Haemopoietic progenitors in the adult mouse omentum: permanent production of B lymphocytes and monocytes

The coelome-associated lympho-myeloid tissues, including the omentum, are derived from early embryo haemopoietic tissue of the splanchnopleura, and produce B lymphocytes and macrophages. They are reactive in pathologies involving coelomic cavities, in which they can expand in situ the cells of inflammatory infiltrates. We have addressed the question of the role of the adult omentum in permanent basal production of early lymphopoietic progenitors (pro-B/pre-B cells), through characterisation of omentum cells ex vivo, and study of their in vitro differentiation. We have shown that the murine omentum produces early haemopoietic progenitors throughout life, including B-cell progenitors prior to the Ig gene recombination expressing RAG-1 and 5, as well as macrophages. Their production is stroma-dependent. The omentum stroma can supply in vitro the cytokines (SDF-1, Flt3 ligand and IL-7) and the molecular environment required for generation of these two cell lineages. Omentum haemopoietic progenitors are similar to those observed in foetal blood cell production, rather than to progenitors found in the adult haemopoietic tissue in the bone marrow—in terms of phenotype expression and differentiation capacity. We conclude that a primitive pattern of haemopoiesis observed in the early embryo is permanently preserved and functional in the adult omentum, providing production of cells engaged in nonspecific protection of abdominal intestinal tissue and of the coelomic cavity.Supported by CNPq, FINEP and PADCT grants of the Brazilian Ministry of Science and Technology, and a FAPERJ grant of the Rio de Janeiro State Government     

T cell phenotype and intracellular IFN-gamma production in peritoneal exudate cells and gut intraepithelial lymphocytes during acute Toxoplasma gondii infection in mice

Although there are many reports on the splenic (systemic) T cell response after Toxoplasma gondii infection, little information is available regarding the local T cell responses of peritoneal exudate cells (PEC) and gut intraepithelial lymphocytes (IEL) following peroral infection with bradyzoites. Mice were infected with 40 cysts of the 76K strain of T. gondii, and then sacrificed at days 0, 1, 4, 7 and 10 postinfection (PI). The cellular composition and T cell responses of PEC and IEL were analyzed. The total number of PEC and IEL per mouse increased after infection, but the ratio of increase was higher in IEL. Lymphocytes were the major component of both PEC and IEL. The relative percentages of PEC macrophages and neutrophils/eosinophils increased significantly at day 1 and 4 PI, whereas those of IEL did not change significantly. The percentage of PEC NK1.1 and gamma delta T cells peaked at day 4 PI (p < 0.0001), and CD4 and CD8 alpha T cells increased continuously after infection. The percentages of IEL CD8 alpha and gamma delta T cells decreased slightly at first, and then increased. CD4 and NK1.1 T cells of IEL did not change significantly after infection. IFN-gamma-producing PEC NK1.1 T cells increased significantly from day 1 PI, but the other T cell subsets produced IFN-gamma abundantly thereafter. The proportion of IEL IFN-gamma-producing CD8 alpha and gamma delta T cells increased significantly after infection, while IEL NK1.1 T cells had similar IFN-gamma production patterns. Taken together, CD4 T cells were the major phenotype and the important IFN-gamma-producing T cell subsets in PEC after oral infection with T. gondii, whereas CD8 alpha T cells had these roles in IEL. These results suggest that PEC and IEL comprise different cell differentials and T cell responses, and according to infection route these factors may contribute to the different cellular immune responses.     

RF nonlinear interactions in living cells--II: detection methods for spectral signatures

The presence of harmonic products due to possible nonlinear interaction of amplitude modulated RF signals in living cells is best detected by using a cavity with high quality factor. Harmonic products generated by elementary oscillators can be trapped and accumulated in a cavity, permitting detection sensitivity much greater than in an open environment, where they would be radiated in all directions. The experimental method described herein is a systematic approach to detection of the non-Planck RF energy (if any) emitted by an exposed sample of living cells. Balzano and Sheppard [Balzano and Sheppard (2003): Bioelectromagnetics 24:473-482] classified the non-Planck RF emissions from living cells as coming from (1). nonlinear interactions and (2). inelastic interactions. Nonlinear harmonic products would appear in the band at twice the frequency of an amplitude modulated RF carrier. Inelastic interaction products resulting from the interaction between the incident RF energy and normally occurring mechanical vibrations are found in the band immediately adjacent to the carrier. Detection of the latter signals is difficult because of this close spectral proximity, for example, 1 part in 10(7) for 100 Hz modulation of a GHz carrier. Modern audio spectrum analyzers have excellent selectivity, providing 60 dB rejections only 2 kHz away from the carrier. By judicious selection of the amplitude modulation (AM) frequency, frequency of the RF carrier, and size of the biological sample, it is possible to achieve very high sensitivity (about -90 dBm) with commercially available instrumentation. The presence (or absence) of harmonics in the band adjacent to the amplitude modulated RF carrier would establish (or negate) the existence of coherent interactions between mechanical vibrations in the cell ensemble and the incident RF signal.     

