Both processes, macro- and microevolution need horizontal gene transfer, which is particularly important for the development of pathogenic microorganisms. Plasmids, bacteriophages and so-called pathogenicity islands (PAIs) play a crucial role in the evolution of pathogens. During microevolution, genome variability of pathogenic microbes leads to new phenotypes, which play an important role in the acute development of an infectious disease. Infections due to Staphylococcus epidermidis, Candida albicans and Escherichia coli will be described with special emphasis on processes of microevolution. In contrast, the development of PAIs is a process involved in macroevolution. PAIs are especially important in processes leading to new pathotypes or even species. In this review, particular attention will be given to the fact that the evolution of pathogenic microbes can be considered as a specific example for microbial evolution in general.

Candida albicans↗

Interaction of 4-hydroxybiphenyl with cyclodextrins: effect of complex structure on spectroscopic and photophysical properties.

The structures, spectroscopic and photophysical properties of the inclusion complexes between 4-OH-biphenyl and cyclodextrins (CD) in water were studied theoretically and experimentally. The complex structures were predicted using a dynamic Monte Carlo procedure including solvation effects and analyzed via computed and experimentally determined circular dichroism. The interpretation of the induced circular dichroism spectra indicated the formation of stable 2:2 complexes between the probe and alpha-CD, while 1:1 structures account for the circular dichroism induced by beta- and gamma-CD. Fluorescence emission quantum yields and lifetimes, measured as a function of the CD concentration, confirmed the formation of this higher order complex with alpha-CD. Prototropic equilibration in the singlet excited state was found to be depressed in 2:2 complexes due to the hydrophobic environment of the OH groups, while it remained unperturbed in 1:1 complexes, where the substituent is exposed to the aqueous environment. Triplet-triplet absorption and triplet quenching data supported this interpretation. The photophysical properties of both the 1:1 and the 2:2 complexes are characterized by a significant reduction of the nonradiative decay rates.

Biphenyl Compounds↗

Phase I study in melanoma patients of a vaccine with peptide-pulsed dendritic cells generated in vitro from CD34(+) hematopoietic progenitor cells.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that can be used for vaccination purposes, to induce a specific T-cell response in vivo against melanoma-associated antigens. We have shown that the sequential use of early-acting hematopoietic growth factors, stem cell factor, IL-3 and IL-6, followed by differentiation with IL-4 and granulocyte-macrophage colony-stimulating factor allows the in vitro generation of large numbers of immature DCs from CD34(+) peripheral blood progenitor cells. Maturation to interdigitating DCs could specifically be induced within 24 hr by addition of TNF-alpha. Here, we report on a phase I clinical vaccination trial in melanoma patients using peptide-pulsed DCs. Fourteen HLA-A1(+) or HLA-A2(+) patients received at least 4 i.v. infusions of 5 x 10(6) to 5 x 10(7) DCs pulsed with a pool of peptides including either MAGE-1, MAGE-3 (HLA-A1) or Melan-A, gp100, tyrosinase (HLA-A2), depending on the HLA haplotype. A total of 83 vaccinations were performed. Clinical side effects were mild and consisted of low-grade fever (WHO grade I-II). Clinical and immunological responses consisted of anti-tumor responses in 2 patients, increased melanoma peptide-specific delayed-type hypersensitivity reactions in 4 patients, significant expansion of Melan-A- and gp100-specific cytotoxic T lymphocytes in the peripheral blood lymphocytes of 1 patient after vaccination and development of vitiligo in another HLA-A2(+) patient. Our data indicate that the vaccination of peptide-pulsed DCs is capable of inducing clinical and systemic tumor-specific immune responses without provoking major side effects.

Adolescent↗

A molecular genetic system for the pathogenic yeast Candida dubliniensis.

