Formation of spheroids by dental pulp cells in the presence of hypoxia and hypoxia mimetic agents.

AIM: To evaluate the impact of hypoxia and hypoxia mimetic agents (HMA) on the formation and activity of spheroids by dental pulp cells (DPC). METHODOLOGY: DPC on agarose-coated plates were treated with hypoxia and the HMA dimethyloxallyl glycine (DMOG), desferrioxamine (DFO) and L-mimosine (L-MIM). Images of spheroids were taken directly after seeding and at 6 h and 24 h. Spheroid sizes were quantified by area measurement with ImageJ software. Viability was assessed with Live-Dead staining, MTT and resazurin-based toxicity assay. Production of VEGF, IL-8 and SDF-1 was evaluated using immunoassays. Data were analysed using Kruskal-Wallis test and post hoc Mann-Whitney U-test. RESULTS: DPC formed spheroids in the presence of hypoxia, HMA and combined treatment with hypoxia and HMA. No pronounced difference in spheroid size was found in the groups treated with hypoxia, DMOG, DFO, L-MIM and the combination of hypoxia and the HMA relative to their normoxic controls (P > 0.05). Spheroids appeared vital in Live-Dead and MTT staining and the resazurin-based toxicity assay. Evaluation of protein production with immunoassays revealed significantly enhanced levels of VEGF and IL-8 (P < 0.05), but there was no significant effect on SDF-1 production (P > 0.05). Treatment with a combination of hypoxia and HMA did not further boost VEGF and IL-8 production (P > 0.05). CONCLUSIONS: Pre-conditioning with hypoxia and HMA increased the pro-angiogenic capacity of spheroids whilst not interfering with their formation. Pre-clinical studies will reveal whether pre-conditioning of spheroids with hypoxia and HMA can effectively improve the efficiency of cell transplantation approaches for regenerative endodontics.

Difference in release kinetics of unwashed and washed platelet-released supernatants from bone substitute materials: the impact of platelet preparation modalities.

BACKGROUND AND OBJECTIVE: In regenerative dentistry, platelet preparations are applied to stimulate bone healing and periodontal regeneration. Here, we pursue a strategy where bone substitutes are used as carriers for platelet-released supernatants. The mitogenic capacity and release kinetics of loaded bone substitutes were assessed. MATERIAL AND METHODS: Platelet-released supernatants of washed platelets (washed PRS) and platelet-released supernatants of unwashed platelets (unwashed PRS) were lyophilized onto the bone substitutes deproteinized bovine bone mineral, hydroxyapatite and ß-tricalcium phosphate. Scanning electron microscopy images were taken. Supernatants of bone substitutes were collected at hours 1, 3, 6, 24, and 48 and medium was replaced. We evaluated the protein content with the bicinchoninic acid assay and the effect on proliferation using bioassays with human periodontal fibroblasts. Release of growth factors from the loaded bone substitutes was measured based on the platelet-derived growth factor isoform (PDGF-BB) and thrombin immunoassays. Furthermore, we assessed DNA and RNA content of washed PRS and unwashed PRS. RESULTS: Unwashed PRS showed higher total protein concentrations than washed PRS, while the concentration of PDGF-BB, thrombin, DNA, RNA and their mitogenic effect was not significantly different. The bone substitute materials adsorbed protein over time but no significant changes in overall appearance was found. Supernatants collected from unwashed PRS-loaded bone substitute after 1 h induced a potent mitogenic response in periodontal fibroblasts. This pro-mitogenic capacity of the supernatants decreased over the observation period. Supernatants of washed PRS-loaded bone substitutes did not induce a substantial mitogenic effect. Levels of PDGF-BB, thrombin and protein were higher in supernatants of unwashed PRS-loaded bone substitutes than of washed PRS-loaded bone substitutes. CONCLUSION: Bone substitutes loaded with unwashed PRS, but not bone substitutes loaded with washed PRS show continuously declining release kinetics. These data suggest that plasma components in platelet preparations can modify the release kinetics profile.

L-mimosine increases the production of vascular endothelial growth factor in human tooth slice organ culture model.

