The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and become cytolytic

Natural killer cells are important cytolytic cells in innate immunity. We have characterized human NK cells of spleen, lymph nodes, and tonsils. More than 95% of peripheral blood and 85% of spleen NK cells are CD56(dim)CD16(+) and express perforin, the natural cytotoxicity receptors (NCRs) NKp30 and NKp46, as well as in part killer cell Ig-like receptors (KIRs). In contrast, NK cells in lymph nodes have mainly a CD56(bright)CD16(-) phenotype and lack perforin. In addition, they lack KIRs and all NCR expression, except low levels of NKp46. The NK cells of tonsils also lack perforin, KIRs, NKp30, and CD16, but partially express NKp44 and NKp46. Upon IL-2 stimulation, however, lymph node and tonsilar NK cells up-regulate NCRs, express perforin, and acquire cytolytic activity for NK-sensitive target cells. In addition, they express CD16 and KIRs upon IL-2 activation, and therefore display a phenotype similar to peripheral blood NK cells. We hypothesize that IL-2 can mobilize the NK cells of secondary lymphoid tissues to mediate natural killing during immune responses. Because lymph nodes harbor 40% and peripheral blood only 2% of all lymphocytes in humans, this newly characterized perforin(-) NK cell compartment in lymph nodes and related tissues probably outnumbers perforin(+) NK cells. These results also suggest secondary lymphoid organs as a possible site of NK cell differentiation and self-tolerance acquisition.     

Genome-wide analysis of epigenetic dynamics across human developmental stages and tissues

Background

Epigenome is highly dynamic during the early stages of embryonic development. Epigenetic modifications provide the necessary regulation for lineage specification and enable the maintenance of cellular identity. Given the rapid accumulation of genome-wide epigenomic modification maps across cellular differentiation process, there is an urgent need to characterize epigenetic dynamics and reveal their impacts on differential gene regulation.

Methods

We proposed DiffEM, a computational method for differential analysis of epigenetic modifications and identified highly dynamic modification sites along cellular differentiation process. We applied this approach to investigating 6 epigenetic marks of 20 kinds of human early developmental stages and tissues, including hESCs, 4 hESC-derived lineages and 15 human primary tissues.

Results

We identified highly dynamic modification sites where different cell types exhibit distinctive modification patterns, and found that these highly dynamic sites enriched in the genes related to cellular development and differentiation. Further, to evaluate the effectiveness of our method, we correlated the dynamics scores of epigenetic modifications with the variance of gene expression, and compared the results of our method with those of the existing algorithms. The comparison results demonstrate the power of our method in evaluating the epigenetic dynamics and identifying highly dynamic regions along cell differentiation process.

     

Immunophenotypic characterization of primary and secondary lymphoid follicles

The need for an immunophenotypical referential framework relative to lymphoid follicle has led us to apply a panel of monoclonal and polyclonal antibodies, by means of a sensitive immunostaining method. Lymphoid follicle is an immunophenotypically complex structure made up of three lymphoid populations (B, being its bulk, and a few T and NK cells), dendritic reticulum cells (DRCs) and Flemming's macrophages. Follicular B population is To 15 +, B1+, OKB 7 +, HLA-DR + and C3bR +. In secondary follicles there are differential characteristic reactivities for each topographic compartment: Mantle zone is positive for OKB 2 and surface IgM (sIgM) and IgD (sIgD); germinal center (GC) clear zone (with centrocytic predominance) for OKT 9, sIgM and weakly for OKB 2; and GC dark zone (with centroblastic predominance) only for OKT 9. In sections, OKT 10 allows one to see immunoblasts and plasma cells, the latter being with lymphoplasmacytoid cells the only intracytoplasmic immunoglobulin holders. 10% of GC lymphocytes are T cells, almost exclusively T-helper (Leu 3a +). Another 10% to 15% of lymphoid cells are Leu 7 (HNK-1) +. In histological sections, DRCs are specifically marked with R4/23 and Flemming's macrophages with anti-alpha1-antitrypsin and anti-alpha1-antichymotrypsin antibodies, both populations being negative to OKM 1 and OKM 5.     

