Separation of mitogen-induced suppressor cells of human antibody-producing cells

Human antibody-forming cells were demonstrated by a plaque in agar technique following in vitro stimulation with either pokeweed mitogen or Cowan I strain of protein A-positive Staphylococcus aureus bacteria. We evaluated the effects on this antibody formation caused by the addition of cells which had been stimulated with PH A or Con A. Both Con A and PHA cells harvested after 3 days showed strong inhibition of pokeweed-induced plaque formation. The majority of the suppression could be accounted for by a blast fraction separated on 1g sedimentation gradients from the Con A or PHA cultures. Small cells from such cultures showed inhibition of PFC when added at high ratios (1:2), but this suppressive activity diluted out much more rapidly than that of the blast cells. No helper activity was noted with either small cells or blasts. Our studies indicate a T-cell blast as the suppressive fraction in Con A- or PHA-stimulated human lymphoid cells. While this T-cell suppression applies to T-dependent responses such as antibody stimulation with pokeweed mitogen, it does not have a substantial effect on Cowan I-induced plaque-forming responses. The finding that Cowan I-induced plaques could not be inhibited by Con A or PHA blasts indicates the T independence of this response.     

Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies

The promyelocytic leukaemia (PML) tumour suppressor protein epitomizes the PML-nuclear body (PML-NB) and is crucially required for the proper assembly of this macromolecular nuclear structure. Unlike other, more specialized subnuclear structures such as Cajal and Polycomb group bodies, PML-NBs are functionally promiscuous and have been implicated in the regulation of diverse cellular functions. PML-NBs are dynamic structures that favour the sequestration and release of proteins, mediate their post-translational modifications and promote specific nuclear events in response to various cellular stresses. Recent data suggest that PML-NBs may be heterogeneous in composition, mobility and function.     

Downstream of tyrosine kinases-1 and Src homology 2-containing inositol 5'-phosphatase are required for regulation of CD4+CD25+ T cell development

The adaptor protein, downstream of tyrosine kinases-1 (Dok-1), and the phosphatase SHIP are both tyrosine phosphorylated in response to T cell stimulation. However, a function for these molecules in T cell development has not been defined. To clarify the role of Dok-1 and SHIP in T cell development in vivo, we compared the T cell phenotype of wild-type, Dok-1 knockout (KO), SHIP KO, and Dok-1/SHIP double-knockout (DKO) mice. Dok-1/SHIP DKO mice were runted and had a shorter life span compared with either Dok-1 KO or SHIP KO mice. Thymocyte numbers from Dok-1/SHIP DKO mice were reduced by 90%. Surface expression of both CD25 and CD69 was elevated on freshly isolated splenic CD4(+) T cells from SHIP KO and Dok-1/SHIP DKO, suggesting these cells were constitutively activated. However, these T cells did not proliferate or produce IL-2 after stimulation. Interestingly, the CD4(+) T cells from SHIP KO and Dok-1/SHIP DKO mice produced higher levels of TGF-beta, expressed Foxp3, and inhibited IL-2 production by CD3-stimulated CD4(+)CD25(-) T cells in vitro. These findings suggest Dok-1 and SHIP function in pathways that influence regulatory T cell development.     

Comparing image (fractal analysis) and electrochemical (impedance spectroscopy and electrolyte leakage) techniques for the assessment of the freezing tolerance in olive

Olive growth and productivity are limited by low temperatures mainly during winter, but sometimes also in spring and fall. The most effective way to avoid these damages in areas subjected to these climatic conditions is to select least susceptible varieties, but the choice of the right method to determine cold hardiness is extremely difficult. The aims of the work were (1) to assess LT₅₀ (lethal temperature at which 50% of damage in plants subjected to low temperatures occurs) of some olive varieties in two seasons (summer and winter) and (2) to assess the reliability of different methods to evaluate cold hardiness. LT₅₀ was determined on 21 different olive (Olea europaea L.) Italian varieties by leaf and shoot electrolyte leakage, shoot impedance spectroscopy and leaf color determination of fractal spectrum. All the experiments were conducted on non-acclimated and cold-acclimated plants. Our results showed that all the three methods were able to detect damages on olive plants after exposure to low temperatures, with leaves appearing more sensitive to cold stress than shoots. Among these methods, fractal analysis could be very useful in assessing cold hardiness of plants on the basis of visible injury, without sophisticated or expensive instruments and in a reliable and cost-effective way, using only a scanning device, a personal computer and dedicated freeware software.     