AIM: To assess the pro-angiogenic and pro-inflammatory capacity of the dentine-pulp complex in response to the prolyl hydroxylase inhibitor L-mimosine in a tooth slice organ culture model. METHODOLOGY: Human teeth were sectioned transversely into 600-µm-thick slices and cultured in medium supplemented with serum and antibiotics. Then, pulps were stimulated for 48 h with L-mimosine. Pulps were subjected to viability measurements based on formazan formation in MTT assays. In addition, histological evaluation of pulps was performed based on haematoxylin and eosin staining. Culture supernatants were subjected to immunoassays for vascular endothelial growth factor (VEGF) to determine the pro-angiogenic capacity and to immunoassays for interleukin (IL)-6 and IL-8 to assess the pro-inflammatory response. Interleukin-1 served as pro-inflammatory control. Echinomycin was used to inhibit hypoxia-inducible factor-1 (HIF-1) alpha activity. Data were analysed using Student's t-test and Mann-Whitney U test. RESULTS: Pulps within tooth slices remained vital upon L-mimosine stimulation as indicated by formazan formation and histological evaluation. L-mimosine increased VEGF production when normalized to formazan formation in the pulp tissue of the tooth slices (P < 0.05). This effect on VEGF was reduced by echinomycin (P < 0.01). Changes in normalized IL-6 and IL-8 levels upon treatment with L-mimosine did not reach the level of significance (P > 0.05), whilst treatment with IL-1, which served as positive control, increased IL-6 (P < 0.05) and IL-8 levels (P < 0.05). CONCLUSIONS: The prolyl hydroxylase inhibitor L-mimosine increased VEGF production via HIF-1 alpha in the tooth slice organ culture model whilst inducing no prominent increase in IL-6 and IL-8. Pre-clinical studies will reveal if these in vitro effects translate into dental pulp regeneration.

Prolyl hydroxylase inhibitors increase the production of vascular endothelial growth factor by periodontal fibroblasts.

BACKGROUND AND OBJECTIVE: Pharmacological inhibitors of prolyl hydroxylases (PHDs) can induce a proangiogenic response that favors wound healing and bone regeneration. However, the response of periodontal cells to PHD inhibitors is unknown. MATERIAL AND METHODS: To determine the effects of PHD inhibitors on periodontal cells, we exposed human fibroblasts from the gingiva and the periodontal ligament to dimethyloxallyl glycine, desferrioxamine, l-mimosine and CoCl(2). Viability, proliferation, and protein synthesis were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), [(3)H]thymidine, and [(3)H]leucine incorporation, respectively. The levels of Ki67, hypoxia-inducible factor 1α (HIF-1α), p27, phosphorylated c-Jun N-terminal kinase (JNK) and phosphorylated p38 were determined by immunohistochemistry and western blotting. Vascular endothelial growth factor (VEGF) mRNA levels were measured by quantitative PCR. Protein levels of VEGF and interleukin (IL)-6 were evaluated by immunoassays. RESULTS: We found that PHD inhibitors, while leaving cell viability unchanged, reduced proliferation and protein synthesis. This was paralleled by decreased Ki67 levels and increased p27 levels, suggesting that PHD inhibitors provoke growth arrest. Independently from this response, PHD inhibitors stabilized HIF-1α and increased the production of VEGF. This increase of VEGF was observed in the presence of proinflammatory IL-1 and pharmacological inhibitors of JNK and p38 signaling. Moreover, PHD inhibitors did not modulate expression of IL-6 and the phosphorylation of JNK and p38. CONCLUSION: These results suggest that PHD inhibitors enhance the production of VEGF in periodontal fibroblasts, even in the presence of proinflammatory IL-1. The data further suggest that PHD inhibitors do not provoke a significant proinflammatory or anti-inflammatory response in this in vitro setting.

The response of dental pulp-derived cells to zoledronate depends on the experimental model.

