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H Franke

Publications and source records attributed to H Franke.

At least 37 records · Page 2Linked to original sources
PubMed2000

Primary cultures of brain microvessel endothelial cells: a valid and flexible model to study drug transport through the blood-brain barrier in vitro.

Studies on drug entry into the brain and permeation of the blood-brain barrier start to gain more and more importance in neuropharmaceutical research in order to develop new drugs for the therapy of central nervous system diseases. Procedures that provide quick access to permeation properties of those drugs with high throughput are difficult to achieve with animal models. Although various useful cell culture models approaching this issue have been described, results are often not comparable among each other unless determined with an equal experimental setup. Reproducibility of cell culture methods as well as corresponding findings gathered with these tools are often impeded due to the lack of details in experimental manuals. Here we present a precise manual for preparation and maintenance of porcine brain microvessel endothelial cells, serving as a culture model of the blood-brain barrier. Furthermore experimental details for blood-brain barrier transport investigations are presented. Validation of this model was carried out by determination of bioelectric properties and permeation experiments using various marker molecules reflecting paracellular and transcellular blood-brain barrier penetration. Results obtained with our model are closely resembling the in vivo-situation although astrocytes are not included. This simplification of the system is one of the major advantages towards robot derived cell cultures necessary for high throughput screening.

Animals↗
PubMed2000

Inhibition by adenosine A(2A) receptors of NMDA but not AMPA currents in rat neostriatal neurons.

Whole-cell patch clamp experiments were used to investigate the transduction mechanism of adenosine A(2A) receptors in modulating N-methyl-D-aspartate (NMDA)-induced currents in rat striatal brain slices. The A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) inhibited the NMDA, but not the (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) current in a subset of striatal neurons. Lucifer yellow-filled pipettes in combination with immunostaining of A(2A) receptors were used to identify CGS 21680-sensitive cells as typical medium spiny striatal neurons. Dibutyryl cyclic AMP and the protein kinase A activator Sp-cyclic AMPs, but not the protein kinase A inhibitors Rp-cyclic AMPS or PKI(14 - 24)amide abolished the inhibitory effect of CGS 21680. The phospholipase C inhibitor U-73122, but not the inactive structural analogue U-73343 also interfered with CGS 21680. The activation of protein kinase C by phorbol 12-myristate 13-acetate or the blockade of this enzyme by staurosporine did not alter the effect of CGS 21680. Heparin, an antagonist of inositol 1, 4,5-trisphosphate (InsP(3)) and a more efficient buffering of intracellular Ca(2+) by BAPTA instead of EGTA in the pipette solution, abolished the CGS 21680-induced inhibition. The calmodulin antagonist W-7 and cytochalasin B which enhances actin depolymerization also prevented the effect of CGS 21680; the calmodulin kinase II inhibitors CaM kinase II(281 - 309) and KN-93 but not the inactive structural analogue KN-92 were also effective. The calcineurin inhibitor deltamethrin did not interfere with CGS 21680. It is suggested that the transduction mechanism of A(2A) receptors to inhibit NMDA receptor channels is the phospholipase C/InsP(3)/calmodulin and calmodulin kinase II pathway. The adenylate cyclase/protein kinase A and phospholipase C/protein kinase C pathways do not appear to be involved.

Adenylyl Cyclases↗
PubMed2000

Pharmacokinetics of single-dose telmisartan 120 mg given during and between hemodialysis in subjects with severe renal insufficiency: comparison with healthy volunteers.

The pharmacokinetics of oral telmisartan 120 mg evaluated in subjects with severe renal insufficiency between dialyses and during hemodialysis were compared with those observed in healthy male subjects. Between dialyses and during dialysis, the plasma concentration-time curves of subjects with renal insufficiency were lower than those of healthy subjects. The mean plasma protein binding of telmisartan was 99.5% in healthy subjects, compared with mean values of 99.1% between dialyses and 98.8% during dialysis. Only very small amounts of telmisartan were removed by dialysis. Single doses of telmisartan 120 mg were well tolerated in subjects with severe renal insufficiency when administered either between dialyses or during dialysis, and no clinically relevant changes in vital signs were detected. In conclusion, the maximum plasma concentrations of telmisartan and areas under the plasma concentration-time curves in subjects with severe renal insufficiency were markedly reduced compared with healthy subjects. The fraction of telmisartan not bound to plasma proteins was increased approximately twofold. Changes in the pharmacokinetic profile in subjects with renal insufficiency did not affect the safety profile of telmisartan, which was well tolerated in these subjects.

Adult↗
PubMed1999

Neuroprotection by ATP-dependent potassium channels in rat neocortical brain slices during hypoxia.