Involvement of somatolactin in background adaptation of the cichlid fish Cichlasoma dimerus

Somatolactin (SL) is a pituitary hormone present exclusively in fish that is involved in different physiological processes. The role of SL was evaluated in Cichlasoma dimerus (Teleostei, Perciformes) exposed for 10 days to a black and white background (BB and WB). Changes in alpha-melanophore stimulating hormone (alphaMSH) and melanin concentrating hormone (MCH) cells were also analyzed for comparison with SL. A melanin dispersing effect was observed in fish exposed to a BB, while a concentrating one was observed in those exposed to a WB. By Western blot, three SL-immunoreactive (ir) bands (32, 28 and 23.5 kD) were evidenced. Pituitary SL-ir levels were 2.66- and 2.67-fold greater in the 32 Kd and 28 kD bands, respectively, in BB fish compared with those of WB fish. The SL-ir 23.5 Kd band was not included in the analysis because of its unknown identity. In addition, SL-ir cell number and area were significantly higher in the BB condition (BB 22.73+/-1.46, WB 7.37+/-0.54 and BB 27.39+/-1.00 microm2; WB: 16.61+/-0.65 microm2). No significant differences were observed in the number of the hypothalamic MCH-ir neurons. However, a significant difference was observed in their nuclear area (BB 11.61+/-0.42 microm2, WB 17.80+/-0.84 microm2). alphaMSH-ir cells showed a marked increased in number (BB 35.96+/-1.22, WB 24.36+/-1.04), but no significant differences were observed in the cell area. In conclusion, this study presented clear evidence towards a possible involvement of SL in the adaptation to background colors in teleost together with alphaMSH and MCH.     

Co-aggregation of FcgammaRII with FcepsilonRI on human mast cells inhibits antigen-induced secretion and involves SHIP-Grb2-Dok complexes

Signaling through the high affinity IgE receptor FcepsilonRI on human basophils and rodent mast cells is decreased by co-aggregating these receptors to the low affinity IgG receptor FcgammaRII. We used a recently described fusion protein, GE2, which is composed of key portions of the human gamma1 and the human epsilon heavy chains, to dissect the mechanisms that lead to human mast cell and basophil inhibition through co-aggregation of FcgammaRII and FcepsilonRI. Unstimulated human mast cells derived from umbilical cord blood express the immunoreceptor tyrosine-based inhibitory motif-containing receptor FcgammaRII but not FcgammaRI or FcgammaRIII. Interaction of the mast cells with GE2 alone did not cause degranulation. Co-aggregating FcepsilonRI and FcgammaRII with GE2 1) significantly inhibited IgE-mediated histamine release, cytokine production, and Ca(2+) mobilization, 2) reduced the antigen-induced morphological changes associated with mast cell degranulation, 3) reduced the tyrosine phosphorylation of several cellular substrates, and 4) increased the tyrosine phosphorylation of the adapter protein downstream of kinase 1 (p62(dok); Dok), growth factor receptor-bound protein 2 (Grb2), and SH2 domain containing inositol 5-phosphatase (SHIP). Tyrosine phosphorylation of Dok was associated with increased binding to Grb2. Surprisingly, in non-stimulated cells, there were complexes of phosphorylated SHIP-Grb2-Dok that were lost upon IgE receptor activation but retained under conditions of Fcepsilon-Fcgamma co-aggregation. Finally, studies using mast cells from Dok-1 knock-out mice showed that IgE alone triggers degranulation supporting an inhibitory role for Dok degranulation. Our results demonstrate how human FcepsilonRI-mediated responses can be inhibited by co-aggregation with FcgammaRIIB and implicate Dok, SHIP, and Grb2 as key intermediates in regulating antigen-induced mediator release.