Monocyte-derived dendritic cells display a highly activated phenotype and altered function in patients with familial Mediterranean fever.

Dendritic cells (DCs) are sentinels of the immune system that bridge innate and adaptive immunity. By capturing antigens in peripheral tissue, processing and presenting them with concurrent expression of co-stimulatory molecules and cytokine secretion they control and modulate immune reactions. Through pattern recognition receptors, DCs sense molecules that are associated with infection or tissue damage, frequently resulting in the formation of inflammasomes upon intracellular stimulation. The inherited autoinflammatory familial Mediterranean fever (FMF) is associated with deregulated activity of the pyrin inflammasome leading to acute inflammatory episodes. However, differentiation and function of DCs in this disease are as yet unclear. Therefore, we first determined DC subpopulation frequency in peripheral blood of a cohort of FMF patients. Joint evaluation without classification according to specific patient characteristics, such as mutational status, did not disclose significant differences compared to healthy controls. For the further examination of phenotype and function, we used immature and mature monocyte-derived DCs (imMo-DCs, mMo-DCs) that were generated in vitro from FMF patients. Immunophenotypical analysis of imMo-DCs revealed a significantly elevated expression of CD83, CD86 and human leukocyte antigen D-related (HLA-DR) as well as a significant down-regulation of CD206, CD209 and glycoprotein NMB (GPNMB) in our FMF patient group. Furthermore, FMF imMo-DCs presented a significantly higher capacity to migrate and to stimulate the proliferation of unmatched allogeneic T cells. Finally, the transition towards a more mature, and therefore activated, phenotype was additionally reinforced by the fact that peripheral blood DC populations in FMF patients exhibited significantly increased expression of the co-stimulatory molecule CD86.

Gate-Controlled WSe2 Transistors Using a Buried Triple-Gate Structure.

In the present paper, we show tungsten diselenide (WSe2) devices that can be tuned to operate as n-type and p-type field-effect transistors (FETs) as well as band-to-band tunnel transistors on the same flake. Source, channel, and drain areas of the WSe2 flake are adjusted, using buried triple-gate substrates with three independently controllable gates. The device characteristics found in the tunnel transistor configuration are determined by the particular geometry of the buried triple-gate structure, consistent with a simple estimation of the expected off-state behavior.

Differences in genome-wide gene expression response in peripheral blood mononuclear cells between young and old men upon caloric restriction.

BACKGROUND: Caloric restriction (CR) is considered to increase lifespan and to prevent various age-related diseases in different nonhuman organisms. Only a limited number of CR studies have been performed on humans, and results put CR as a beneficial tool to decrease risk factors in several age-related diseases. The question remains at what age CR should be implemented to be most effective with respect to healthy aging. The aim of our study was to elucidate the role of age in the transcriptional response to a completely controlled 30 % CR diet on immune cells, as immune response is affected during aging. Ten healthy young men, aged 20-28, and nine healthy old men, aged 64-85, were subjected to a 2-week weight maintenance diet, followed by 3 weeks of 30 % CR. Before and after 30 % CR, the whole genome gene expression in peripheral blood mononuclear cells (PBMCs) was assessed. RESULTS: Expression of 554 genes showed a different response between young and old men upon CR. Gene set enrichment analysis revealed a downregulation of gene sets involved in the immune response in young but not in old men. At baseline, immune response-related genes were higher expressed in old compared to young men. Upstream regulator analyses revealed that most potential regulators were controlling the immune response. CONCLUSIONS: Based on the gene expression data, we theorise that a short period of CR is not effective in old men regarding immune-related pathways while it is effective in young men. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00561145.

Evaluation of cyclosporine-sparing effects of polyunsaturated fatty acids in the treatment of canine atopic dermatitis.

