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Biomedical subjects

C Hölscher

Publications and source records attributed to C Hölscher.

At least 19 recordsLinked to original sources
PubMed2005

Rats are able to navigate in virtual environments.

Virtual reality (VR) systems are useful tools that enable users to alter environmental settings and the location of landmarks in an accurate and fast way. Primates have been shown to be able to navigate in virtual environments. For rodents, however, all previous attempts to develop VR systems in which rats behave in the same way as in corresponding 3-D environments have failed. The question arises as to whether, in principle, rodents can be trained to navigate in a properly designed virtual environment (VE), or whether this peculiarity is limited to primates and humans. We built a virtual reality set-up that takes the wide-angle visual system of rats into account. We show for the first time that rats learn spatial tasks in this VE quite readily. This set-up opens up new opportunities for investigations of information processing in navigation (e.g. the importance of optic flow or vestibular input).

Animals↗
PubMed2003

Effects of the isolation methodology on protein profiles of blood trypomastigotes of Trypanosoma cruzi.

Blood trypomastigotes of Trypanosoma cruzi were isolated from infected athymic rnu/rnu rats and purified by an improved procedure of DEAE-Sephacel ion-exchange chromatography. Elution into a buffer supplemented with bovine serum albumin avoided column-induced changes on the surface of the parasites. Biotin-labelled bovine serum albumin, fluorescence microscopy, flow cytometry and Western blot analysis revealed a very intense binding of albumin to the parasite. Incubation and washing of cells without protein supplementation did not result in any damage or lysis of parasites but it did cause extensive shedding of cellular and surface proteins into the supernatant which could be prevented by using the protein-supplemented buffer. A decreasing yield of high molecular weight cellular proteins in relation to centrifugal force was a general phenomenon observed in scanning densitometry of SDS gels after isolation in either protein-supplemented buffer or protein-free buffer. The quantity of shed cellular components increased with increasing centrifugal force. In contrast, quantities of high molecular weight, biotin-labelled surface proteins increased with greater centrifugal force, indicating labelling of otherwise inaccessible residues. These data emphasize the importance of protein supplementation of buffers with proteins and of choosing low centrifugation forces (<400 g) during investigations of T. cruzi.

Animals↗
PubMed2001

A protective and agonistic function of IL-12p40 in mycobacterial infection.

IL-12p35(-/-)p40(-/-) mice are highly susceptible to Mycobacterium bovis bacillus Calmette-Guérin (BCG) or Mycobacterium tuberculosis infection. In this study IL-12p35(-/-) mice, which are able to produce endogenous IL-12p40, cleared M. bovis BCG and showed reduced susceptibility to pulmonary M. tuberculosis infection, which was in striking contrast to the outcome of mycobacterial infection in IL-12p35(-/-)p40(-/-) mice. Resistance in wild-type and IL-12p35(-/-) mice was accompanied by protective granuloma formation and Ag-specific delayed-type hypersensitivity responses, which were impaired in susceptible IL-12p35(-/- )p40(-/-) mice. Furthermore, IL-12p35(-/-) mice, but not IL-12p35(-/-)p40(-/-) mice, mounted Ag-specific Th1 and cytotoxic T cell responses. In vivo therapy with rIL-12p40 homodimer restored the impaired delayed-type hypersensitivity responses in M. bovis BCG-infected IL-12p35(-/-)p40(-/-) mice and reverted them to a more resistant phenotype. Together, these results show evidence for a protective and agonistic role of endogenous and exogenous IL-12p40 in mycobacterial infection, which is independent of IL-12p70.

Animals↗
PubMed2001

IL-12-independent IFN-gamma production by T cells in experimental Chagas' disease is mediated by IL-18.