Candida dubliniensis is a recently described pathogenic yeast of the genus Candida that is closely related to Candida albicans but differs from it in several phenotypic and genotypic characteristics, including putative virulence traits, which may explain differences in the spectrum of diseases caused by the two species. In contrast to C. albicans, a molecular genetic system to study virulence of C. dubliniensis is lacking. We have developed a system for the genetic transformation of C. dubliniensis that is based on the use of the dominant selection marker MPA(R) from C. albicans that confers resistance to mycophenolic acid (MPA). Using this transformation system, a GFP (green fluorescent protein) reporter gene that was genetically engineered for functional expression in C. albicans and placed under control of the inducible C. albicans SAP2 (secreted aspartic proteinase) promoter was integrated into the C. dubliniensis genome. MPA-resistant transformants containing the SAP2P-GFP fusion fluoresced under SAP2-inducing conditions but not under SAP2-repressing conditions. These results demonstrate that the MPA(R) selection marker is useful for transformation of C. dubliniensis wild-type strains, that the GFP reporter gene is functionally expressed in C. dubliniensis, and that the C. albicans SAP2 promoter can be used for controlled gene expression in C. dubliniensis. These genetic tools will allow the dissection of the differences in virulence characteristics between the two pathogenic yeast species at the molecular level.

Aspartic Acid Endopeptidases↗

IL-4 and IL-10 antagonize IL-12-mediated protection against acute vaccinia virus infection with a limited role of IFN-gamma and nitric oxide synthetase 2.

Resistance or susceptibility to most infectious diseases is strongly determined by the balance of type 1 vs type 2 cytokines produced during infection. However, for viruses, this scheme may be applicable only to infections with some cytopathic viruses, where IFN-gamma is considered as mandatory for host defense with little if any participation of type 2 responses. We studied the role of signature Th1 (IL-12, IFN-gamma) and Th2 (IL-4, IL-10) cytokines for immune responses against vaccinia virus (VV). IL-12-/- mice were far more susceptible than IFN-gamma-/- mice, and primary CTL responses against VV were absent in IL-12-/- mice but remained intact in IFN-gamma-/- mice. Both CD4+ and CD8+ T cells from IL-12-/- mice were unimpaired in IFN-gamma production, although CD4+ T cells showed elevated Th2 cytokine responses. Virus replication was impaired in IL-4-/- mice and, even more strikingly, in IL-10-/- mice, which both produced elevated levels of the proinflammatory cytokines IL-1alpha and IL-6. Thus, IL-4 produced by Th2 cells and IL-10 produced by Th2 cells and probably also by macrophages counteract efficient anti-viral host defense. Surprisingly, NO production, which is considered as a major type 1 effector pathway inhibited by type 2 cytokines, appears to play a limited role against VV, because NO sythetase 2-deficient mice did not show increased viral replication. Thus, our results identify a new role for IL-12 in defense beyond the induction of IFN-gamma and show that IL-4 and IL-10 modulate host protective responses to VV.

Acute Disease↗

Enzyme-mimetic model compounds: conformational analysis and far-IR study of Cu(TAAB)2+.

Many enzymes occurring in nature like superoxide dismutase are systems rather too big to be accessible for vibrational and quantum chemical investigations. Thus, enzyme-mimetic model compounds consisting of a biological active metal centre surrounded by a macrocyclic ligand are used to shed light on binding properties of the active metal centre. Far- and mid-range IR spectroscopic investigations and a conformational analysis with the semi-empirical ZINDO/1 method of superoxide dismutase-mimetic complex Cu[TAAB]2+ are performed (TAAB = [b,f,j,n][1,5,9,13]tetra-aza-cyclohexadecine (tetra-anhydroamino benzaldehyde)). A distorted tetrahedral copper(II) centre with slightly twisted phenyl subunits is determined as the most stable conformation. Calculated mid- and far-IR spectra are in good agreement with the experimental data and confirm the proposed structure. A harmonic normal-coordinate analysis is used to assign the vibrational modes of the observed spectra.

Binding Sites↗

Specific Helicobacter pylori antigens unable to distinguish nonucler dyspepsia or peptic ulcer cases from asymptomatic seropositive controls: a nested case-control study in employees of a large company.

We investigated in a large industrial population the antibody response to specific H. pylori antigens (CagA and seven others) in relation to peptic ulcer and nonulcer dyspepsia (NUD). The two groups consisted of 37 and 39 employees, respectively, with endoscopically proven peptic ulcer and NUD. Age- and gender-matched controls were H. pylori seropositive employees without abdominal complaints or history of ulcer disease. IgG antibodies against CagA and other antigens were analyzed by western immunoblot. Relative percentages of CagA-positive individuals were 89 and 76% for ulcer cases and their controls (P = 0.22) and 77% and 74% for NUD cases and their controls. The corresponding percentages of VacA-positive individuals were 87 and 76% for ulcer cases and controls and 64% and 77% for NUD cases and controls, respectively. Analysis of other H. pylori-specific antigens was not particularly helpful in discriminating between symptomatic and asymptomatic seropositive individuals for either disease group. In conclusion, assessment of IgG response against specific H. pylori antigens was not predictive of peptic ulcer or NUD case status in this active employee population and would not appear to be useful in routine clinical practice.