AIM: To investigate whether zoledronate (ZOL) can cause a cytotoxic response in dental pulp-derived cells (DPCs) in vitro. METHODOLOGY: Cell activity was assessed utilizing MTT tests, (3) [H]thymidine, and (3) [H]leucine incorporation assays in human DPCs in response to ZOL. Cell activity assays were also preformed on calcium phosphate-coated plates. Cell death was analysed with annexin V/propidium iodide, trypan blue staining and Western blot analysis. RESULTS: Micromolar concentrations of ZOL were required to decrease the activity of DPCs. The decreased activity of DPCs was associated with the occurrence of apoptosis and necrosis. No adverse effects were observed when DPCs were cultured on calcium phosphate-coated plates with ZOL. CONCLUSION: High concentrations of soluble ZOL were required to cause adverse effects in vitro. These adverse effects are abolished when the bisphosphonate was bound to a mineralized surface. However, the clinical relevance of these results remains to be determined.

First-line daratumumab shows high efficacy and tolerability even in advanced AL amyloidosis: the real-world experience.

BACKGROUND: Daratumumab was the first monoclonal CD38 antibody with single-agent activity approved for the treatment of multiple myeloma. Moreover, daratumumab demonstrated high response rates in relapsed immunoglobulin light-chain (AL) amyloidosis. PATIENTS AND METHODS: In our single-center retrospective real-life case series, we analyzed the efficacy and safety of daratumumab as first-line treatment. Daratumumab was administered with low-dose dexamethasone alone or in combination with other multiple myeloma therapeutics RESULTS: Fourteen patients were eligible, including nine patients with cardiac stage IIIa or IIIb. Overall hematologic response rate was 100%, with 64.3% achieving complete response after a median of 16 cycles of treatment. Median time to hematologic response was 1.4 months. Organ response rates were 45.5% after a median of 4.0 months and 66.7% after a median of 10.0 months, for heart and kidney involvement, respectively. After a median follow-up of 20.5 months, two patients underwent successful autologous stem cell transplantation (ASCT), while another three patients were in preparation for ASCT. Three patients remained on daratumumab at the last follow-up. There were no unexpected toxicities and no grade III or IV adverse events, although more than half of our patients were in stage IIIa or IIIb. CONCLUSION: Daratumumab proved to be highly effective in newly diagnosed AL amyloidosis with excellent hematologic and organ response rates, a remarkable safety profile, and good tolerability even in patients with advanced stage of disease.

Activated platelets increase proliferation and protein synthesis of human dental pulp-derived cells.

AIM: To evaluate the impact of activated platelets on the mitogenic expansion of human dental pulp-derived cells (DPC) in vitro. METHODOLOGY: The effect of supernatants released from activated platelets (PRS) and the corresponding platelet membranes (MEM) on proliferation and protein synthesis of DPC was evaluated. The contributions of the phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK) pathways to the response of DPC were assessed using kinase inhibitors. Also examined was whether the presence of calcium hydroxide or the inflammatory mediators tumour necrosis factor alpha, interleukin-1, interleukin-6 and lipopolysaccharide of Escherichia coli modulated the expansion of DPC. RESULTS: Physiologic concentrations of PRS and MEM stimulated proliferation and protein synthesis by 18.4-fold (P < 0.01) and 2.9-fold (P < 0.01), respectively. This mitogenic expansion was paralleled by activation of AKT and the MAP kinases extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. Inhibitor studies revealed that the mitogenic response of DPC involved PI3K/AKT, JNK and p38 signalling (P < 0.05). Calcium hydroxide and inflammatory factors did not significantly modulate the mitogenic response of DPC to PRS and MEM. CONCLUSION: Supernatants released from activated platelets and the corresponding platelet membranes stimulated DPC proliferation and protein synthesis involving PI3K/AKT and MAPK signalling. These findings may serve as a basis for preclinical studies that address the role of activated platelets in dental pulp repair.

Elevated tryptase levels selectively cluster in myeloid neoplasms: a novel diagnostic approach and screen marker in clinical haematology.