IL-12p35-deficient (IL-12p35(-/-)) mice were highly susceptible to Trypanosoma cruzi infection and succumbed during acute infection, demonstrating the crucial importance of endogenous IL-12 in resistance to experimental Chagas' disease. Delayed immune responses were observed in mutant mice, although comparable IFN-gamma and TNF-alpha blood levels as in wild-type mice were detected 2 wk postinfection. In vivo and in vitro analysis demonstrated that T cells, but not NK cells, were recruited to infected organs. Analysis of mice double deficient in the recombinase-activating gene 2 (RAG2) and IL-12p35, as well as studies involving T cell depletion, identified CD4(+) T cells as the cellular source for IL-12-independent IFN-gamma production. IL-18 was induced in IL-12p35(-/-) mice and was responsible for IFN-gamma production, as demonstrated by in vivo IL-18 neutralization studies. In conclusion, evidence is presented for an IL-12-independent IFN-gamma production in experimental Chagas' disease that is T cell and IL-18 dependent.

Acute Disease↗
PubMed2001

Prion protein contains a second endoplasmic reticulum targeting signal sequence located at its C terminus.

Prion protein (PrP) is synthesized at the membrane of the endoplasmic reticulum (ER) in three different topological forms as follows: a fully translocated one ((sec)PrP) and two with opposite orientations in the membrane ((Ntm)PrP and (Ctm)PrP). We asked whether other signal sequences exist in the PrP, other than the N-terminal signal sequence, that contribute to its topological diversity. In vitro translocation assays showed that PrP lacking its N-terminal signal sequence could still translocate into ER microsomes, although at reduced efficiency. Deletion of each of the two hydrophobic regions in PrP revealed that the C-terminally located hydrophobic region (TM2) can function as second signal sequence in PrP. Translocation mediated by the TM2 alone can occur post-translationally and yields mainly (Ctm)PrP, which is implicated in some forms of neurodegeneration in prion diseases. We conclude that, in vitro, PrP can insert into ER membranes co- and post-translationally and can use two different signal sequences. We propose that the unusually complex topology of PrP results from the differential utilization of two signal sequences in PrP.

Animals↗
PubMed2001

Development of high numbers of blood trypomastigotes of Trypanosoma cruzi in nude rats.

The development and pathogenicity of a Trypanosoma cruzi strain ("Chile 5") of low virulence were studied after infection of nude rats with different doses of blood trypomastigotes (10-10(7) parasites/rat). Peak parasitemias were correlated with the infection dose, which also influenced the mean survival times (26-36 days post-infection). Within 26 or 27 days, a subcutaneous injection of 10(7) blood trypomastigotes developed to about 8-20 x 10(7) parasites/ml.

Animals↗
PubMed2000

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↗
PubMed1999

Synaptic plasticity and learning and memory: LTP and beyond.

Long-term potentiation (LTP) of synaptic activity is by far the most popular and widely researched model of synaptic plastic changes that might occur during learning. Numerous recent reports, however, have not found a correlation between the inducibility of LTP in the hippocampus and the ability of animals to learn hippocampus-dependent tasks. For example, some experiments with gene deletion (knockout) mice strains have shown that in some strains LTP is not inducible in the dentate gyrus, in area CA3, or CA1, but the animals are still able to learn spatial tasks. This apparent mismatch has rejuvenated the discussion concerning whether LTP is a good model for mechanisms that underlie memory formation in the nervous system. This review analyzes the conditions under which LTP is induced or learning takes place and suggests reasons for the mismatches that can occur and what we can learn from them. High-frequency stimulation protocols and in vitro assays cannot be seen to resemble natural firing patterns or conditions found in the brain. More physiological experimental conditions, especially in vivo recording in awake animals, could lead the way to the development of improved models of learning mechanisms that better correlate with learning abilities of animals.

Animals↗
PubMed1999

Long term substitution and specific immune responses after transfer of bovine peripheral blood lymphocytes into severe combined immunodeficient mice.