A randomised, double-blinded, placebo-controlled multicentre trial was conducted in 36 dogs with atopic dermatitis to evaluate the cyclosporine-sparing effect of polyunsaturated fatty acids. Dogs were stable on their individual cyclosporine dosage and received either a mainly omega-3 fatty acid product with a minor omega-6 fatty acid fraction or placebo, orally for 12 weeks. Dogs were examined every 4 weeks and the Canine Atopic Dermatitis Extent and Severity Index (CADESI-03) was determined by a clinician. Pruritus, quality of life, global condition and coat quality were scored by the owner. If the dog's CADESI-03 and/or pruritus score improved by at least 25% compared with the previous visit, the cyclosporine dosage was decreased by approximately 25%. If the scores deteriorated by at least 25%, the cyclosporine dosage was increased by the same percentage. The median daily cyclosporine dosage/kg bodyweight decreased in the active group from 4.1 mg to 2.6 mg and in the placebo group from 3.5 mg to 3.3 mg over the study period. The difference between the two groups was significant (P = 0.009). The improvement in median pruritus score from inclusion to completion was significantly greater in the active group than in the placebo group (P = 0.04). There was no significant difference in CADESI-03 changes between groups (P = 0.38). The results of this study indicate a cyclosporine-sparing effect of a mainly omega-3 fatty acid supplement in dogs with atopic dermatitis.

[Surgical staging of lung cancer]. / Thoraxchirurgisches Staging des Lungenkarzinoms.

Proper staging of lung cancer represents the basis for any stage-adapted and optimized treatment. This is today implemented in specialized centers mainly through the use of modern imaging methods and minimally-invasive measures. However, general thoracic surgery has a role not only in the therapeutic management of lung cancer, but offers additional staging information whenever endoscopic or interventional methods fail to achieve representative tissue biopsies of mediastinal lymph nodes or suspect lesions for conclusive diagnosis. The thoracic surgical armentarium comprises of cervical or extended mediastinoscopy, video-assisted mediastinal lymphadenectomy (VAMLA), anterior mediastinotomy (Chamberlain procedure) and video-thoracoscopy (VATS). Indications for any invasive diagnostic methods always have to respect a therapeutic benefit for the patient.

[Pulmonary infection in neutropenia]. / Pulmonale Infektion bei Neutropenie - Fall 1/2015.

UNLABELLED: MEDICAL HISTORY AND CLINICAL COURSE: A 42-year-old patient with hairy cell leukemia had been treated for 3 years by a hematologist in private practice. Initially the patient received 1 course of cladribine upon which the disease went into complete remission. 6 weeks ago a relapse was diagnosed and combination therapy with cladibrin and rituximab was initiated. Now the patient presented to the emergency room with shortness of breath and pain when breathing. INVESTIGATIONS, TREATMENT AND COURSE: In the chest x-ray, patchy infiltrates and pleural effusions were found on both sides. The subsequently performed computed tomography showed bilateral compactions with an Halo suspicious for fungal infiltrates. Upon admission to the hospital, an empirical antibiotic therapy with clarithromycin and piperacillin/tazobactam was initiated, which was later escalated to meropenem and linezolid. Additionally, an antifungal therapy with voriconazole was started and later switched to liposomal amphotericin B. At his admission, a positive aspergillus antigen could be detected in the microbiological laboratory. Under antimycotic treatment the aspergillus antigen was repeatedly negative. The patient presented with pronounced cytopenias and after a switch of therapy to vemurafenib and filgrastim, the hematopoiesis could only be stimulated insufficiently. The patient was transferred to the intensive care unit three days after admission with severe respiratory failure. He died on day 8 after admission. AUTOPSY AND DIAGNOSIS: Diagnosis was consistent with relapse of hairy cell leukemia with positive BRAF mutation and a bone marrow infiltration > 80 %. Autopsy revealed a significant hepato-splenomegaly, a lack of erythro-, granulo- and thrombopoiesis. Clots interspersed with fungal hyphae were found in both lungs and an infarction of the spleen with evidence of fungal hyphae was detected. The cultural findings post mortem on yeast or mold were negative. CONCLUSION: Patients with refractory hairy cell leukemia and prolonged neutropenia are at increased risk for systemic fungal infections. Therefore, prohylactic antimycotic therapy should be considered early in this group of patients. The therapeutic approach of vemurafenib in treatment-refractory hairy cell leukemia is promising and offers an additional treatment option. In the present case, the patient could unfortunately not be stabilized due to the septic complications.