BACKGROUND: Recent data suggest that tryptase, a mast cell enzyme, is expressed in neoplastic cells in myeloid leukaemias. In several of these patients, increased serum tryptase levels are detectable. MATERIALS AND METHODS: We have determined serum tryptase levels in 914 patients with haematological malignancies, including myeloproliferative disorders (n = 156), myelodysplastic syndromes (MDS, n = 241), acute myeloid leukaemia (AML, n = 317), systemic mastocytosis (SM, n = 81), non-Hodgkin's lymphoma (n = 59) and acute lymphoblastic leukaemia (n = 26). Moreover, tryptase was measured in 136 patients with non-neoplastic haematological disorders, 102 with non-haematological disorders and 164 healthy subjects. RESULTS: In healthy subjects, the median serum tryptase was 5.2 ng mL(-1). Elevated serum tryptase levels were found to cluster in myeloid neoplasm, whereas almost all patients with lymphoid neoplasms exhibited normal tryptase. Among myeloid neoplasms, elevated tryptase levels (> 15 ng mL(-1)) were recorded in > 90% of patients with SM, 38% with AML, 34% with CML and 25% with MDS. The highest tryptase levels, often > 1000 ng mL(-1), were found in advanced SM and core-binding-factor leukaemias. In most patients with non-neoplastic haematological disorders and non-haematological disorders analysed in our study, tryptase levels were normal, the exception being a few patients with end-stage kidney disease and helminth infections, in whom a slightly elevated tryptase was found. CONCLUSIONS: In summary, tryptase is a new diagnostic marker of myeloid neoplasms and a useful test in clinical haematology.

Mixed-lineage eosinophil/basophil crisis in MDS: a rare form of progression.

BACKGROUND: Basophilic crisis and eosinophilia are well recognized features of advanced chronic myeloid leukaemia. In other myeloid neoplasms, however, transformation with marked basophilia and eosinophilia is considered unusual. DESIGN: We examined the long-term follow-up of 322 patients with de novo myelodysplastic syndromes (MDS) to define the frequency of basophilic, eosinophilic and mixed lineage (basophilic and eosinophilic) transformation. RESULTS: Of all patients, only one developed mixed lineage crisis (>or= 20% basophils and >or= 20% eosinophils). In this patient, who initially suffered from chronic myelomonocytic leukaemia, basophils increased to 48% and eosinophils up to 31% at the time of progression. Mixed lineage crisis was not accompanied by an increase in blast cells or organomegaly. The presence of BCR/ABL and other relevant fusion gene products (FIP1L1/PDGFRA, AML1/ETO, PML/RAR alpha, CBF beta/MYH11) were excluded by PCR. Myelomastocytic transformation/myelomastocytic leukaemia and primary mast cell disease were excluded by histology, KIT mutation analysis, electron microscopy and immunophenotyping. Basophils were thus found to be CD123+, CD203c+, BB1+, KIT- cells, and to express a functional IgE-receptor. Among the other patients with MDS examined, 4(1.2%) were found to have marked basophilia (>or= 20%) and 7(2.1%) were found to have massive eosinophilia ( >or= 20%), whereas mixed-lineage crisis was detected in none of them. CONCLUSIONS: Mixed basophil/eosinophil crisis may develop in patients with MDS but is an extremely rare event.

Effects of platelet-derived growth factor isoforms on plasminogen activation by periodontal ligament and gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Platelet-derived growth factor isoforms and components of the plasminogen activator system are expressed at higher levels during periodontal regeneration. Recombinant platelet-derived growth factor-BB is approved for the treatment of periodontal defects. In the present study we investigated the effect of platelet-derived growth factor isoforms on the plasminogen activator system in periodontal fibroblasts. MATERIAL AND METHODS: Human periodontal ligament fibroblasts and gingival fibroblasts were exposed to platelet-derived growth factor isoforms. Changes in urokinase-type plasminogen activator, tissue-type plasminogen activator, plasminogen activator inhibitor-1 and plasminogen activator inhibitor-2 transcript levels by platelet-derived growth factor-BB were monitored with a quantitative reverse transcription-polymerase chain reaction. Urokinase-type plasminogen activator and plasminogen activator inhibitor-1 protein levels were assessed by immunoassays. The effects of platelet-derived growth factor-BB on mitogen-activated protein kinase and phosphoinositol-3 kinase/Akt signaling were investigated by western blot and inhibitor studies. Casein zymography and kinetic assays revealed the size and activity, respectively, of the plasminogen activators. RESULTS: We found that incubation of periodontal ligament fibroblasts and gingival fibroblasts with platelet-derived growth factor-BB resulted in enhanced levels of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 transcripts, but not of tissue-type plasminogen activator and plasminogen activator inhibitor-2. Platelet-derived growth factor-BB also increased urokinase-type plasminogen activator and plasminogen activator inhibitor-1 release into the culture medium. Phosphorylation of extracellular signal-regulated kinase, p38, c-Jun N-terminal kinase and Akt was observed in fibroblasts of both origin. Inhibition of phosphoinositol-3 kinase signaling abrogated the platelet-derived growth factor-BB effect on plasminogen activator inhibitor-1 production. Casein zymography revealed enzymatic activity of the urokinase-type plasminogen activator in cell-conditioned media and lysates of periodontal ligament fibroblasts and gingival fibroblasts. Exposure of gingival fibroblasts, but not of periodontal ligament fibroblasts, to platelet-derived growth factor isoforms moderately increased total plasminogen activation in the medium. CONCLUSION: These findings suggest that periodontal ligament fibroblasts attempt to maintain an equilibrium of the plasminogen activator system in the presence of platelet-derived growth factor isoforms.