Neutrophils and monocytes transport tumor cell antigens from the peritoneal cavity to secondary lymphoid tissues

Antigen-transporting cells take up pathogens, and then migrate from sites of inflammation to secondary lymphoid tissues to induce an immune response. Among antigen-transporting cells, dendritic cells (DCs) are believed to be the most potent and professional antigen-presenting cells that can stimulate naïve T cells. However, the cells that transport antigens, tumor cell antigens in particular, have not been clearly identified. In this study we have analyzed what types of cells transport tumor cell antigens to secondary lymphoid tissues. We show that neutrophils, monocytes and macrophages but not DCs engulf X-irradiated P388 leukemic cells after their injection into the peritoneal cavity, and that neutrophils and monocytes but not macrophages migrate to the parathymic lymph nodes (pLN), the blood, and then the spleen. The monocytes in the pLN comprise Gr-1⁻ and Gr-1⁺ ones, and some of these cells express CD11c. Overall, this study demonstrates that neutrophils and monocytes transport tumor cell antigens from the peritoneal cavity to secondary lymphoid tissues.     

Formation of primary microvessels in the early stages of prenatal human morphogenesis

By means of light and electron microscopy, the most general mechanisms of formation and development of primary blood microvessels in functionally different organs (adenohypophysis, thyroid, thymus, liver, spleen, small and large intestine) have been studied in human embryos 4-8 weeks of age. Ultrastructure of cells in the extra- and intraorganic mesenchyme is described; to the latter belongs the leading role in organization the pathways of the prevascular microcirculation. The primary microvessels are formed as a result of canalization of the intercellular clefts, lining with mesenchymal cells, that gradually transfer into primordial endotheliocytes. Basing on ultrastructural analysis, certain stages of differentiation of protocapillary endotheliocytes have been defined and described in different organs. The change of the prevascular microcirculation into the intraorganic protocapillary bed (the primary blood bed) is an essential and necessary stage of the organogenesis.     

The effect of primary and secondary immunization on the lymphoid tissues of the carp, Cyprinus carpio L

Mirror carp immunized with human gamma globulin (HGG) in Freund's complete adjuvant (FCA) show a proliferative response involving cells whose cytoplasm stains deep red with methyl green-pyronin (pyroninophilic cells). This response occurs particularly in the haemopoietic parenchyma of the pronephros and mesonephros. It peaks at week 3, with the formation of clusters of pyroninophilic cells in the pronephros. Immunization with Aeromonas salmonicida elicited a less intense pyroninophilic response but caused a larger increase in pigment-containing cells. After a secondary immunization with HGG in FCA, a distinct response was observed in the spleen: Pyroninophilic cells collected within the ellipsoid sheaths in large numbers and formed nodules. The reticulum of such nodules acquired spherical proportions and resembled the white pulp reticulum of the tetrapod spleen. The roles of such pyroninophilic cells and the possibility that aggregations of them may be functionally analogous to homoiotherm germinal centres are discussed.     

The role of lymphotoxin in development and maintenance of secondary lymphoid tissues

Secondary lymphoid organs provide the necessary microenvironment for the cooperation of antigen-specific T- and B-lymphocytes and antigen-presenting cells in order to initiate an efficient immune response. Remarkable progress in understanding of the mechanisms of lymphoid organogenesis was achieved due to the analysis of various gene-targeted mice. This review primarily focuses on the role of lymphotoxin (LT) in development, maturation and maintenance of secondary lymphoid organs.     

Fungal and algal gene expression in early developmental stages of lichen-symbiosis

How plants and microbes recognize each other and interact to form long-lasting relationships remains one of the central questions in cellular communication. The symbiosis between the filamentous fungus Cladonia grayi and the single-celled green alga Asterochloris sp. was used to determine fungal and algal genes upregulated in vitro in early lichen development. cDNA libraries of upregulated genes were created with suppression subtractive hybridization in the first two stages of lichen development. Quantitative PCR subsequently was used to verify the expression level of 41 and 33 candidate fungal and algal genes respectively. Induced fungal genes showed significant matches to genes putatively encoding proteins involved in self and non-self recognition, lipid metabolism, and negative regulation of glucose repressible genes, as well as to a putative d-arabitol reductase and two dioxygenases. Upregulated algal genes included a chitinase-like protein, an amino acid metabolism protein, a dynein-related protein and a protein arginine methyltransferase. These results also provided the first evidence that extracellular communication without cellular contact can occur between lichen symbionts. Many genes showing slight variation in expression appear to direct the development of the lichen symbiosis. The results of this study highlight future avenues of investigation into the molecular biology of lichen symbiosis.     