Resistance to EGFR targeting therapies in lung cancer.

The treatment of advanced non-small cell lung cancer (NSCLC) by therapies targeting the epidermal growth factor receptor (EGFR) pathway represents one of the most important advances in thoracic oncology. Reversible EGFR tyrosine kinase inhibitors (TKIs), like gefitinib and erlotinib, are able to achieve dramatic responses in a subset of patients. However, most patients treated with TKIs eventually develop resistance against these drugs. Here we review the physiology and pathology of EGFR activation in NSCLC, the clinical experience with TKIs, the mechanisms of resistance against TKIs, and discuss various approaches to treat resistance against TKIs.

Dissection of the IgNAR V domain: molecular scanning and orthologue database mining define novel IgNAR hallmarks and affinity maturation mechanisms.

The shark antigen-binding V(NAR) domain has the potential to provide an attractive alternative to traditional biotherapeutics based on its small size, advantageous physiochemical properties, and unusual ability to target clefts in enzymes or cell surface molecules. The V(NAR) shares many of the properties of the well-characterised single-domain camelid V(H)H but is much less understood at the molecular level. We chose the hen-egg-lysozyme-specific archetypal Type I V(NAR) 5A7 and used ribosome display in combination with error-prone mutagenesis to interrogate the entire sequence space. We found a high level of mutational plasticity across the V(NAR) domain, particularly within the framework 2 and hypervariable region 2 regions. A number of residues important for affinity were identified, and a triple mutant combining A1D, S61R, and G62R resulted in a K(D) of 460 pM for hen egg lysozyme, a 20-fold improvement over wild-type 5A7, and the highest K(D) yet reported for V(NAR)-antigen interactions. These findings were rationalised using structural modelling and indicate the importance of residues outside the classical complementarity determining regions in making novel antigen contacts that modulate affinity. We also located two solvent-exposed residues (G15 and G42), distant from the V(NAR) paratope, which retain function upon mutation to cysteine and have the potential to be exploited as sites for targeted covalent modification. Our findings with 5A7 were extended to all known NAR structures using an in-depth bioinformatic analysis of sequence data available in the literature and a newly generated V(NAR) database. This study allowed us to identify, for the first time, both V(NAR)-specific and V(NAR)/Ig V(L)/TCR V(alpha) overlapping hallmark residues, which are critical for the structural and functional integrity of the single domain. Intriguingly, each of our designated V(NAR)-specific hallmarks align precisely with previously defined mutational 'cold spots' in natural nurse shark cDNA sequences. These findings will aid future V(NAR) engineering and optimisation studies towards the development of V(NAR) single-domain proteins as viable biotherapeutics.

Activin tunes GABAergic neurotransmission and modulates anxiety-like behavior.

Activin, a member of the transforming growth factor-beta superfamily, affords neuroprotection in acute brain injury, but its physiological functions in normal adult brain are largely unknown. Using transgenic (tg) mice expressing a dominant-negative activin receptor mutant under the control of the CaMKIIalpha promoter in forebrain neurons, we identified activin as a key regulator of gamma-aminobutyric acid (GABA)ergic synapses and anxiety-like behavior. In the open field, wild-type (wt) and tg mice did not differ in spontaneous locomotion and exploration behavior. However, tg mice visited inner fields significantly more often than wt mice. In the light-dark exploration test, tg mice made more exits, spent significantly more time on a well-lit elevated bar and went farther away from the dark box as compared to wt mice. In addition, the anxiolytic effect of diazepam was abrogated in tg mice. Thus the disruption of activin receptor signaling produced a low-anxiety phenotype that failed to respond to benzodiazepines. In whole-cell recordings from hippocampal pyramidal cells, enhanced spontaneous GABA release, increased GABA tonus, reduced benzodiazepine sensitivity and augmented GABA(B) receptor function emerged as likely substrates of the low-anxiety phenotype. These data provide strong evidence that activin influences pre- and postsynaptic components of GABAergic synapses in a highly synergistic fashion. Given the crucial role of GABAergic neurotransmission in emotional states, anxiety and depression, dysfunctions of activin receptor signaling could be involved in affective disorders: and drugs affecting this pathway might show promise for psychopharmacological treatment.