Clinical and prognostic significance of histamine monitoring in patients with CML during treatment with imatinib (STI571).

BACKGROUND: Although imatinib is highly effective in chronic myeloid leukemia (CML), drug-resistance may occur. Therefore, monitoring of minimal residual disease (MRD) during treatment with imatinib is important. However, most MRD-parameters are expensive and require special technology. We determined the value of histamine as MRD-marker in CML. PATIENTS AND METHODS: Histamine levels were measured serially in whole blood samples before and during imatinib therapy in 80 CML patients by radioimmunoassay. RESULTS: Histamine levels were highly upregulated in CML at diagnosis compared to healthy controls, and correlated with the presence of basophils. During treatment with imatinib, histamine levels decreased and returned to normal levels in those achieving a complete cytogenetic response (CCR). Loss of CCR during therapy was invariably accompanied by an increase in histamine. Moreover, a histamine level of >100 ng/ml three or six months after start of imatinib was associated with a significantly reduced probability of survival (p<0.05). Whereas basophils were found to correlate well with histamine during imatinib, no correlations were found between histamine and Ph+ metaphases or histamine and BCR/ABL. CONCLUSION: Histamine-monitoring during treatment with imatinib is of prognostic significance.

The tryptase positive compact round cell infiltrate of the bone marrow (TROCI-BM): a novel histopathological finding requiring the application of lineage specific markers.

AIMS: Compact tryptase-positive round cell infiltrates of the bone marrow (TROCI-BM) are very rare histopathological findings and may pose challenging problems with regard to the cell type involved (either mast cells or basophilic granulocytes) and the exact diagnosis. METHODS: A selected panel of immunohistochemical markers against mast cell and basophil related antigens, including CD25, CD34, CD117/Kit, and the 2D7 antigen (which is found only in basophilic granulocytes) on a total of 410 routinely processed bone marrow biopsy specimens (including 88 cases of systemic mastocytosis (SM), 20 cases of chronic myeloid leukaemia (CML), 92 cases of myeloid neoplasms other than CML, and 210 controls with normal/reactive bone marrows). RESULTS: In total, 17 cases with TROCI-BM could be identified: 11 SM (including two cases of well-differentiated SM and two mast cell leukaemias; MCL), 2 myelomastocytic leukaemia (MML), 2 CML with excess of basophils (secondary basophilic leukaemia (CMLba)), and 2 tryptase positive acute myeloid leukaemia (AML). Regarding the cell types involved, TROCI-BM cells were found to express CD117/Kit in all cases of SM and MCL. In MML and tryptase postitive AML, TROCI-BM cells were found to coexpress CD34 and Kit. The basophil specific antigen 2D7 was only detected in CD34/Kit negative TROCI-BM cells in two patients with CMLba. The activating point mutation D816V was detected in 8/11 patients with SM but not in any of the other haematological malignancies. CONCLUSIONS: In summary, a total of six rare myeloid neoplasms may present with a novel immunohistochemical phenomenon tentatively termed TROCI-BM.

Enumeration and immunohistochemical characterisation of bone marrow basophils in myeloproliferative disorders using the basophil specific monoclonal antibody 2D7.