Morphological changes induced by 30 min of hypoxia (incubation in medium saturated with 95% N2-5% CO2 instead of the normal 95% O2-5% CO2) were investigated in neurons (layers II/III of the parietal cortex) of rat neocortical brain slices. The cells were identified as intact, reversibly or irreversibly injured. As expected, hypoxia decreased the number of intact cells and increased the number of irreversibly injured cells. Pretreatment of slices with diazoxide (300 microM), an agonist of ATP-dependent potassium (KATP) channels completely prevented the morphological damage induced by hypoxia, whereas tolbutamide (300 microM), an antagonist of KATP channels, was ineffective when given alone. However, tolbutamide (300 microM) co-applied with diazoxide (300 microM), partly reversed the neuroprotective effect of this agonist during hypoxia. In conclusion, KATP channels appear to be present on neocortical neurons and their opening counteracts hypoxia-induced cell injury.

ATP-Binding Cassette Transporters↗
PubMed1999

An improved low-permeability in vitro-model of the blood-brain barrier: transport studies on retinoids, sucrose, haloperidol, caffeine and mannitol.

Primary cultures of porcine brain capillary endothelial cells grown on collagen coated polycarbonate membranes were used to build up an in vitro-model for the blood-brain barrier. Improved cultivation techniques allowed cell-storage and experiments under serum-free conditions. We employed this model to perform permeability studies in vitro with the radioactively labelled marker substances sucrose, retinoic acid, retinol, haloperidol, caffeine, and mannitol. Permeability values obtained with this blood-brain barrier model (1. 0x10-6 cm/s for sucrose, 6.2x10-6 cm/s for retinoic acid, 4.8x10-6 cm/s for retinol, 49.5x10-6 cm/s for haloperidol, 62.4x10-6 cm/s for caffeine, and 1.8x10-6 cm/s for mannitol) show a good correlation to data which are already known from in vivo-experiments. As judged by the sucrose permeability our blood-brain barrier model is less permeable than numerous other models published so far. Therefore it represents a powerful tool for in vitro-prediction of blood-brain barrier permeability of drugs and offers the possibility to scan a large quantity of drugs for their potential to enter the brain.

Animals↗
PubMed1999

How many myeloid post-progenitor cells have to be transplanted to completely abrogate neutropenia after peripheral blood progenitor cell transplantation? Results of a computer simulation.

Although hematopoietic recovery following high-dose chemotherapy (HD-CT) and peripheral blood progenitor cell (PBPC) transplantation is rapid, there is still a 5- to 7-day period of severe neutropenia which, theoretically, might be abrogated by an additional transplantation of more differentiated myeloid post-progenitor cells (MPPC). However, both the number of MPPC required to abrogate neutropenia as well as the optimum scheduling of MPPC infusions are currently unknown. Therefore, these questions were addressed by applying a computer model of human granulopoiesis. First, model calculations simulating varying levels of chemotherapy dose intensity were performed and compared with typical clinical neutrophil recovery curves. Using this approach, the data for HD-CT without PBPC transplantation could be reproduced by assuming a reduction of stem cells, committed granulopoietic progenitors and proliferating precursors to about 0.001% of normal. PBPC-supported HD-CT was reproduced by increasing the starting values to at least 0.1%, which corresponded to about 1 to 2 x 10(5)/kg transplanted CFU-GM. Interestingly, reproduction of PBPC-supported HD-CT data could be observed for a wide range of starting values (0.1%-10% of normal), thus confirming the clinical observation that hematopoietic recovery after PBPCT cannot be improved by increasing the dose of transplanted cells over a certain threshold. Using the same simulation model, we then studied the effects of an additional MPPC transplantation. The results showed, that at least 5.7 X 10(8) MPPC/kg have to be provided in addition to the normal PBPC graft to avoid neutropenia <100/microL, and that MPPC are best transplanted on days 0 and 6 after HD-CT. Assuming a 100- to 120-fold cellular ex-vivo expansion rate and MPPC representing about 70% of total expanded cells, 5.7 X 10(8) MPPC/kg could be generated starting from 1 to 2 leukapheresis preparations with about 7 to 8 x 10(6) CD34+ PBPC/kg. Considering furthermore, that only a fraction of ex-vivo generated cells will seed and effectively produce neutrophils in-vivo, the required number of MPPC is most likely even higher and, therefore, might be difficult to be achieved clinically. However, the validity of the model results remains to be proven in appropriate clinical studies.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1999

Endogenous thrombopoietin serum levels during multicycle chemotherapy.

