Defective function of dendritic cells (DC) in cancer has been recently described and may represent one of the mechanisms of tumor evasion from immune system control. We have previously shown in vitro that vascular endothelial growth factor (VEGF), produced by almost all tumors, is one of the tumor-derived factors responsible for the defective function of these cells. In this study, we investigated whether in vivo infusion of recombinant VEGF could reproduce the observed DC dysfunction. Continuous VEGF infusion, at rates as low as 50 ng/h (resulting in serum VEGF concentrations of 120 to 160 pg/mL), resulted in a dramatic inhibition of dendritic cell development, associated with an increase in the production of B cells and immature Gr-1+ myeloid cells. Infusion of VEGF was associated with inhibition of the activity of the transcription factor NF-κB in bone marrow progenitor cells. Experiments in vitro showed that VEGF itself, and not factors released by VEGF-activated endothelial cells, affected polypotent stem cells resulting in the observed abnormal hematopoiesis. These data suggest that VEGF, at pathologically relevant concentrations in vivo, may exert effects on pluripotent stem cells that result in blocked DC development as well as affect many other hematopoietic lineages.

AimsThe distinction between benign and malignant thyroid nodules has important therapeutic implications. Our objective was to develop an assay that could classify indeterminate thyroid nodules as benign or suspicious, using routinely prepared fine needle aspirate (FNA) cytology smears.MethodsA training set of 375 FNA smears was used to develop the microRNA-based assay, which was validated using a blinded, multicentre, retrospective cohort of 201 smears. Final diagnosis of the validation samples was determined based on corresponding surgical specimens, reviewed by the contributing institute pathologist and two independent pathologists. Validation samples were from adult patients (≥18 years) with nodule size >0.5 cm, and a final diagnosis confirmed by at least one of the two blinded, independent pathologists. The developed assay, RosettaGX Reveal, differentiates benign from malignant thyroid nodules, using quantitative RT-PCR.ResultsTest performance on the 189 samples that passed quality control: negative predictive value: 91% (95% CI 84% to 96%); sensitivity: 85% (CI 74% to 93%); specificity: 72% (CI 63% to 79%). Performance for cases in which all three reviewing pathologists were in agreement regarding the final diagnosis (n=150): negative predictive value: 99% (CI 94% to 100%); sensitivity: 98% (CI 87% to 100%); specificity: 78% (CI 69% to 85%).ConclusionsA novel assay utilising microRNA expression in cytology smears was developed. The assay distinguishes benign from malignant thyroid nodules using a single FNA stained smear, and does not require fresh tissue or special collection and shipment conditions. This assay offers a valuable tool for the preoperative classification of thyroid samples with indeterminate cytology.

Defective function of dendritic cells (DC) in cancer has been recently described and may represent one of the mechanisms of tumor evasion from immune system control. We have previously shown in vitro that vascular endothelial growth factor (VEGF), produced by almost all tumors, is one of the tumor-derived factors responsible for the defective function of these cells. In this study, we investigated whether in vivo infusion of recombinant VEGF could reproduce the observed DC dysfunction. Continuous VEGF infusion, at rates as low as 50 ng/h (resulting in serum VEGF concentrations of 120 to 160 pg/mL), resulted in a dramatic inhibition of dendritic cell development, associated with an increase in the production of B cells and immature Gr-1+ myeloid cells. Infusion of VEGF was associated with inhibition of the activity of the transcription factor NF-κB in bone marrow progenitor cells. Experiments in vitro showed that VEGF itself, and not factors released by VEGF-activated endothelial cells, affected polypotent stem cells resulting in the observed abnormal hematopoiesis. These data suggest that VEGF, at pathologically relevant concentrations in vivo, may exert effects on pluripotent stem cells that result in blocked DC development as well as affect many other hematopoietic lineages.

Cultured alveolar type II cells and alveolar macrophages were found to secrete colony-stimulating factors (CSF) into the medium. Surfactant protein A (SP-A; 0.1-5 micrograms/ml) and bacterial lipopolysaccharide (LPS; 10-20 micrograms/ml) were found to upregulate the secretion of CSF (seven-fold) from these cells. However, a reversal of the stimulatory effect was observed when the two agents were added simultaneously to the cells. SP-A-enhanced phagocytosis of bacteria by alveolar macrophages was also inhibited by simultaneous addition of SP-A and LPS. Thus some biological activities attributed to either SP-A or LPS are inhibited in the simultaneous presence of the two agents. We therefore investigated the possibility of interaction and binding between SP-A and LPS molecules. Our biochemical data that include immunoblots and enzyme-linked immunosorbent assay support the notion that SP-A is capable of binding LPS, and this interaction is time and concentration dependent. The binding was partially inhibited (60%) by antibody to SP-A. The binding was calcium independent and was not affected by excess carbohydrates such as methyl alpha-D-mannopyranoside or heparin. Lipid A, the hydrophobic component of LPS, however, inhibited the SP-A-LPS interaction and also caused a partial reversal of the binding. Thus these results indicate that lipid A is associated with this binding. The biological implication of SP-A-LPS interaction, especially during inflammatory responses, is discussed.

