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  • 1
    Abstract: BACKGROUND: Reactivation of latent viruses such as human cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naive T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs ("SmyleDCpp65") that accelerated antigen-specific T cell development. METHODS: Here we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-alpha and the cytomegalovirus antigen pp65) that were cryopreserved and thawed. RESULTS: Upscaling and standardized production of one lot of IDLV and three lots of SmyleDCpp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCpp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCpp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCpp65 was validated. CONCLUSION: These results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.
    Type of Publication: Journal article published
    PubMed ID: 26198406
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  • 2
    Keywords: THERAPY ; PROTEIN ; T-CELLS ; leukemia ; NATURAL-KILLER-CELLS ; CANCER-IMMUNOTHERAPY ; PROTOONCOGENE ; CYTOTOXIC ACTIVITY ; LINE NK-92 ; ERBB-2 RECEPTOR
    Abstract: Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3? signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. ?-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy.Molecular Therapy (2014); doi:10.1038/mt.2014.219.
    Type of Publication: Journal article published
    PubMed ID: 25373520
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  • 3
    Keywords: LUNG-CANCER ; T-CELLS ; COLORECTAL-CANCER ; CERVICAL-CANCER ; NATURAL-KILLER-CELLS ; RECEPTORS ; CLINICAL-APPLICATIONS ; NKG2D ligands ; MEDIATED CYTOLYSIS ; MULTICELLULAR SPHEROIDS
    Abstract: BACKGROUND: The complex cellular networks within tumors, the cytokine milieu, and tumor immune escape mechanisms affecting infiltration and anti-tumor activity of immune cells are of great interest to understand tumor formation and to decipher novel access points for cancer therapy. However, cellular in vitro assays, which rely on monolayer cultures of mammalian cell lines, neglect the three-dimensional architecture of a tumor, thus limiting their validity for the in vivo situation. METHODS: Three-dimensional in vivo-like tumor spheroid were established from human cervical carcinoma cell lines as proof of concept to investigate infiltration and cytotoxicity of NK cells in a 96-well plate format, which is applicable for high-throughput screening. Tumor spheroids were monitored for NK cell infiltration and cytotoxicity by flow cytometry. Infiltrated NK cells, could be recovered by magnetic cell separation. RESULTS: The tumor spheroids were stable over several days with minor alterations in phenotypic appearance. The tumor spheroids expressed high levels of cellular ligands for the natural killer (NK) group 2D receptor (NKG2D), mediating spheroid destruction by primary human NK cells. Interestingly, destruction of a three-dimensional tumor spheroid took much longer when compared to the parental monolayer cultures. Moreover, destruction of tumor spheroids was accompanied by infiltration of a fraction of NK cells, which could be recovered at high purity. CONCLUSION: Tumor spheroids represent a versatile in vivo-like model system to study cytotoxicity and infiltration of immune cells in high-throughput screening. This system might proof useful for the investigation of the modulatory potential of soluble factors and cells of the tumor microenvironment on immune cell activity as well as profiling of patient-/donor-derived immune cells to personalize cellular immunotherapy.
    Type of Publication: Journal article published
    PubMed ID: 25933805
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  • 4
    ISSN: 1432-1440
    Keywords: Epithelial transport ; Kidney ; Lactate transport ; Electrolyte transport ; Epithelialer Transport ; Niere ; Laktattransport ; Elektrolyttransport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Zusammenfassung Resorption bzw. Sekretion im proximalen Nierentubulus läuft einmal passiv auf dem parazellulären Weg, d.i. zwischen den Zellen hindurch, ab, zum anderen aktiv, transzellulär, durch die Zellen hindurch. Der transzelluläre aktive Transport ist in der Regel sekundär aktiv. Er verläuft gekoppelt an den Fluß von Na+-Ionen, wobei ein transzellulärer Gradient von Na+-Ionen, der seinerseits durch die kontraluminal gelegene (Na+-K+)-ATPase geschaffen wird, die Triebkraft liefert. Einmal in der Zelle, verlassen die Substanzen die kontraluminale Zellseite vermittels Karrier, die Na+-unabhängig sind. Mit Hilfe von Mikroperfusions- und elektrophysiologischen Techniken sowie mit Hilfe von Bürstensaumvesikeln wurde der Na+-Kotransport von Aminosäuren, Phosphat, Sulfat, Thiosulfat, Gallensäuren, aliphatischen und aromatischen Monokarboxylsäuren (Laktat) sowie der von Dikarboxylsäuren untersucht. Besonderes Augenmerk wurde dem bidirektionalen Transport von Thiosulfat sowie der Spezifität des Mono- und Dikarboxylsäure-Transportsystems gewidmet.