Little is known about the behaviour of endogenous thrombopoietin (TPO) serum levels during rapid sequences of dose-intensified chemotherapy. To characterize the relationship between TPO levels and platelet counts in this setting we serially measured both parameters over the entire treatment period of patients receiving multicycle polychemotherapy. We found TPO and platelet responses to be generally antagonistic through all cycles. However, a cross-correlation analysis indicated that TPO responses preceded platelet responses by approximately one day in all patients. The cumulative severity of thrombocytopenia observed over successive cycles was accompanied by an increasing TPO response which tended to grow overproportionally in relation to the degree of peripheral thrombocytopenia. These findings are consistent with a model suggesting that both platelet and megakaryocyte mass contribute to a receptor-dependent consumption process regulating the endogenous TPO level. In order to develop optimal schedules for exogenous TPO administration it might be important to consider endogenous TPO response characteristics.

Adult↗
PubMed1999

P2 receptor-mediated proliferative effects on astrocytes in vivo.

Astrogliosis in response to injury usually represents up-regulation of glial fibrillary acidic protein (GFAP), hypertrophy, and proliferation. Following pathological events in brain tissue, purine nucleotides and nucleosides are released into the extracellular space. The (patho)physiological importance and molecular mechanisms of the purinoceptor-mediated effects are nearly unknown. In the present study, the involvement of extracellular ATP in astrogliotic processes via stimulation of P2 receptors was investigated. The structural analogue, 2-methylthio ATP (2-MeSATP) and its antagonists reactive blue 2 and pyridoxal-phosphate-6-azophenyl-2,4-disulphonic acid (PPADS) were microinfused in situ into the rat nucleus accumbens. The reaction of astrocytes in the nucleus accumbens was investigated by GFAP- and 5-bromo-2'-deoxyuridine (BrdU)- immunocytochemistry. Tissue injury due to the microinjection procedure caused astrogliosis, which was increased further by 2-MeSATP. Up-regulation of GFAP-immunoreactivity, hypertrophy of astrocytes, and an increase in the number of GFAP-positive and of GFAP-/BrdU-double labeled cells were observed. Reactive blue 2 and PPADS decreased the consequences of tissue injury on astrocytic proliferation when given alone. In addition, both antagonists counteracted the 2-MeSATP-induced astrogliosis, supporting the hypothesis that purine nucleotides are involved in these processes via stimulation of P2 receptors in vivo.

Adenosine Triphosphate↗
PubMed1998

Hydrocortisone reinforces the blood-brain properties in a serum free cell culture system.

The increasing number of newly developed drugs demands for functional in vitro models of the blood-brain barrier to determine their brain uptake. Cultured cerebral capillary endothelial cells are considered to be such a model, however in serum containing media they exhibit low electrical resistances and high permeabilities compared to the in vivo situation. Here we report the establishment of a serum-free cell culture model. Withdrawal of serum already caused a twofold increase of transendothelial resistance (TER), which in presence of serum is about 100-150 Omega x cm2. We tested several supplements and found that hydrocortisone is a potent stimulator for the formation of barrier properties. TERs up to 1000 Omega x cm2 were measured in the presence of physiological relevant hydrocortisone concentrations. In correspondence to the TER increase hydrocortisone decreased cell monolayer permeability for sucrose down to 5x10(-7) cm/s, which is close to the in vivo value of 1.2x10(-7) cm/s and by a factor of five lower compared to cultures without hydrocortisone and in presence of serum.

Animals↗
PubMed1998

Hydrocortisone reinforces the blood-brain barrier properties in a serum free cell culture system.

The increasing number of newly developed drugs demands for functional in vitro models of the blood-brain barrier to determine their brain uptake. Cultured cerebral capillary endothelial cells are considered to be such a model, however in serum containing media they exhibit low electrical resistances and high permeabilities compared to the in vivo situation. Here we report the establishment of a serum-free cell culture model. Withdrawal of serum already caused a twofold increase of transendothelial resistance (TER), which in presence of serum is about 100-150 omega.cm2. We tested several supplements and found that hydrocortisone is a potent stimulator for the formation of barrier properties. TERs up to 1000 omega.cm2 were measured in the presence of physiological relevant hydrocortisone concentrations. In correspondence to the TER increase hydrocortisone decreased cell monolayer permeability for sucrose down to 5 x 10(-7) cm/s, which is close to the in vivo value of 1.2 x 10(-7) cm/s and by a factor of five lower compared to cultures without hydrocortisone and in presence of serum.

Animals↗
PubMed1998

Differential age-dependent expression of alpha2 adrenoceptor- and P2 purinoceptor-functions in rat locus coeruleus neurons.