BACKGROUND: Basophils are highly specialised granulocytes that express a unique profile of antigens and increase in myeloproliferative disorders (MPD). In chronic myeloid leukaemia (CML), basophilia is a diagnostic and prognostic determinant. So far, however, no reliable approach for routine detection and enumeration of bone marrow basophils has become available. OBJECTIVE: To detect and enumerate basophils in bone marrow sections in patients with CML and other MPD. METHODS: The anti-basophil antibody 2D7 was applied to paraffin embedded bone marrow sections from normal/reactive subjects (n = 31), patients with CML (chronic phase, n = 37; accelerated phase, n = 9), and other MPD (chronic idiopathic myelofibrosis (CIMF), n = 20; polycythaemia vera (PV), n = 20; essential thrombocythaemia (ET), n = 20; indolent systemic mastocytosis (ISM), n = 7). RESULTS: As assessed by serial section staining, 2D7(+) cells were found to co-express myeloperoxidase, histidine decarboxylase, CD9, and CD43, but did not express B cell or T cell restricted antigens. 2D7(+) bone marrow cells were found to increase in CML compared with normal/reactive bone marrow and other MPD (median numbers of 2D7(+) cells/mm(2): CML, 33; normal/reactive bone marrow, 6; CIMF, 10; PV, 6; ET, 5; ISM, 3; p<0.05). The highest basophil counts were recorded in accelerated phase CML (115/mm(2)). CONCLUSIONS: A novel immunohistochemical procedure has been established for basophil detection in normal bone marrow and MPD. This approach should help in the quantification of bone marrow basophils at diagnosis and during anti-leukaemic treatment.

Immunophenotypic and functional characterization of human tonsillar mast cells.

Mast cells (MC) are proinflammatory immune cells residing in various organs. Tissue-specific heterogeneity of MC has been described. The aim of this study was to establish the phenotype and functional profile of human tonsillar mast cells (ToMC) and to compare ToMC with lung-, skin-, and uterus MC. Tonsillar tissue was obtained from 23 patients suffering from hyperplastic tonsils and dispersed by enzymatic digestion. With the use of a combined toluidine blue/immunofluorescence staining technique, isolated ToMC were found to react with monoclonal antibodies (mAb) to immunoglobulin E, CD9, CD43, CD44, CD46, CD54, CD55, and CD59, as well as mAb to stem cell factor (SCF) receptor (CD117/c-kit). ToMC were not recognized by mAb to other cytokine receptors or mAb to CD3, CD11b, CD14, CDw17, the skin MC marker CD88 (C5aR) or CD89 (Fc alphaR). Activation of ToMC by recombinant human (rh) SCF or anti-IgE resulted in histamine secretion, whereas no effects were seen with rhC5a, rh granulocyte-macrophage colony-stimulating factor, or rh interleukin-1 through -10. In summary, ToMC exhibit functional and phenotypic properties similar to lung- or uterus MC. Unlike skin MC, ToMC lack C5aR and are unresponsive to rhC5a.

Stem cell factor-induced downregulation of c-kit in human lung mast cells and HMC-1 mast cells.

Recent data suggest that local overexpression of the tissue-hormone c-kit ligand (stem cell factor [SCF]) is associated with accumulation of mast cells (MCs) and a decrease in expression of c-kit in the accumulated MCs [28]. In the present study, the effects of recombinant human (rh) SCF on expression of c-kit mRNA and c-kit protein in isolated human MCs and a human mast cell line, HMC-1, were analyzed. Incubation of isolated lung MC with rhSCF (100 ng/mL) for 120 minutes resulted in decreased expression of c-kit mRNA (optical density [OD], control: 100% vs. rhSCF: 37%). Almost identical results were obtained with HMC-1 cells (OD, control: 100% vs. rhSCF: 40 to 45%). As assessed by flow cytometry and monoclonal antibodies (mAbs) to c-kit, the SCF-induced decrease of c-kit mRNA in HMC-1 was associated with a substantial decrease in surface expression of c-kit (MFI, control: 100 +/- 21%, vs. MFI in cells incubated with rhSCF [100 ng/mL at 37 degrees C for 12 hours]: 8 +/- 2%, vs. MFI in cells incubated with rhSCF, 100 ng/mL, at 4 degrees C: 34 +/- 3%). The effects of rhSCF on c-kit expression in HMC-1 cells were dose- and time-dependent with maximum effects observed with 10-100 ng/mL of rhSCF after 4 to 12 hours. The SCF-dependent loss of c-kit was also accompanied by a decreased chemotactic response to rhSCF (control: 100%; rhSCF: 71 +/- 2%). This study shows that exposure of human lung MC and HMC-1 cells to recombinant SCF results in downregulation of c-kit mRNA and surface c-kit expression. These data may explain the partial loss of c-kit on MCs in areas of SCF overexpression.