The long term immune responsiveness of bovine peripheral blood lymphocytes engrafted into severe combined immunodeficient mice (bovine PBL SCID mice) was analyzed. After intraperitoneal transfer (i.p.) of 2x10(7) bovine PBL into SCID mice, FACS analysis revealed successful engraftment of bovine CD4 and CD8+ T cells in the peritoneal cavity, the peripheral blood, spleen, lymph nodes, bone marrow, and thymus of reconstituted mice for up to 13 weeks. As shown by immunocytochemistry in sections of spleens from SCID mice 16 weeks after substitution, bovine T and B cells were localized perivasculary forming pseudofollicular structures. Nevertheless, histopathology of spleen and liver from bovine PBL SCID mice revealed pathological alterations indicating rejection of xenogenic cells or graft versus host disease (GVHD). On the functional level, i.p. transfer of bovine PBL into SCID mice induced increasing levels of bovine IgM and IgG in the sera of recipients. Bovine Ig could be detected up to 20 weeks. Immunization of SCID mice reconstituted with PBL of normal donors with dinitrophenol (DNP)-edestin induced a weak specific bovine antibody response in recipient mice. In contrast, a secondary specific bovine IgG response was observed after antigen restimulation of SCID mice reconstituted with PBL from calves preimmunized either with DNP-edestin or keyhole limpet hemocyanin (KLH) showing functional T cell-independent and -dependent antibody responses of bovine PBL SCID mice. Our data demonstrate that transfer of bovine PBL into SCID mice leads to a long term engraftment of bovine cells in lymphatic and non-lymphatic organs inducing a functional substitution of T and B cell immune response of SCID mice. Therefore, bovine PBL SCID chimera can serve as a small animal model for the analysis of bovine lymphopoiesis and infectious diseases of cattle.

Animals↗
PubMed1999

Control of glycosylation of MHC class II-associated invariant chain by translocon-associated RAMP4.

Protein translocation across the membrane of the endoplasmic reticulum (ER) proceeds through a proteinaceous translocation machinery, the translocon. To identify components that may regulate translocation by interacting with nascent polypeptides in the translocon, we used site-specific photo-crosslinking. We found that a region C-terminal of the two N-glycosylation sites of the MHC class II-associated invariant chain (Ii) interacts specifically with the ribosome-associated membrane protein 4 (RAMP4). RAMP4 is a small, tail-anchored protein of 66 amino acid residues that is homologous to the yeast YSY6 protein. YSY6 suppresses a secretion defect of a secY mutant in Escherichia coli. The interaction of RAMP4 with Ii occurred when nascent Ii chains reached a length of 170 amino acid residues and persisted until Ii chain completion, suggesting translocational pausing. Site-directed mutagenesis revealed that the region of Ii interacting with RAMP4 contains essential hydrophobic amino acid residues. Exchange of these residues for serines led to a reduced interaction with RAMP4 and inefficient N-glycosylation. We propose that RAMP4 controls modification of Ii and possibly also of other secretory and membrane proteins containing specific RAMP4-interacting sequences. Efficient or variable glycosylation of Ii may contribute to its capacity to modulate antigen presentation by MHC class II molecules.

Amino Acid Sequence↗
PubMed1999

Metabotropic glutamate receptor activation and blockade: their role in long-term potentiation, learning and neurotoxicity.

Metabotropic glutamate receptors represent a fairly recent addition to the family of glutamate receptors. These receptors have the distinguishing feature of being coupled to G-proteins rather than ion channels and they appear to have a variety of functional characteristics. These receptors play a vital role, for example, in the induction and maintenance of long-term potentiation, the most popular current model of the biological correlates of learning and memory. Blockade of metabotropic glutamate receptors prevents long-term potentiation induction and learning in a variety of tasks in different species. Chronic metabotropic glutamate receptor activation is also associated with neurodegeneration and selective neuronal loss when agonists of these receptors are injected in high concentrations directly into the brain. Metabotropic glutamate receptors also play a role in the normal development of the nervous system and these sites within the central nervous system offer possible routes for drug therapies; selective receptor antagonists, for example, may prove to have the very desirable feature of endowing neuroprotection during ischaemic episodes whilst allowing normal excitatory neurotransmission to occur.

Animals↗
PubMed1999

Stress impairs performance in spatial water maze learning tasks.