    Notes: Summary The transport through the epithelial cell layer of the renal proximal tubule proceeds in principle by passive paracellular and active transcellular transport. The active transcellular transport is mostly secondary active. This means it proceeds coupled with the flux of Na+ ions, where-by the transcellular gradient of sodium, created by the (Na++K+)-ATPase, located at the contraluminal cell side, provides the main driving force. Once in the cell the substances leave the other cell side by a Na+-independent, but carrier-mediated transport system. Using microperfusion and electrophysiological techniques as well as brush border membrane vesicle preparation the Na+-H+ countertransport and the Na+-cotransport of amino acids, phosphate, sulfate, thiosulfate, bile acids, aliphatic-aromatic monocarboxylic acids (lactate) and dicarboxylic acids was studied. Special emphasis will be given to the bidirectional transport of thiosulfate as well as to the specificity of the monocarboxylic acid and dicarboxylic acid transport system.
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  • 5
    ISSN: 1432-2013
    Keywords: Renal Microperfusion ; Isotonic Reabsorption ; Tracer Permeability ; Glomerulo Tubular Balance ; Renale Mikroperfusion ; Isotone Resorption ; Tracerpermeabilität ; Glomerulotubuläre Balance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary In the first experimental series proximal convolutions of the rat kidney were perfused with a modified Ringer solution and the isotonic fluid absorption was measured. In a second series the tubule was perfused with equilibrium solution which contained36Cl and the chloride permeability was determined. By the recollection method each individual tubule was perfused twice either at constant luminal diameter but different perfusion rates (10:30 or 6:16 nl/min) or at constant perfusion rates but different luminal diameters (20:30 μ). The perfusate was recollected at two different sites which were at least 500 μ distant from the infusion site. The isotonic fluid absorption as well as the36Cl permeability was unchanged when the tubule was distended from 20–30 μ. Both, however, increased about 20% when the perfusion rate was increased 3-fold. The data led to the following conclusions: 1. It is unlikely that there is a flow reactor type dependence of proximal tubular transport on flow rate. 2. The tubular distension cannot be responsible for the glomerulo-tubular balance. 3. It is more advantageous to relate permeability data of the rat nephron to tubular length. 4. In microperfusion experiments non steady sampling does not affect transepithelial fluxes per unit tubular length, provided that the pump delivery is constant.
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Pflügers Archiv 364 (1976), S. 223-228 
    ISSN: 1432-2013
    Keywords: Renal calcium transport ; Renal calcium permeability ; Sodium dependence ; H+ transport ; Ouabain
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Using the stop flow microperfusion technique with simultaneous capillary perfusion the rate of active Ca2+ reabsorption was evaluated by measuring the static head electrochemical potential difference as well as the permeability of the tubular wall for Ca2+ ions. Under control conditions the active Ca2+ transport was calculated to be 3.35×10−13 mol/cm·s. It declined toward zero if the ambient Na+ was replaced by choline or lithium. Parallel experiments in the golden hamster showed that active Ca2+ transport, vanished completely if active Na+ transport was blocked by ouabain (1 mM). These data indicate that the active Ca2+ reabsorption from the proximal tubule depends on the active reabsorption of Na2+ presumably via a Na+−Ca2+ countertransport at the contraluminal cell membrane. The static head electrochemical potential difference of Ca2+ is the same in late and early proximal tubules. It is also not affected by the presence of acetazolamide (10−4 M) by the absence of bicarbonate or glycodiazine buffer or by the absence or presence of phosphate (2 mM).