Adult↗

"Tubular Spitz's nevus" an artifact of fixation?

BACKGROUND: Spitz's nevi may contain numerous tubule-like structures. This phenomenon has been described as "tubular Spitz's nevus" and has been attributed to either apoptosis in the center of nests of melanocytes or tubular metaplasia of melanocytes. METHODS: In order to study this peculiar phenomenon, we reviewed 31 Spitz's nevi from our teaching collection. RESULTS: Empty spaces vaguely resembling tubules were found in 15 Spitz's nevi, all of which were examples of the epithelioid-cell type of Spitz's nevus. Histopathologically and immunohistochemically, there was no evidence of apoptosis within or around tubule-like structures. Empty; spaces in the center of nests ranged from distinctive tubule-like structures to only slight separation of melanocytes from one another. Furthermore, empty spaces were also seen around nests, often in combination with distinctive tubule-like changes. CONCLUSIONS: Our findings suggest that so-called "tubular" structures in epithelioid Spitz's nevi result from retraction of tissue secondary to fixation in formalin. "Tubular Spitz's nevus" seems to represent an artifact rather than a distinctive variant of Spitz's nevus.

Artifacts↗

Tumor necrosis factor alpha-mediated toxic shock in Trypanosoma cruzi-infected interleukin 10-deficient mice.

Using interleukin-10 (IL-10)-deficient (IL-10(-/-)) mice, previous studies revealed a pathological immune response after infection with Trypanosoma cruzi that is associated with CD4(+) T cells and overproduction of proinflammatory cytokines. In this study we further investigate the pathology and potential mediators for the mortality in infected animals. T. cruzi-infected IL-10(-/-) mice showed reduced parasitemia accompanied by increased systemic release of gamma interferon (IFN-gamma), IL-12, and reactive nitrogen intermediates and overproduction of tumor necrosis factor alpha (TNF-alpha). Despite this early resistance, IL-10(-/-) mice died within the third week of infection, whereas all control mice survived acute infection. The clinical manifestation with weight loss, hypothermia, hypoglycemia, hyperkalemia, and increased liver-derived enzymes in the blood together with hepatic necrosis and intravascular coagulation in moribund mice indicated a toxic shock-like syndrome, possibly mediated by the systemic TNF-alpha overproduction. Indeed, high production of systemic TNF-alpha significantly correlated with mortality, and moribund mice died with critically high TNF-alpha concentrations in the blood. Consequent treatment with anti-TNF-alpha antiserum attenuated pathological changes in T. cruzi-infected IL-10(-/-) mice and significantly prolonged survival; the mice died during the fourth week postinfection, again with a striking correlation between regaining high systemic TNF-alpha concentrations and the time of death. Since elevated serum IL-12 and IFN-gamma concentrations were not affected by the administration of antiserum, these studies suggest that TNF-alpha is the direct mediator of this toxic shock syndrome. In conclusion, induction of endogenous IL-10 during experimentally induced Chagas' disease seems to be crucial for counterregulating an overshooting proinflammatory cytokine response resulting in TNF-alpha-mediated toxic shock.

Animals↗

Activation of the multiple drug resistance gene MDR1 in fluconazole-resistant, clinical Candida albicans strains is caused by mutations in a trans-regulatory factor.