Preemptive analgesia by lornoxicam--an NSAID--significantly inhibits perioperative platelet aggregation.

BACKGROUND AND OBJECTIVE: To investigate whether preemptive administered lornoxicam changes perioperative platelet function during thoracic surgery. METHODS: A total of 20 patients scheduled for elective thoracic surgery were randomly assigned to receive either lornoxicam (16 mg, i.v.; n = 10) or placebo (n = 10) preoperatively. All patients underwent treatment of solitary lung metastasis and denied any antiplatelet medication within the past 2 weeks. Blood samples were drawn via an arterial catheter directly into silicone-coated Vacutainer tubes containing 0.5 mL of 0.129 M buffered sodium citrate 3.8% before, 15 min, 4 h and 8 h after the study medication was administered. Platelet aggregation curves were obtained by whole blood electrical impedance aggregometry (Chrono Log). RESULTS: Platelet aggregation was significantly reduced 15 min, 4 h and 8 h after lornoxicam administration compared to placebo (P < 0.05) for collagen, adenosine diphosphate and arachidonic acid as trigger substances. Adenosine diphosphate-induced platelet aggregation decreased by 85% 15 min after lornoxicam administration, and remained impaired for 8 h. CONCLUSION: Platelet aggregation assays are impaired for at least 8 h after lornoxicam application. Therefore perioperative analgesia by use of lornoxicam should be carefully administered under consideration of subsequent platelet dysfunction.

Effects of various statins on cytokine-dependent growth and IgE-dependent release of histamine in human mast cells.

BACKGROUND: Statins are inhibitors of hydroxymethylglutaryl coenzyme A (HMG CoA) reductase, a key enzyme in mevalonic acid (MVA)-dependent signaling. Recent data suggest that statins exhibit profound inhibitory effects on growth and function of various immune cells. In the present study, we examined the in vitro effects of five different statins on primary human mast cells (MCs), MC progenitors, and the human MC line HMC-1. METHODS: Histamine release experiments were conducted on isolated MCs using statins and an anti-immunoglobulin E (IgE) antibody. Culture experiments were performed with stem cell factor (SCF) and interleukin (IL)-6, and cord blood-derived progenitors. RESULTS: Preincubation of primary lung MCs with cerivastatin or atorvastatin (1-50 microM) for 24 h resulted in inhibition of anti-IgE-induced release of histamine. The effects of both statins were dose-dependent. Moreover, both statins, and to a lesser degree lovastatin, were found to inhibit the SCF-induced differentiation of MCs from their progenitors. The other statins tested (simvastatin, pravastatin) did not affect mediator release or growth of MCs. CONCLUSIONS: Cerivastatin and atorvastatin act as inhibitors of growth and function of human MCs.

Delivery of tumor-derived RNA for the induction of cytotoxic T-lymphocytes.