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  • 7
    ISSN: 1432-2013
    Keywords: Dicarboxylate transport ; Sulfate transport ; Benzoyl compounds ; Phenoxy compounds ; Valproate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In order to study the specificity of the contraluminal para-aminohippurate (PAH) transport system, the inhibitory potency of monocarboxylates on the3H-PAH influx from the interstitium into cortical tubular cells in situ has been determined. The following was found: if a homologous series of fatty acids with increasing chain length is tested, inhibition of contraluminal PAH influx is first seen with valerate (app.K i 1.4 mmol/l), increasing up to nonanoate (app.K i 0.06 mmol/l) and remaining in this range up to duodecanoate, the last compound of this series which is sufficiently water-soluble. Similarly, the inhibitory potency of aromatic monocarboxylates increases with increasing hydrophobicity. If the fatty acids are esterified, their inhibitory potency is lost. If they are transformed to the respective aldehydes their inhibitory potency is preserved at a reduced degree. Introduction of a hydrophobic methyl-, ethyl-, or propyl-group increases the inhibitory potency. A β-, but not an α-oxo-group augments the inhibitory potency of phenylpropionate analogs, an OH group diminishes it, and a NH2 group abolishes it. Among phenyl-fatty acids an increase in affinity is observed from phenyl- 〈 benzoylamine-〈 phenoxy- 〈 benzoyl-acetate and-propionate. All monocarboxylate compounds, so far tested, do not inhibit contraluminal sulfate and Na+/succinate influx. The data indicate that the PAH transporter interacts with monocarboxylates and also with aldehydes which have a hydrophobic moiety. An additional oxo-group facilitates the interaction. Thus, the benzoyl compounds show the highest affinity observed.
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  • 8
    ISSN: 1432-2013
    Keywords: Proximal Kidney Tubule ; Mercurials ; SH Reagents ; Site Group Reagents ; Transtubular Transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The effects of site group reagents were tested on the following transport processes of the proximal convolution. Isotonic Na+ absorption, evaluated by the shrinking droplet procedure, histidine and glucose transport, evaluated by measuring the respective transtubular concentration difference at zero substance and water net flux. The test substances were applied either by continuous microperfusion of the peritubular capillaries or by luminal perfusion prior to the transport tests or by addition to the luminal test solution. The SH reagents (0.2 mM) N-ethylmaleimide,p-chloromercuribenzoate (pCMB) 3,6-bis-(acetatomercurimethyl)dioxane and Mersalyl (Salyrgane) caused 50% inhibition of the isotonic Na+ absorption in approximately 1.5 min when applied to the capillary perfusate. The same effect was reached in 2–3 min by 0.2 mMp-chloromercuriphenylsulfonate, benzamido-4-iodo-acetylstilbene-2,5-disulfonate and 2,2′-dihydroperoxy-2,2′-dibutylperoxide. However, the large molecular SH reagentspCMB-dextran T10 and benzoxanthene-3,4-dicarboxylic-N-iodoacetyloligoprolyl-2-aminoethylimid, did not inhibit the isotonic Na+ absorption. If an inhibitory effect was observed on the Na+ transport its onset was faster, when the substance was applied from the blood site than when it was given from the tubular lumen. Because SH reagents inhibit the isotonic Na transport faster when applied from the blood side, and because SH reagents with MW up to 690 are inhibitory whereas larger ones with MW over 1700 are not, it seems that they exert their inhibitory action on SH groups located a) predominantly on the blood side and b) deep within the membrane and not at the surface. Histidine- and glucose transport was inhibited only when the sodium transport was inhibited considerably. The oxygen consumption of teased kidney slices is not inhibited by 0.2 mMpCMB or Mersalyl within 10 min, but it is inhibited considerably by 1 mM of these substances in the same period of incubation time. The COOH reagents N,N′-carbonyl-diimidazole and N-ethyl-N′-(3-dimethyl-aminopropyl)carbodiimid (10 mM) and the NH2 reagents 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid, 2 Na+ (SITS) (1 mM) as well as danslychloride (applied from the lumen at 5 mM in paraffin oil) did not inhibit the isotonic Na+ absorption.