Resistance of Candida albicans against the widely used antifungal agent fluconazole is often due to active drug efflux from the cells. In many fluconazole-resistant C. albicans isolates the reduced intracellular drug accumulation correlates with constitutive strong expression of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not detectably expressed in vitro in fluconazole-susceptible isolates. To elucidate the molecular changes responsible for MDR1 activation, two pairs of matched fluconazole-susceptible and resistant isolates in which drug resistance coincided with stable MDR1 activation were analyzed. Sequence analysis of the MDR1 regulatory region did not reveal any promoter mutations in the resistant isolates that might account for the altered expression of the gene. To test for a possible involvement of trans-regulatory factors, a GFP reporter gene was placed under the control of the MDR1 promoter from the fluconazole-susceptible C. albicans strain CAI4, which does not express the MDR1 gene in vitro. This MDR1P-GFP fusion was integrated into the genome of the clinical C. albicans isolates with the help of the dominant selection marker MPA(R) developed for the transformation of C. albicans wild-type strains. Integration was targeted to an ectopic locus such that no recombination between the heterologous and resident MDR1 promoters occurred. The transformants of the two resistant isolates exhibited a fluorescent phenotype, whereas transformants of the corresponding susceptible isolates did not express the GFP gene. These results demonstrate that the MDR1 promoter was activated by a trans-regulatory factor that was mutated in fluconazole-resistant isolates, resulting in deregulated, constitutive MDR1 expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Trichostatin A modulates expression of p21waf1/cip1, Bcl-xL, ID1, ID2, ID3, CRAB2, GATA-2, hsp86 and TFIID/TAFII31 mRNA in human lung adenocarcinoma cells.

Lung adenocarcinoma cells treated for 16 h with trichostatin A (TSA), an inhibitor of histone deacetylases, and untreated cells were analyzed with respect to differential gene expression. Complex hybridization of cDNA arrays revealed repression of Bcl-xL, CRAB2 and TFIID/TAFII31 as well as induction of p21waf1/cip1, GATA-2, hsp86, ID1, ID2 and ID3 mRNA expression, which could be verified by Northern blotting. ID2 induction was further confirmed by Taqman realtime quantitative RT-PCR. The described alterations of gene expression due to TSA renders the lung adenocarcinoma cells susceptible to induction of apoptosis.

Adenocarcinoma↗

Trichostatin A-mediated regulation of gene expression and protein kinase activities: reprogramming tumor cells for ribotoxic stress-induced apoptosis.

Recently we described a new signal transduction-based tumor therapeutic strategy involving first sensitization of tumor cells by trichostatin A (TSA), an inhibitor of histone deacetylation, and thereafter efficient apoptotic triggering by ribotoxic agents, which activate stress-activated protein kinases. In the present work we investigate the molecular basis of the sensitization step in this therapeutic model system and describe TSA-induced changes in mRNA and protein expression of several candidate genes identified previously by complex hybridization. Furthermore, activities of 15 different protein kinases were followed after TSA application, using a new filter-based technique (PhosphoSpots-Assay). The obtained data suggest that TSA induces pro-apoptotic genes like ID1, ID2, ID3, and down-regulates anti-apoptotic genes like Hsp27 and Bcl-xL, thereby shifting the cellular equilibrium from life to death. Furthermore, activities of calcium/calmodulin-dependent kinase II and protein kinase C, which have been assigned pro-apoptotic function in other systems, are induced.

Apoptosis↗

Cutting edge: lymphoproliferative disease in the absence of CTLA-4 is not T cell autonomous.

Mice deficient for the expression of CTLA-4 develop a lethal lymphoproliferative syndrome and multiorgan inflammation leading to death at about 4 wk of age. Here we show that RAG2-deficient mice reconstituted with CTLA-4-deficient bone marrow do not develop a lymphoproliferative syndrome despite lymphocyte infiltration mainly into pericardium and liver. Moreover, RAG2-deficient mice reconstituted with a mixture of normal and CTLA-4-deficient bone marrow remain healthy and do not develop any disease. Thus, the lethal disease observed in CTLA-4-deficient mice is not T cell autonomous and can be prevented by factors produced by normal T cells.

Abatacept↗

Differences between IL-4- and IL-4 receptor alpha-deficient mice in chronic leishmaniasis reveal a protective role for IL-13 receptor signaling.