Dendritic cells (DC) are professional antigen-presenting cells playing a central role in the induction of antigen-specific cytotoxic T-lymphocytes (CTL). We analyzed the efficiency of tumor RNA transfection into DC using different sources of RNA as well as delivery strategies including electroporation, lipofection and CD71-receptor-based delivery. To evaluate the sensitivity of these approaches, we utilized in vitro transcribed enhanced green fluorescence protein (EGFP)-RNA and whole tumor RNA from EGFP-transfected renal cell carcinoma cell line N43. We demonstrate that electroporation was the most effective way yielding about 30% EGFP positive cells while less than 1% of DC expressed EGFP using the transferrin receptor transfection system. Delivery of RNA with liposomes resulted in 17.5% of EGFP positive cells depending on the RNA amount. However, when these approaches were applied to transduce DC with RNA derived from the A498 cell line for T-cell priming, tumor-specific CTL could be induced using all delivery strategies suggesting that this technology has the potential to induce cytotoxic T-cell response even when low level of antigen is delivered. Furthermore, we demonstrate that amplification of whole tumor messenger RNA (mRNA) as well as the use of total instead of purified mRNA can be utilized for stimulating tumor-specific CTL responses.

Lipopeptide adjuvants: monitoring and comparison of P3CSK4- and LPS-induced gene transcription.

Bacteria-derived synthetic lipoproteins constitute potent macrophage activators in vivo and are effective stimuli, enhancing the immune response especially with respect to low or non-immunogenic compounds. N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)3-lysine (P3CSK4), exhibiting one of the most effective lipopeptide derivatives, represents a highly efficient immunoadjuvant in parenteral, oral, nasal and genetic immunization either in combination with or after covalent linkage to antigen. In order to further elucidate its molecular mode of action with respect to the transcriptional level, we focused our investigations on the P3CSK4-induced modulation of gene transcription. We could show that P3CSK4 activates/represses an array of at least 140 genes partly involved in signal transduction and regulation of the immune response. P3CSK4 activates the expression of tumor suppressor protein p53 (p53), c-rel, inhibitor of nuclear factor kappa B (NFkappaB) alpha (IkappaB alpha), type 2 (inducible) nitric oxide (NO) synthase (iNOS), CD40-LR, intercellular adhesion molecule-1 (ICAM-1) and interleukin 1/6/15 (IL-1/6/15). We detected no activation of heat shock protein (HSP) 27, 60, 84 and 86, osmotic stress protein 94 (Osp 94), IL-12, extracellular signal-regulated protein kinase 1 (ERK1), p38 mitogen activated protein (MAP)-kinase (p38), c-Jun NH2-terminal kinase (JNK), signal transducer and activator of transcription 1 (STAT1), CD14 and caspase genes. Furthermore, we monitored inhibition of STAT6, Janus kinase 3 (Jak3) and cyclin D1/D3 gene transcription after stimulating bone marrow-derived macrophages (BMDM) with lipopeptide. In addition, we monitored significant differences after lipopeptide and lipopolysaccharide (LPS) stimulation of bone marrow-derived murine macrophages. Our findings are of importance for further optimizing both conventional and genetic immunization, and for the development of novel synthetic vaccines.

Immunostimulation by the synthetic lipopeptide P3CSK4: TLR4-independent activation of the ERK1/2 signal transduction pathway in macrophages.

Synthetic lipopeptides based on bacterial lipoprotein are efficient activators for monocytes/macrophages inducing the release of interleukin (IL)-1, IL-6, tumour necrosis factor-alpha (TNF-alpha), reactive oxygen/nitrogen intermediates, and the translocation of nuclear factor kappaB (NFkappaB). In this report we investigate the signal transduction pathways involved in leucocyte activation by the synthetic lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)3-lysine (P3CSK4). We show that P3CSK4 activates mitogen-activated protein (MAP)-kinases ERK1/2 and MAP kinase (MAPK)-kinases MEK1/2 in bone-marrow-derived macrophages (BMDM) and in the macrophage cell line RAW 264.7. Additionally, we could detect differences between the P3CSK4 and lipopolysaccharide (LPS)-induced phosphorylation of MAP kinases: Different levels in phosphorylation were found both in kinetics and dose-response using RAW 264.7 cells or BMDM from BALB/c and LPS responder mice (C57BL/10ScSn) or LPS non-responder mice (C57BL/10ScCr). The lipopeptide activated the MAPK-signalling cascade in both LPS responder and non-responder macrophages, whereas LPS induced the MAPK signalling pathway only in macrophages derived from LPS responder mice. An approximately 70% decrease of lipopeptide induced NFkappaB translocation and an about 50% reduction of nitric oxide (NO) release was observed in the presence of anti-CD14. These data correspond to the reduction of phosphorylation of ERK1/2 after stimulation with P3CSK4 in the presence of anti-CD14 antibodies. Inhibition of MEK1/2 by PD98059 completely reduced the lipopeptide-induced phosphorylation of ERK1/2 indicating that MEK1/2 are solely responsible for the phosphorylation of the downstream-located MAP kinases ERK1/2.