The water maze task has been developed to test spatial learning abilities in rats or mice, and is widely used. Though it has been reported before that numerous cognitive abilities are of importance for learning this task, poor performance is usually interpreted as an impairment of spatial memory formation. Previous investigations that tried to correlate long-term potentiation (LTP) of synaptic transmission with spatial learning abilities in rats reported that injection of drugs or specific gene deletions which blocked the expression of LTP correlated with learning impairments of spatial tasks in a water maze. Recent studies, however, have shown that pretraining enables these animals to learn such spatial tasks even though LTP was still found to be blocked. I investigated to what degree altered fear condition and stress perception could account for the impaired spatial learning when no pretraining is given. In a fear habituation task, unhandled rats preferred a dark over a well lit chamber more than handled animals did, but unhandled rats favoured the lit chamber more in an active avoidance task. They also performed poorly in a spatial water maze task compared with handled rats. Rats pretrained in a radial arm maze performed better in a water maze than non-pretrained rats. No difference between groups was found in a non-spatial water maze task. On the other hand, when pretrained in a water maze, rats performed only marginally better in a radial arm maze compared to non-pretrained animals. Since animals have to be handled to learn a radial arm maze, the difference in this task was not due to stress but most probably due to getting accustomed to the room dimensions prior to learning the spatial task. The results suggest that impaired learning of spatial tasks in the water maze can be due to increased stress and decreased fear conditioning without actually affecting spatial learning abilities. These results question the interpretations of the results of some previously published results of spatial water maze tasks.

Animals↗
PubMed1999

Nitric oxide is required for expression of LTP that is induced by stimulation phase-locked with theta rhythm.

Long-term potentiation (LTP) can be induced by giving only one burst (five stimuli at 200 Hz) on the positive phase of sensory-induced theta rhythm in awake or anaesthetized rats, a stimulation protocol that mimics naturally occurring neuronal activity. Nitric oxide has been discussed as an important neuronal messenger in the induction of LTP. However, experiments testing inhibitors of nitric oxide synthase (NOS) in vitro produced contradictory results. The non-specific NOS inhibitor Nitro-L-arginine (L-NARG) impaired LTP induced by high-frequency stimulation (HFS) [from 155 +/- 7% to 122 +/- 8%), but completely blocked theta-dependent LTP induction (161 +/- 8% to 102 +/- 5%). NOS inhibitors, e.g. 7-nitro indazole (7-NI) or 1-(2-trifluoromethylphenyl) imidazole (TRIM) that are more selective for neuronal NOS and affect blood pressure less also impaired HFS-induced LTP (186 +/- 11% to 135 +/- 9% for TRIM) but completely blocked theta-dependent LTP (154 +/- 7 to 91 +/- 8). L-Arginine reversed the effects of the NOS inhibitors tested. Therefore, NO appears to be a modulator that is important for synaptic plasticity in this more physiological stimulation technique in vivo. NO is not released in slice preparations in sufficient quantities or at the right timing. Instead, the unphysiologically strong HFS protocol appears to induce an NO-independent type of LTP in some cases.

Anesthesia↗
PubMed1998

Beta-amyloid induced reduction in synaptic transmission is reversed by inhibitors of nitric oxide synthase.

beta-Amyloid has been shown to be neurotoxic in vivo and in vitro. Free radical production and subsequent lipid oxidation after beta-amyloid application have been observed in vitro and are considered to be factors that contribute to the neurotoxicity. Field recordings in the area CA1 for 3 weeks showed a dose-dependent effect on amplitude after intracerebroventricular (i.c.v.) injections of 1, 5 or 10 nmol beta-amyloid (25-35). The nitric oxide synthase inhibitors 7-nitro indazole (30 mg/kg, i.p.) and 1-(2-trifluoromethylphenyl)imidazole (150 nmol, i.c.v.) which preferentially inhibit the neuronal isoform prevented this beta-amyloid-induced decay of synaptic transmission. The protective effect of these inhibitors was reversed by L-arginine (200 mg/kg, i.p.). The results support the theory that nitric oxide production contributes to beta-amyloid-induced neuronal degeneration or reduction of neurotransmission.

Amyloid beta-Peptides↗
PubMed1998

Possible causes of Alzheimer's disease: amyloid fragments, free radicals, and calcium homeostasis.

Alzheimer's disease (AD) is a form of dementia in which patients develop neurodegeneration and complete loss of cognitive abilities and die prematurely. No treatment is known for this condition. Evidence points toward beta-amyloid as one of the main causes for cytotoxic processes. The cascade of biochemical events that lead to neuronal death appears to be interference with intracellular calcium homeostasis via activation of calcium channels, intracellular calcium stores, and subsequent production of free radicals by calcium-sensitive enzymes. The glutamatergic system seems to be implicated in mediating the toxic processes. Several strategies promise amelioration of neurodegenerative developments as judging from in vitro experiments. Glutamate receptor-selective drugs, antioxidants, inhibitors of nitric oxide synthase, calcium channel antagonists, receptor or enzyme inhibitors, and growth factors promise help. Especially combinations of drugs that act at different levels might prolong patients' health.