IL-4 receptor alpha-chain-deficient (IL-4Ralpha-/-) mice were generated by homologous and site-specific recombination, using the Cre/loxP system in BALB/c-derived embryonic stem cells. In vitro analysis of cells from these mice revealed impaired IL-4- and IL-13-mediated functions, demonstrating that the IL-4Ralpha-chain is an essential component of both the IL-4 and the IL-13 receptor. Whereas Leishmania major-infected BALB/c mice developed fatal progressive disease with type 2 Ab responses within 3 mo, both IL-4Ralpha-/- and IL-4-/- BALB/c mice contained infection with reduced footpad swelling, parasite load, moderate histopathology, and type 1 Ab responses during this time period. Conclusively, these results demonstrate an IL-4-dependent mechanism of susceptibility in BALB/c mice. Nevertheless, in contrast to mutant mice, infected C57BL/6 mice healed completely within 3 mo, indicating that additional factors are necessary for subsequent healing and elimination of the pathogen. During the further course of infection, IL-4Ralpha-/- mice developed progressive disease with massive footpad swelling. Lesions became ulcerative and necrotic with subsequent destruction of connective tissue and bones, as well as dissemination into organs and consequent mortality within the monitored 6 mo of chronic infection. In striking contrast, IL-4-/- mice maintained control of infection on a moderate level, but were unable to clear the pathogen. The distinct phenotypes of the BALB/c embryonic stem cell-derived IL-4-/- and IL-4Ralpha-/- mouse strains identify previously unsuspected mechanisms for maintaining host immunity to chronic infection with L. major, mediated by a functional IL-13 receptor.

Animals↗

Suppression of metastasis formation by a recombinant single chain antibody-toxin targeted to full-length and oncogenic variant EGF receptors.

Cytotoxic strategies which are directed to tumor-associated antigens might be most beneficial for cancer patients with minimal tumor load such as in an adjuvant setting after initial therapy. We have recently described a highly potent single chain antibody-toxin, scFv(14E1)-ETA, which consists of the variable domains of the antibody 14E1 genetically fused to a truncated form of Pseudomonas exotoxin A. ScFv(14E1)-ETA specifically recognizes the human epidermal growth factor receptor (EGFR) and the oncogenically activated receptor variant EGFRvIII, which have been implicated in the development of various human malignancies. Here we have investigated the antimetastatic activity of bacterially expressed scFv(14E1)-ETA and its disulfide-stabilized derivative ds-scFv(14E1)-ETA in a novel model for disseminated disease which is based on murine renal carcinoma cells subsequently transfected with the E. coli beta-galactosidase gene, and human full-length or variant EGFR cDNAs. Intravenous injection of these Renca-lacZ/EGFR and Renca-lacZ/EGFRvIII cells in syngenic Balb/c mice led to the formation of pulmonary metastases which were readily detectable upon excision of the lungs and X-gal staining. Systemic treatment of mice with scFv(14E1)-ETA resulted in the complete suppression of Renca-lacZ/EGFRvIII metastasis formation and drastically reduced the number of pulmonary Renca-lacZ/EGFR tumor nodules. The ds-scFv(14E1)-ETA derivative where the antibody variable regions are connected by an artificial disulfide bond displayed improved thermal stability at physiological temperature but due to reduced cytotoxic activity was less potent than the original scFv(14E1)-ETA in metastasis suppression.

ADP Ribose Transferases↗

Leukostasis followed by hemorrhage complicating the initiation of chemotherapy in patients with acute myeloid leukemia and hyperleukocytosis: a clinicopathologic report of four cases.

BACKGROUND: Pulmonary and cerebral leukostasis, or parenchymal hemorrhage in these organs, are well-known early complications developing in patients with acute myeloid leukemia (AML), particularly when myelomonocytic features, hyperleukocytosis, and/or a coagulation disorder are initially present. Commonly, these complications arise during increasing leukocyte counts (WBCs). METHODS: The authors describe four patients with AML and hyperleukocytosis who developed leukostasis followed by parenchymal hemorrhage. RESULTS: Bleeding in all patients occurred while their WBCs were decreasing following cytosine-arabinoside chemotherapy, and in the absence of disseminated intravascular coagulation or severe thrombocytopenia. Radiologic and histopathologic findings underscoring possible mechanisms are presented in the article. CONCLUSIONS: Alterations of cell adhesion associated with chemotherapy-induced blast lysis or cellular differentiation are possible factors contributing to this particular sequence (cytosine arabinoside-based chemotherapy, leukostasis, and subsequent hemorrhage). Prophylactic measures for managing this early complication of AML treatment include leukapheresis to reduce the WBC prior to the initiation of chemotherapy.

Acute Disease↗