[Neoadjuvant chemotherapy and chemoradiotherapy in surgically treated non-small-cell stage III bronchial carcinoma]. / Neoadjuvante Chemotherapie und Chemoradiotherapie beim operiertem nicht-kleinzelligen Bronchialkarzinom Stadium III.

Patients with unfavorable stages of lung cancer are rarely cured with local treatment modalities alone. Aim of our phase II trial was to investigate the effectivity of a multimodality treatment. Ninety-four patients with NSCLC (stage IIIA/IIIB) were treated preoperatively with chemoradiotherapy (cisplatin and etoposide, 45 Gy hyperfractionated accelerated radiotherapy). After repeat mediastinoscopy patients underwent surgery. Complete resection (R0) was achieved in 53% of all patients with NSCLC. Two patients died of sepsis preoperatively and four postoperatively (90-days lethality: 6.4%). The median survival time was 20 months for IIIA and 18 months for IIIB. Calculated survival rates at 6 years were 34% for IIIA and 17% for IIIB. This multimodality treatment demonstrates high efficacy in prognostically unfavorable NSCLC compared with historical controls.

Autoantibody reactivity in a case of Schnitzler's syndrome: evidence for a Th1-like response and detection of IgG2 anti-FcepsilonRIalpha antibodies.

Schnitzler's syndrome is a rare disease characterized by chronic urticaria, monoclonal IgM, and clinical and laboratory signs of inflammation. In a subset of patients, the urticarial lesions cause pruritus. However, the pathophysiology of the disease and the biochemical basis of urticaria are not known. We describe a female patient with Schnitzler's syndrome suffering from chronic urticaria associated with pruritus. The patient's serum was found to contain IgG antibodies recognizing cellular components of the microvasculature. In particular, IgG3 antibodies directed against proteins (14-100 kD) expressed in cultured dermal microvascular endothelial cells and mast cells, were found by immunoblotting. Moreover, IgG2 antibodies specific for the alpha-chain of the FcepsilonRI were detectable. However, the autoantibodies did not mediate histamine release in mast cells or basophils. In patients with IgM paraproteinemia who did not have Schnitzler's syndrome, antibodies against endothelial/mast cells or FcepsilonRI were not detectable. In summary, we describe subclass-specific IgG reactivity against microvascular endothelial cells and mast cells indicating Th1 autoimmunity in a patient with Schnitzler's syndrome. Whether such autoantibodies are recurrently produced in patients with Schnitzler's syndrome and play a role in the pathophysiology of the disease remains to be determined.

Multiplex PCR for the detection of Lactobacillus pontis and two related species in a sourdough fermentation.

A specific multiplex PCR assay based on the amplification of parts of the 16S rRNA molecule was designed. Primers derived from variable regions of the 16S rRNA provided a means of easily differentiating the species Lactobacillus pontis and Lactobacillus panis. They could be clearly discriminated from the phylogenetically related species Lactobacillus vaginalis, Lactobacillus oris, and Lactobacillus reuteri and from other lactobacilli commonly known to be present in sourdough. Other strains isolated together with L. pontis from an industrial sourdough fermentation could be clearly separated from these species by comparative sequence analysis and construction of a specific PCR primer. For a fast identification a DNA isolation protocol based on the ultrasonic lysis of cells from single colonies was developed. To demonstrate the potential of such techniques for tracking these organisms in a laboratory-scale fermentation, we combined the specific PCR assay with direct DNA extraction from the organisms in the sourdough without previous cultivation.