Alzheimer Disease↗
PubMed1998

Defective nitric oxide effector functions lead to extreme susceptibility of Trypanosoma cruzi-infected mice deficient in gamma interferon receptor or inducible nitric oxide synthase.

Trypanosoma cruzi, the causative agent of Chagas' disease, induces an innate and adaptive host immune response during the acute phase of infection. These responses were analyzed by comparing mouse lines deficient for the gamma interferon (IFN-gamma) receptor (IFN-gammaR(-/-)) or deficient for inducible nitric oxide synthase (iNOS(-/-)). Both lines were highly susceptible, with similar and dramatically increased parasite burdens and severe histopathology and were incapable of surviving even very low doses, exhibiting similar mortality kinetics. This pathophysiological correlation has a common cause, since both mutant mouse strains were unable to respond to infection by producing nitric oxide (NO) with the consequence that mutant macrophages had impaired trypanocidal activities. These in vivo and subsequent in vitro studies further demonstrated that an IFN-gamma-dependent pathway of iNOS induction is crucial for efficient NO production and mandatory for resisting acute infection with T. cruzi. Despite this defect, both mutant mouse strains had a rather normal proinflammatory cytokine response (interleukin-12 [IL-12], IFN-gamma, IL-6), with the exception of an impaired tumor necrosis factor alpha and IL-1alpha response in IFN-gammaR(-/-) mice, demonstrating that only the latter two cytokines are dependent on IFN-gamma activation. Moreover, polarization of T cells in type 1 and type 2 T-helper (Th1/Th2) and cytotoxic T (Tc1/Tc2) cells as well as T. cruzi-specific antibody responses were normal in IFN-gammaR(-/-) mice, demonstrating that IFN-gamma is not necessary for the promotion of T-cell differentiation and T. cruzi-specific antibody responses.

Animals↗
PubMed1998

Overexpression of nonconvertible PrPc delta114-121 in scrapie-infected mouse neuroblastoma cells leads to trans-dominant inhibition of wild-type PrP(Sc) accumulation.

One hallmark of prion diseases is the accumulation of the abnormal isoform PrP(Sc) of a normal cellular glycoprotein, PrPc, which is characterized by a high content of beta-sheet structures and by its partial resistance to proteinase K. It was hypothesized that the PrP region comprising amino acid residues 109 to 122 [PrP(109-122)], which spontaneously forms amyloid when it is synthesized as a peptide but which does not display significant secondary structure in the context of the full-length PrPc molecule, should play a role in promoting the conversion into PrP(Sc). By using persistently scrapie-infected mouse neuroblastoma (Sc+-MNB) cells as a model system for prion replication, we set out to design dominant-negative mutants of PrPc that are capable of blocking the conversion of endogenous, wild-type PrPc into PrP(Sc). We constructed a deletion mutant (PrPc delta114-121) lacking eight codons that span most of the highly amyloidogenic part, AGAAAAGA, of PrP(109-122). Transient transfections of mammalian expression vectors encoding either wild-type PrPc or PrPc delta114-121 into uninfected mouse neuroblastoma cells (Neuro2a) led to overexpression of the respective PrPc versions, which proved to be correctly localized on the extracellular face of the plasma membrane. Transfection of Sc+-MNB cells revealed that PrPc delta114-121 was not a substrate for conversion into a proteinase K-resistant isoform. Furthermore, its presence led to a significant reduction in the steady-state levels of PrP(Sc) derived from endogenous PrPc. Thus, we showed that the presence of amino acids 114 to 121 of mouse PrPc plays an important role in the conversion process of PrPc into PrP(Sc) and that a deletion mutant lacking these codons indeed behaves as a dominant-negative mutant with respect to PrP(Sc) accumulation. This mechanism could form a basis for a new gene therapy and/or a prevention concept for prion diseases.

Animals↗