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  • 9
    ISSN: 1432-2013
    Keywords: Renal tubule ; Phosphate transport ; pH dependence ; Micropuncture ; Microperfusion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Early loops of the proximal convoluted tubule of parathyroidectomized rats (PTX-rats) were microperfused with a phosphate (4 mM) containing perfusate. With a perfusion solution of pH around 7.45 as estimated as anion deficit theP i reabsorption was two times greater than with a perfusion solution of pH around 6.85. TheP i reabsorption is reduced in PTX-rats made chronic alkalotic (PTX-cA-rats) but the same pH dependence ofP i reabsorption was found. The data indicate that the divalent phosphate is preferentially reabsorbed.
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  • 10
    ISSN: 1432-2013
    Keywords: Renal tubule ; H+ ion secretion ; Na+ coupled transport ; Ouabain ; SITS
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The rate of active transport by the proximal renal tubule of amino acid (l-histidine), sugar (α-methyl-d-glycoside), H+ ions (glycodiazine), phosphate and para-aminohippurate was evaluated by measuring the zero net flux concentration difference (Δc) of these substances. In the case of calcium the electrochemical potential differenceΔc +zFci Δϕ/RT) was the criterion employed. The rate of isotonic Na+-absorption (JNa) was measured with the shrinking droplet method. The effect of ouabain on the transport of these substances was tested in the golden hamster and the effect of SITS (4-acetamido-4′isothiocyanatostilbene 2,2′-disulfonic acid) was observed in rats. Ouabain (1 mM) applied peritubularly incompletely inhibited JNa (80%), but in combination with acetazolamide (0.2 mM) the inhibition was almost complete (93%). In addition, ouabain inhibited the sodium coupled (secondary active) transport processes ofl-histidine, α-methyl-d-glycoside, calcium and phosphate by more than 75%. It did not affect H+ (glycodiazine) transport and PAH transport was only slightly affected. When SITS (1 mM) was applied from both sides of the cell it inhibited H+ (glycodiazine) transport by 72% and reduced JNa by 38% when given from only the peritubular cell side. SITS (1 mM), however, had no significant affect on H+ secretion and sodium reabsorption if it was applied from only the luminal side. Furthermore it had no affect on the other transport processes tested, regardless of the cell side to which it was applied. When the HCO 3 − buffer or physically related buffers were omitted from the perfusate the absorption of Na+ was reduced by 66%, phosphate by 44%, andl-histidine by 15%. All the other transport processes tested were not significantly affected. The data are consistent with the hypothesis that the active transport processes of histidine, α-methyl-d-glycoside and phosphate, which are located in the brush border, are driven by a sodium gradient which is abolished by ouabain. This may also apply to the Na+-Ca2+ countertransport located at the contraluminal cell side. The residual Na+ transport remaining in the presence of ouabain is likely to be passively driven by the continuing H+ transport which probably is driven directly by ATP. SITS seems to inhibit the exit step of HCO 3 − from the cell and secondary to that, the luminal H+-Na+ exchange and consequently the Na+ reabsorption. In the absence of HCO 3 − buffer in the perfusates the luminal H+-Na+ exchange seems to be affected and the pattern of inhibition of the other transport processes is almost the same as with SITS. The different effects onP i reabsorption observed under these conditions might be explained by possible variations in intracellular pH.
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