Purification of human basophils and mast cells by multistep separation technique and mAb to CDw17 and CD117/c-kit.

Basophils and mast cells represent distinct cell lineages within the hemopoietic system. Based on the unique cell surface antigen profile of both cells, we have established methods which allow the reproducible purification to homogeneity (> 99%) of normal human basophil granulocytes from the peripheral blood and of mast cells from human dispersed tissues. Basophils (n = 9) were purified by current counterflow elutriation followed by depletion of monocytes with CD14 mAb conjugated to magnetic beads, and subsequent cell sorting for CD217+ cells. Basophil purity was 99.5 +/- 0.4% (range 98.7-99.9%). Mast cells were obtained from lung (n = 6), uterus (n = 1), mastocytosis bone marrow (n = 2), and human foreskin (n = 2). Mast cells were purified by collagenase digestion followed by current counterflow elutriation and sorting with CD117/c-kit mAb. Mast cell purity was 99.4 +/- 0.7% (range: 97.5-99.9%). Purified cells were more than 90% viable and were able to release histamine on induction with IgE plus anti-IgE. Furthermore, the PCR technique could be applied on pure cells and confirmed expression of high affinity IgE receptor (Fc epsilon R1) alpha chain mRNA. Thus, by combining isolation techniques including elutriation, magnetic cell depletion and cell sorting with mAb, functionally intact normal human basophils and mast cells can be enriched to homogeneity.

Inhalation scintigraphy with iodine-123-labeled interferon gamma-1b: pulmonary deposition and dose escalation study in healthy volunteers.

UNLABELLED: Recent studies have suggested that recombinant interferon gamma (IFNg) may be useful in the treatment of various respiratory diseases, such as chronic inflammatory disease. This study was undertaken to investigate the dose response of escalating doses of inhaled 123I-labeled IFNg (123I-IFNg) and its safety, biodistribution and radiation absorbed doses in healthy volunteers. METHODS: IFNg was labeled with 123I to produce a specific activity of 1800 MBq/mg of IFNg. The biological activities of 123I-IFNg, nebulized 123I-IFNg and unlabeled IFNg were evaluated in various functional in vitro tests. Ten healthy volunteers were enrolled in the in vivo dose escalation study (180 MBq of 123I-IFNg diluted with 0.1-2 mg of INFg). Inhalation scintigraphy, using a Pari-Master nebulizer, was performed for up to 37 min, during which dynamic posterior images of the lungs were obtained. Whole-body scanning was performed at various time points up to 24 hr postinjection, for biodistribution and dosimetry purposes. Blood, urine and feces were also collected over this 24-hr period. Lung perfusion scintigraphy with 99mTc-microspheres was performed at the end of the study for attenuation correction. RESULTS: Inhaled nebulized IFNg showed a uniform deposition pattern in the lungs with deposition ratios of 0.74 (central-to-peripheral) and 0.78 (upper-to-lower). The lung deposition of IFNg was time-dependent, with a deposition half-time between 1 and 5 min. Despite a large interindividual variation, the total lung deposition was proportional to the nebulizer charge and was 53 +/- 12% of the inhaled dose and 19 +/- 7% of the initial nebulizer charge (between 0.1 and 2 mg of IFNg). The biological half-life in the lung could be fitted to a biexponential function, with resultant half-lives of 1 and 11 hr. Blood activity was maximal at 3.5 hr after inhalation and was due to free iodine. The radioactivity was excreted through both the urinary and intestinal tracts. Plasma IFNg levels did not significantly increase over time, and no significant HLA-DR induction on peripheral blood cells was detected. The highest radiation absorbed doses of 0.14 and 0.19 mGy/MBq were determined for the trachea and the lower intestines, respectively. The effective dose equivalent was 0.05 mSv/MBq. CONCLUSION: After inhalation with the Pari-Master nebulizer, IFNg deposits normally in the lungs and shows no systemic effects in healthy volunteers.

