A cytomorphological and immunohistochemical profile of aggressive B-cell lymphoma: high clinical impact of a cumulative immunohistochemical outcome predictor score

We analyzed morphological and immunohistochemical features in 174 aggressive B-cell lymphomas of nodal and extranodal origin. Morphological features included presence or absence of a follicular component and cytologic criteria according to the Kiel classification, whereas immunohistochemical studies included expression of CD10, BCL-2, BCL-6, IRF4/MUM1, HLA-DR, p53, Ki-67 and the assessment of plasmacytoid differentiation. Patients were treated with a CHOP-like regimen. While the presence or absence of either CD10, BCL-6 and IRF4/MUM1 reactivity or plasmacytoid differentiation did not identify particular cytomorphologic or site-specific subtypes, we found that expression of CD10 and BCL-6, and a low reactivity for IRF4/MUM1 were favourable prognostic indicators. In contrast, BCL-2 expression and presence of a monotypic cytoplasmic immunoglobulin expression was associated with an unfavourable prognosis in univariate analyses. Meta-analysis of these data resulted in the development of a cumulative immunohistochemical outcome predictor score (CIOPS) enabling the recognition of four distinct prognostic groups. Multivariate analysis proved this score to be independent of the international prognostic index. Such a cumulative immunohistochemical scoring approach might provide a valuable alternative in the recognition of defined risk types of aggressive B-cell lymphomas.     

N-domain of human adhesion/growth-regulatory galectin-9: Preference for distinct conformers and non-sialylated N-glycans and detection of ligand-induced structural changes in crystal and solution

Human tandem-repeat-type galectin-9 is a potent adhesion/growth-regulatory effector via lectin capacity of its N- and C-terminal domains. This bioactivity prompted further crystallographic study of the N-domain, combined with analysis in solution. Binding of lactose markedly increased the N-domain's resistance to thermal denaturation. Crystallography revealed its intimate contact profile, besides detecting an extension of the β-sandwich fold by an antiparallel β-strand F0 aligned to the C-terminal F1 strand. Ligand accommodation in its low-energy conformation leads to a movement of Arg87's side chain. As consequence, the ligand's glucose moiety and Arg87 become hydrogen bonded. The resulting predictions for spatial parameters in solution were verified by determining (a) the pattern of magnetization transfer from the protein to protons of lactose and Forssman disaccharide by NMR spectroscopy and (b) the ellipticity changes at wavelengths characteristic for Trp/Tyr residues in near-UV CD spectroscopy. Whereas solid-phase assays confirmed a previously noted tendency for homo- and heterotypic aggregation, gel filtration and ultracentrifugation disclosed monomeric status in solution, in line with crystallographic data. Using cell mutants with defects in glycosylation, this lectin domain was shown to preferentially bind N-glycans without α2,3-sialylation. Since proximal promoter sequences were delineated to diverge markedly among galectin genes and resulting differences in expression profiles were exemplarily documented immunohistochemically, the intrafamily diversification appears to have assigned this protein to a characteristic expression and activity profile among galectins. Our data thus take the crystallographic information to the level of the lectin in solution and in tissues by a strategic combination of spectroscopic and cell/histochemical assays.     

Unique sequence and expression profiles of rat galectins-5 and -9 as a result of species-specific gene divergence

Presence of species-specific gene divergence in a protein family prompts to thoroughly study structural aspects and expression profiles of the products. We herein focus on two members of an adhesion/growth-regulatory group of endogenous lectins, i.e. galectins-5 and -9. After first ascertaining species specificity of occurrence of galectin-5, constituted by a short section of rat galectin-9's N-terminal part and its C-terminal carbohydrate recognition domain, by database mining, we next detected and defined sequence differences in the proximal promoter region between the two genes. The ensuing hypothesis for distinct expression profiles was tested first by RT-PCR and then by immunohistochemistry. For the latter purpose, we employed antibodies rigorously controlled for absence of cross-reactivity including assays with various other galectins and, if necessary, refined by chromatographic removal of bi- or oligospecific activities. Indeed, the galectins have non-identical expression profiles, qualitative differences, e.g. seen for galectin-5-positive bone marrow and erythrocytes or for hitherto unknown expression in cells of the theca folliculi and galectin-9-positive skin epidermis and esophageal epithelium. Lack of hepatocyte or renal cortex staining separates these two expression profiles in rat from localization of galectin-9 in mouse. Interspecies extrapolation in a case of a galectin involved in unique gene divergence may thus not be valid. The presented results on galectin-5 relative to galectin-9 intimate distinct functions especially in erythropoiesis and imply currently unknown mechanisms to compensate its absence from the galectin network in other mammals.     