Detection of Pythium violae and Pythium sulcatum in carrots with cavity spot using competition ELISA

Conventional methods indicated that Pythium violae was most commonly isolated from carrot cavity spot samples from 14 UK sites. For one site the most frequently isolated species was Pythium sulcatum. Results of similar isolation work were compared with the assay of cavity spot lesions using polyclonal antibodies, raised to P. violae or P. sulcatum, in competition ELISA. Where lesions were artificially induced the test confirmed which pathogen was causal. With cavities developed on the field-grown carrots P. violae again predominated and the ELISA confirmed this. In one sample P. sulcatum was also isolated from a small number of lesions and was not detected in ELISA. The competition ELISA did not indicate presence of either Pythium in a range of non-cavity spot lesions from which attempts at isolation were negative.     

Tyrosinase-related protein-2 and -1 are trafficked on distinct routes in B16 melanoma cells

Tyrosinase related protein (TRP)-1 and -2 regulate the main steps in melanin synthesis and are immune targets in skin cancer or autoimmune pigmentary disorders. We found that ionophore monensin (Mon) and the quaternary amine chloroquine (CQ) discriminate between the traffic routes of TRP-2 and TRP-1. TRP-2 N-glycan processing is interrupted by Mon between ER and trans-Golgi, whereas this process continues for TRP-1. Mature TRP-2 is diverted by CQ treatment to a degradation pathway which depends on functional vacuolar ATPases. Conversely, the subcellular distribution and stability of TRP-1 were not affected by CQ. We propose that TRP-2 is sorted and trafficked in the early secretory pathway with a cargo which does not include TRP-1; post Golgi, TRP-2 intersects the endocytic pathway following a route via early endosomes, possibly by rapid recycling from the plasma membrane. These data show that highly structural homologous glycoproteins use distinct trafficking pathways in the same cell.     

Distribution profile of inositol 1,4,5-trisphosphate receptor isoforms in adrenal chromaffin cells

Given the importance of inositol 1,4,5-trisphosphate receptor (IP(3)R)/Ca(2+) channels in the control of intracellular Ca(2+) concentrations, we determined the relative concentrations of the IP(3)R isoforms in subcellular organelles, based on serially sectioned electron micrographs. The endoplasmic reticulum (ER) was estimated to contain 15-20% of each of the three IP(3)R isoforms while secretory granules contained 58-69%. The nucleus contained approximately 15% each of IP(3)R-1 and -2, but 25% of IP(3)R-3, whereas the plasma membrane contained approximately 1% or less of each. These suggested that secretory granules, the nucleus and ER are at the center of IP(3)-dependent intracellular Ca(2+) control mechanisms in chromaffin cells.     

Sorting of Von Willebrand factor to lysosome-related granules of haematopoietic cells

The aim of this work was to investigate sorting mechanisms of von Willebrand factor (VWF) when expressed in haematopoietic cells. The processing and sorting of both the wild-type VWF and a multimerization defective propeptide-mutant (VWF(m)) were investigated after expression in the 32D cell line. Normal proteolytic processing was observed for both proteins, however the processing of VWF(m) was much slower and a large portion was unprocessed. Results from subcellular fractionation and immunoelectron microscopy confirmed that a part of VWF, but not VWF(m), was targeted to lysosome-related granules. Partial constitutive secretion was also observed for all forms of VWF and VWF(m). Inhibition of acidification by chloroquine blocked VWF processing but allowed unprocessed pro-VWF targeting to dense organelles. In conclusion, our observations are consistent with VWF multimerization being of importance in cellular retention and targeting to lysosome-related organelles in haematopoietic cells, suggesting a role of protein aggregation for sorting in these cells.     

Cyclooxygenase inhibitors induce apoptosis in oral cavity cancer cells by increased expression of nonsteroidal anti-inflammatory drug-activated gene

We have investigated whether NAG-1 is induced in oral cavity cancer cells by various NSAIDs and if apoptosis induced by NSAIDs can be linked directly with the induction of NAG-1. NAG-1 expression was increased by diclofenac, aceclofenac, indomethacin, ibuprofen, and sulindac sulfide, in the order of NAG-1 induction, but not by acetaminophen, piroxicam or NS-398. Diclofenac was the most effective NAG-1 inducer. Incubation with diclofenac inhibited cell proliferation and induced apoptosis. The expression of NAG-1 was observed in advance of the induction of apoptosis. Conditioned medium from NAG-1-overexpressing Drosophila cells inhibited SCC 1483 cells proliferation and induced apoptosis. In summary, some NSAIDs induce NAG-1 expression in oral cavity cancer cells and the induced NAG-1 protein appears to mediate apoptosis. Therefore, NSAIDs may be considered as a possible chemopreventive agent against oral cavity cancer.     

Proteomic analysis of the proliferative and secretory phases of the human endometrium: protein identification and differential protein expression

Proteomic analyses of the proliferative and secretory phases of the human endometrium were carried out to identify proteins and discover differentially expressed proteins using isotope-coded affinity tags, three stages of chromatographic separation and online tandem mass spectrometry (MS/MS). From an initial list of 346 proteins identified by ProICAT, manual inspection of MS/MS spectra and confirmatory searches pared the list down to 119 positively identified proteins. Only five of the proteins showed consistent differential expression. The utility of some of these proteins as indicators of true differential expression in the endometrium is open to discussion. The two proteins with unquestionable differential expressions in the secretory endometrium are: glutamate NMDA receptor subunit zeta 1 precursor and FRAT1. Some of the proteins that show no differential expression have previously been examined in gene-expression studies with similar conclusions.