Mast cell-lineage versus basophil lineage involvement in myeloproliferative and myelodysplastic syndromes: diagnostic role of cell-immunophenotyping.

Mast cells and blood basophils are distinct hemopoietic cells. They can be distinguished from each other and from all other lymphohemopoietic cells using antibodies against surface receptors or stored cytoplasmic molecules. In patients with myelodysplastic syndromes (MDS) or myeloproliferative syndromes (MPS), an elevation of metachromatically granulated cells (MCS) is frequently seen. These cells can be classified as basophils or mast cells using monoclonal antibodies (mAbs) against leukocyte antigens, including mast cell tryptase, c-kit (= mast cell growth factor [MGF] receptor), interleukin-3 receptor alpha chain (IL-3R alpha = CD123), and CD11b (C3biR). In a stable phase of MDS or MPS, the circulating MCS usually are basophils (histamine+, tryptase-, c-kit-, IL-3R alpha +, CD11b+). In an accelerated or terminal phase of disease, however, mast cell lineage involvement and circulating mast cell precursors (histamine+, tryptase+, c-kit+, IL-3R alpha-, CD11b-) are found in a subset of patients. The use of mAbs against mast cell antigens and granulocyte antigens is diagnostic in these patients.

Activated naive CD4+ peripheral blood T cells in autoimmune thyroid disease.

To better understand the clinical significance of changes in lymphocytes in thyroid disease this study analyzed the proportion of CD19+, CD3+, CD4+ and CD8+ cells among circulating lymphocytes in Graves' disease (GD, n = 34) and autoimmune hypothyroidism (AH, n = 28) vs healthy subjects (n = 15). In addition, the expression of CD25 and CD45 isoforms on CD4+ T cells as well as their modulation by methimazole in patients with GD was measured using three color flow cytometry. It was observed that, irrespective of age, both patients with GD (17.6 +/- 7.0% +/- SD) and those with AH (19.0 +/- 9.5%) had an increased percentage of the CD25+CD45RA+ (naive) subpopulation of helper cells vs healthy subjects (7.9 +/- 2.3%, p < 0.0001). In patients with AH peripheral memory cells and hence overall CD25+ cells were more frequent among helper cells (56.7 +/- 12.2%) than in healthy subjects (40.8 +/- 14.0%, p < 0.001). Patients with GD (46.2 +/- 13.4%) did not differ from normal subjects in this respect. Treatment of GD with MMI reduced the percentage of CD25+CD45RA+ cells among CD4+ cells toward values seen in healthy subjects. In addition, we confirm previous reports that CD8+ cells toward values seen in healthy subjects. In addition, we confirm previous reports that CD8+ cells are significantly reduced in AH (23.9 +/- 4.9%) and untreated Graves' disease (23.2 +/- 6.6%) vs healthy subjects (32.2 +/- 5.9%, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

[Effector cells in allergy: biological principles and new pharmacologic concepts]. / Die Effektorzellen der Allergie: Biologische Grundlagen und neue pharmakologische Konzepte.

The clinical symptoms of allergy are caused by cellular (IgE-triggered) responses to an allergen. Effector cells of allergy include eosinophil and basophil granulocytes, as well as tissue mast cells. Growth and accumulation, as well as IgE-dependent and independent functions of these cells are regulated by distinct proteohormones and peptides. The hemopoietic cytokines IL-3 (interleukin-3), IL-5 and GM-CSF (granulocyte-macrophage colony-stimulating factor) are involved in the regulation of basophils (and eosinophils), whereas the ligand for c-kit, SCF (stem cell factor) is a mast cell-specific agonist. Basophils and mast cells express high-affinity IgE-binding sites. Allergen binding to IgE on mast cells and basophils, and consecutive cross-linking of IgE receptors is followed by production and/or secretion of inflammatory mediator substances. Specific activation and deactivation of mast cells/basophils in vitro has been demonstrated by use of recombinant cytokines and allergens, and specific haptens or by use of novel drugs, and should lead to epitope-specific diagnosis and better management of allergic diseases in the future.