Differential modulation of chemosensitivity to alkylating agents and platinum compounds by DNA repair modulators in human lung cancer cell lines.

PURPOSE: Modulation of DNA repair represents one strategy to overcome cellular drug resistance to alkylating agents and platinum compounds. The effects of different known DNA repair modulators such as O6-benzylguanine (6 microg/ml), fludarabine (25 ng/ml), aphidicolin (8.5 ng/ml), pentoxifylline (1.4 microg/ml) and methoxamine (12.4 microg/ml) on the cytotoxicity of mafosfamide, chlorambucil, 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), cisplatin and carboplatin were tested in human lung cancer cell lines. METHODS: Chemosensitivity of the human adenocarcinoma cell line MOR/P and the cisplatin-resistant subline MOR/CPR as well as the large-cell lung cancer cell line L23/P and its cisplatin-resistant counterpart L23/CPR were evaluated by the MTT colorimetric assay. RESULTS: O6-benzylguanine, an inhibitor of O6-alkylguanine-DNA alkyltransferase, significantly sensitised MOR/P and MOR/CPR cells to the cytotoxic effect of BCNU. Fludarabine, methoxamine and aphidicolin did not change the chemosensitivity of the parental and cisplatin-resistant cell lines to any cytotoxic drug tested. Interestingly, O6-benzylguanine enhanced the chemoresistance of parental and cisplatin-resistant cell lines to platinum compounds. Also, pentoxifylline increased resistance of the MOR cell lines to mafosfamide. CONCLUSIONS: Modulation of DNA repair elicits not only chemosensitisation but may also enhance cellular resistance to DNA-affine drugs.

Simultaneous measurement of cellular P-glycoprotein content and function by multiparametric flow-cytometry.

OBJECTIVE: A multiparametric approach was applied to simultaneously determine expression and function of the drug efflux pump P-glycoprotein (PGP) in multidrug-resistant (MDR) human leukemic lymphoblast cell lines and isolated leukemic blasts using flow-cytometry in a patient with acute myeloid leukemia (AML). METHODS: The antigen was measured by staining PGP using the monoclonal antibody 4e3 which does not inhibit the function of PGP. The 4e3 antibody binds to an external epitope of PGP and can therefore be used for staining living cells. Drug transport, mediated by PGP, was determined simultaneously by measuring rhodamine 123 (rho123) efflux. The MDR cell lines, CEM/VLB10-2 and CEM/VBL100 are 10-fold and 270-fold resistant to vinblastine (VBL), respectively, compared to the human PGP-negative parent cell line CEM/WT and they express different amounts of PGP. Initially, living cells were stained using the 4e3 antibody and a secondary antibody labeled with 7-amino-4-methylcoumarin-3-acetic acid (AMCA). Cells were then incubated for 60 min with rho123 (10 microM) and analyzed for rhodamine and AMCA-derived fluorescence. The decrease in rho123 fluorescence was determined after a further period of 30 min. RESULTS: CEM/VLB100 cells expressed larger amounts of PGP, and rho123 fluorescence after 30 min was 85% lower than the parent cell line. PGP expression and rho123 efflux were also detected in CEM/VLB10-2 cells which display a low degree of resistance, thus reflecting the high sensitivity of this method. PGP-expressing blasts and moderate rho123 efflux were also observed in a specimen derived from a patient with clinically resistant acute myeloid leukemia (AML). CONCLUSION: A multiparametric approach using flow-cytometry allows the reliable and sensitive measurement of both PGP expression and function simultaneously in single cells.