Conformational transition in DNA on a cold surface

The contour length of DNA fragments, deposited and imaged on mica under buffer, was measured as a function of deposition temperature. Extended DNA molecules (on Ni- and silane-treated surfaces) contract rapidly with falling temperature, approaching the contour length of A-DNA at 2°C. The contraction is not unique to a specific sequence and does not occur in solution at 2°C or on a surface at 25°C, indicating that it arises from a combination of low temperature and surface contact. It is probably a consequence of reduced water activity at a cold surface.     

Role of hexagonal structure-forming lipids in diadinoxanthin and violaxanthin solubilization and de-epoxidation

In this study, we have examined the influence of different lipids on the solubility of the xanthophyll cycle pigments diadinoxanthin (Ddx) and violaxanthin (Vx) and on the efficiency of Ddx and Vx de-epoxidation by the enzymes Vx de-epoxidase (VDE) from wheat and Ddx de-epoxidase (DDE) from the diatom Cyclotella meneghiniana, respectively. Our results show that the lipids MGDG and PE are able to solubilize both xanthophyll cycle pigments in an aqueous medium. Substrate solubilization is essential for de-epoxidase activity, because in the absence of MGDG or PE Ddx and Vx are present in an aggregated form, with limited accessibility for DDE and VDE. Our results also show that the hexagonal structure-forming lipids MGDG and PE are able to solubilize Ddx and Vx at much lower lipid concentrations than bilayer-forming lipids DGDG and PC. We furthermore found that, in the presence of MGDG or PE, Ddx is much more solubilizable than Vx. This substantial difference in Ddx and Vx solubility directly affects the respective de-epoxidation reactions. Ddx de-epoxidation by the diatom DDE is saturated at much lower MGDG or PE concentrations than Vx de-epoxidation by the higher-plant VDE. Another important result of our study is that bilayer-forming lipids DGDG and PC are not able to induce efficient xanthophyll de-epoxidation. Even in the presence of high concentrations of DGDG or PC, where Ddx and Vx are completely solubilized, a strongly inhibited Ddx de-epoxidation is observed, while Vx de-epoxidation by VDE is completely absent. This indicates that the inverted hexagonal phase domains provided by lipid MGDG or PE are essential for de-epoxidase activity. We conclude that in the natural thylakoid membrane MGDG serves to solubilize the xanthophyll cycle pigments and furthermore provides inverted hexagonal structures associated with the membrane bilayer, which are essential for efficient xanthophyll de-epoxidase activity.     

The link between lymphocyte deficiency and autoimmunity: roles of endogenous T and B lymphocytes in tolerance

We demonstrate that transfer of OVA-specific DO11 CD4(+) T cells into mice that lack T and B cells and produce secreted OVA as an endogenous self-protein results in a severe systemic autoimmune reaction with skin inflammation, wasting, and death. The transferred DO11 T cells undergo massive expansion and produce IL-2 and IFN-gamma abundantly. Transfer of DO11 cells into OVA-expressing animals in which T cells are absent but B cells are present, leads to mild disease with no death. In this situation, the DO11 cells undergo similar expansion but show poor Th1 differentiation. This regulatory effect of B cells correlates with profound TCR down-regulation. If T cells are present, the DO11 cells fail to expand independent of B cells. These results suggest that both endogenous T and B lymphocytes control T cell tolerance induction and pathogenicity, but at different stages of an anti-self response. Although endogenous T cells prevent expansion and maintain homeostasis, endogenous B cells limit subsequent effector responses of autoreactive CD4(+) T cells.     

Growth response of selected vegetable crops to spent mushroom compost application in a controlled environment

Summary Snap bean, cucumber, radish, spinach, and tomato seedlings and plants were grown in soils amended with 0, 10, 20, 30 and 50% spent mushroom compost (SMC) under greenhouse conditions. While total seedling emergence was not affected by the addition of SMC, the rate of seedling emergence was delayed. Increased growth was observed in the range of 30 to 50% SMC. The elemental content in seedling tissue indicated an antagonism among K, Ca, and Mg for ion uptake. Increased plant growth and yield were obtained with addition of 20 to 30% SMC; those grown at 50% SMC exhibited some stunting. The limiting factor in the use of SMC appeared to be its high soluble salts content.