Apoptosis is a dynamic process in which a characteristic morphological or biochemical event used in an assay as a specific marker of apoptosis may be observed over a limited period of time. Asynchronous involvement of cells in apoptosis results in different proportions of apoptotic cells with blebbed membrane, broken nuclei, modified mitochondrial units or fragmented DNA coexisting in the culture at any single moment. Thus, depending on the method used, the extent of apoptosis determined in the same cell population may vary. In the present study, a microculture kinetic (MiCK) assay was used to monitor apoptosis in HL-60 cells exposed to 1, 2.5, 5, 10, and 20 micromol/L etoposide and cisplatin. Both the extent and timing of apoptotic responses were dependent on the drug and drug concentration. Time-lapse video microscopy (TLVM), flow cytometry analysis of the light scattering properties of cells, morphological studies of Giemsa-stained cells, annexin V binding, and DNA fragmentation assays were performed at multiple times of cell exposure to 10 micromol/L etoposide and 5 micromol/L cisplatin. Steep linear increases in optical density, indicating apoptosis in the MiCK assay, correlated with both linear increases in the proportion of cells with plasma membrane blebbing in TLVM and with increased side scattering properties of apoptotic cells in flow cytometry. During a 24-hour culture period, the MiCK assay and TLVM provided multiple consecutive appraisals of nondisturbed cell microcultures at intervals of 5 and 2.5 minutes, respectively, and thus could be considered as real time kinetic assays. With the three endpoint assays, each of which was applied 12 times at 2-hour intervals, maximum apoptotic responses varied from 22.5 to 72% in etoposide-treated cells and from 30 to 57% in cisplatin-treated cells. With the annexin V binding assay, maximum apoptosis could always be detected 4 to 5 hours earlier than it was seen in Giemsa-stained preparations and 8 hours earlier than it was detected by measuring of DNA fragmentation. Values of the maximum extent of apoptosis varied, being the lowest with annexin V and the greatest with DNA fragmentation assays. The best correlations of both extent and timing of apoptosis were observed between the MiCK, TLVM, and morphological assays. In conclusion, both a maximum apoptotic response and the time at which it was achieved are the obligatory requirements for determining the apoptosis-inducing potency of an agent and for comparing results of studies performed in different laboratories.

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Cultured alveolar type II cells and pulmonary epithelial (PE) cells in long-term culture were found to secrete colony-stimulating factors (CSF) into the medium in similar fashion to alveolar macrophages. CSF activity was determined by using the in vitro assay for myeloid progenitor cells [colony-forming units in culture (CFU-C)]. Both lipopolisaccharide (LPS) and interleukin-1 alpha (IL-1 alpha) were found to upregulate the secretion 6.5- to 8-fold from alveolar type II cells and macrophages. However, no stimulatory effect of these factors was observed in PE cells that release CSF into the medium constitutively, possibly due to the conditions of long-term culture. The CSF activity was partially neutralized (70% inhibition) by antibodies against murine granulocyte/macrophage (GM)-CSF and IL-3, thus indicating the presence of both GM-CSF and IL-3-like factors in the CSF. However, the presence of other cytokines in the CSF is highly probable. Surfactant-associated protein A (SP-A), which is known to play a central role in surfactant homeostasis and function, was also found to upregulate secretion of CSF (at concentrations of 0.1-5 micrograms/ml) from alveolar type II cells and macrophages. Control cells such as rat peritoneal macrophages, alveolar fibroblasts, and 3T3/NIH cell line could not be elicited by SP-A to release CSF. The results are discussed in relation to the possible participation of the alveolar epithelial cells in various intercellular signaling networks. Our studies suggest that alveolar type II cells and SP-A may play an important regulatory role in the modulation of immune and inflammatory effector cells within the alveolar space.

Chemotherapeutic agents exert their antitumor effects by inducing apoptosis. The microculture kinetic (MiCK) assay provides an automated, continuous means of monitoring apoptosis in a cell population. We used the MiCK assay to determine the chemosensitivities of the human promyelocytic HL-60 and lymphoblastic CEM cell lines and leukemia cells freshly isolated from patients with acute nonlymphocytic (ANLL) or acute lymphocytic (ALL) leukemias. Continuous monitoring of apoptosis in the MiCK assay permits determination of the time to the maximum apoptosis (Tm) and its two components which are initiation time (Ti) and development time (Td). Duration of the three timing components of apoptosis varies from hours to days depending on the drug, drug concentration, and type of target cells. In the MiCK assay, the extent of apoptosis is reported in kinetic units of apoptosis. Kinetic units are determined by the slope of the curve created when optical density caused by cell blebbing is plotted as a function of time. Using the leukemia cell lines, we define the relationship between kinetic units determined by the MiCK assay and the percentage of morphologically apoptotic cells in the culture. Flow cytometry analysis of apoptosis in Annexin-V-fluorescein isothiocyanate–labeled preparations of HL-60 and CEM cells was also used to compare with data obtained by the MiCK assay. The feasibility of the MiCK assay of apoptosis as a chemosensitivity test was confirmed by its comparison with a 3H-thymidine incorporation assay. We show that samples from 10 ANLL and ALL patients patients tested for sensitivity to various doses of idarubicin (IDR), daunorubicin (DNR), or mitoxantrone (MTA) gave the same percentages of apoptotic cells when calculated by the MiCK assay as when determined by morphological analysis. The MiCK assay was used for dose-response analyses of the sensitivities to IDR, DNR, and MTA of leukemia cells from 4 other patients (2 ANLL and 2 ALL). The results from both cell lines and patient samples indicate that ANLL cells are more sensitive than ALL cells to all three of these chemotherapeutic agents. However, for individual patients the chemosensitivities varied significantly among the three chemotherapeutic agents. These varying responses to IDR, DNR, and MTA indicate that the MiCK assay results can be of potential use in designing a treatment regimen for a specific patient with acute leukemia. Among several drugs of presumed similar efficacy, the MiCK assay can permit the selection of the specific chemotherapeutic agent that causes the most apoptosis in the patient's leukemic cells. © 1998 by The American Society of Hematology.