Whole-cell patch clamp recordings were made in a pontine slice preparation of the rat brain containing the nucleus locus coeruleus (LC). In a first series of experiments, it was demonstrated that tyrosine hydroxylase-positive LC neurons of young (10-14 days of age) rats are multipolar with numerous dendrites. When pipettes filled with the marker molecule biocytin were used for recording, all cells exhibiting outward current responses to noradrenaline (100 microM) showed the morphology typical for LC neurons. At a holding potential of -80 mV, noradrenaline (100 microM) produced a comparably small outward current both in LC neurons of young (8-14 days) and older (18-23 days) rats. In contrast, 2-methylthio ATP (2-MeSATP; 100 microM) caused a relatively small inward current in the young animals, while inward current responses were much larger in most older animals (8 out of a total of 11). It is suggested that after birth there are probably no functional P2 purinoceptors present at LC neurons. Thereafter, P2 purinoceptor-function increases with age, reaching maturity only in animals older than 18 days.

Adenosine Triphosphate↗
PubMed1998

Expression of tenascin, fibronectin, and laminin in rat liver fibrogenesis--a comparative immunohistochemical study with two models of liver injury.

The aim of this study was to follow semiquantitatively by immunohistochemical means the alterations of the expression of the hepatic glycoproteins tenascin, fibronectin, and laminin in two different models of chronic liver injury, i.e. thioacetamide-induced liver cirrhosis and fibrosis after bile duct ligation. The tenascin distribution pattern observed during cholostasis-induced liver fibrosis showed some similarities, but also some differences in comparison with the results obtained after TAA intoxication. Most importantly, the data show that tenascin staining was detectable in almost all areas of the chronically injured livers up to 3 and 6 months in bile duct-ligated and chemically-injured livers, respectively. Thus, tenascin does not seem to play only a transient role in the fibrogenetic process as previously suggested. Laminin was strongly stained in proliferating ductules, whereas only a weak continuous distribution was observed along the sinusoidal wall. Furthermore, our findings confirm the role of fibronectin as a pacemaker of fibrosis. Regional differences in the kinetics of the expression of the glycoproteins may reflect local differences in their production by parenchymal or non parenchymal cells or regional patterns of proteolytic activity.

Animals↗
PubMed1998

[Not Available].

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Austria↗
PubMed1997

Histological and biochemical changes induced by total bile duct ligation in the rat.

The aim of the present investigation was to assess in a correlated biochemical and morphological study the dynamics of fibrogenesis after bile duct ligation and to compare the time course of alterations with those occurring in thioacetamide induced liver fibrosis. The data show that, after bile duct obstruction, the deposition of connective tissue elements and formation of ductular proliferates rapidly set in. The index of fibroplasia correlated well with the changes of the OH-proline concentration of the liver. Comparing the biliary fibrosis with the thioacetamide induced liver fibrosis, the progress of the former occurred more rapidly, even though in both cases only a few necroses were observed. Therefore, we suggest that in biliary fibrosis other mechanisms are responsible for the rapid onset of production of extracellular material and proliferative processes than in thioacetamide-induced liver fibrosis.

Alanine Transaminase↗
PubMed1996

Model analysis of the contrasting effects of GM-CSF and G-CSF treatment on peripheral blood neutrophils observed in three patients with childhood-onset cyclic neutropenia.

Human cyclic neutropenia (CN) is a rare haematological disorder characterized by regular fluctuation in the serial count of blood neutrophils. The oscillations occur at subnormal levels with a stable 3-week period. To reduce the risk of serious infections during the severe neutropenic nadir phases (< 0.25 x 10(9) neutrophils/l) patients are usually treated with recombinant growth factors. Compared with G-CSF, which has been shown to enhance the amplitudes substantially, the response to GM-CSF is poor: neutrophil numbers are not amplified, the cycles remain unchanged or are dampened. However, two cases with a modest neutrophil increase have been reported in the literature. In a recently published clinical study the different effects of GM-CSF and G-CSF application have been investigated in the same patients. Based on a mathematical model of CN we previously proposed, the detailed neutrophil data measured in this study are analysed by simulation. The contrasting clinical results can be quantitatively explained by the model concept of regulatory control together with possible individual feedback defects, i.e. abnormally reduced mitotic responsiveness of granulopoietic progenitor cells to GM-CSF and G-CSF.

Cell Division↗
PubMed1995

Electrical resistance measurements on cerebral capillary endothelial cells--a new technique to study small surface areas.

We present a new method which allows resistance measurements in selected cell monolayer areas with a size of less than one mm2. Up to now cell-covered macroscopic filters with areas up to 5 cm2 have been used giving reliable results only in the absence of inhomogeneities or contaminating cells. The new measuring device is posed on the microscope optic to allow an optical characterization of the measuring area and enables us to scan the cell-covered filter. This method has been applied to determine a reversible modulation of the tightness of intercellular contacts between epithelial or endothelial cells in culture. Tight junction resistance is modulated by Ca2+ and basic amino acids in cultured porcine cerebral microvascular endothelial cells that represent the blood-brain barrier.

Animals↗