Constitutive versus cell cycle regulation of c-myb mRNA expression correlates with developmental stages in murine B lymphoid tumors.

The expression of c-myb mRNA is differentially regulated in murine B lymphoid tumors such that B cell lymphomas and plasmacytomas contain significantly less c-myb mRNA than pre-B cell lymphomas. To examine the low level of c-myb mRNA expression in the murine B cell lymphoma cell line BCL1, nonessential amino acid starvation was used to block these cells in a G1 state. When BCL1 cells were released from this block, a 7- to 10-fold increase in c-myb mRNA was detected in late G1 and S phase cells relative to that detected in exponentially growing BCL1 cells. This increase was not inhibited by aphidicolin. To determine whether cell cycle regulation of c-myb mRNA expression occurred during exponential growth in both murine pre-B cell lymphoma and B cell lymphoma cell lines, elutriation was used to separate exponentially growing cell populations. An increase in c-myb mRNA expression was seen in late G1 and S phase fractions from B cell lymphoma cell lines. In contrast, c-myb mRNA levels remained constant in elutriation fractions isolated from pre-B cell lymphoma cell lines. Expression of c-myb mRNA was not detected in exponentially growing or in Go serum-stimulated murine fibroblasts. These results indicate that constitutive vs cell cycle regulation of c-myb mRNA expression is related to the state of differentiation in murine B lymphoid tumors and suggest that a switch in regulation may occur during normal B cell development.     

Defining the genetic architecture of human developmental language impairment

Language is a uniquely human trait, which poses limitations on animal models for discovering biological substrates and pathways. Despite this challenge, rapidly developing biotechnology in the field of genomics has made human genetics studies a viable alternative route for defining the molecular neuroscience of human language. This is accomplished by studying families that transmit both normal and disordered language across generations. The language disorder reviewed here is specific language impairment (SLI), a developmental deficiency in language acquisition despite adequate opportunity, normal intelligence, and without any apparent neurological etiology. Here, we describe disease gene discovery paradigms as applied to SLI families and review the progress this field has made. After review the evidence that genetic factors influence SLI, we discuss methods and findings from scans of the human chromosomes, including the main replicated regions on chromosomes 13, 16 and 19 and two identified genes, ATP2C2 and CMIP that appear to account for the language variation on chromosome 16. Additional work has been done on candidate genes, i.e., genes chosen a priori and not through a genome scanning studies, including several studies of CNTNAP2 and some recent work implicating BDNF as a gene x gene interaction partner of genetic variation on chromosome 13 that influences language. These recent developments may allow for better use of post-mortem human brain samples functional studies and animal models for circumscribed language subcomponents. In the future, the identification of genetic variation associated with language phenotypes will provide the molecular pathways to understanding human language.     

Detecting physiological and pesticide-induced apoptosis in early developmental stages of invasive bivalves

In this study, embryos and early larval stages of two invasive bivalves, the Mediterranean mussel (Mytilus galloprovincialis) and zebra mussel (Dreissena polymorpha), were assayed for physiological apoptosis and stress-induced apoptosis post-exposure to a molluscicide, Bayluscide^®. Physiological apoptosis was measured at 4-h intervals and Bayluscide^® exposures were 4 h and 24 h beginning at both 30 min post-fertilization and at 24 h post-fertilization. Apoptosis was detected by the TUNEL assay, and apoptotic values were compared for the different stages present. The two bivalves tested for apoptotic cells displayed variations in physiological and stress-induced apoptotic values despite having the same life stage transitions. However, in the majority of exposures, we found that as pesticide concentrations increased, the apoptotic values decreased in the highest concentrations. We suggest that this change to apoptotic inhibition indicates that there is a threshold at which apoptosis can no longer be used as a protective measure against damaged cells. These concentrations that cause changes in apoptotic pattern should be further tested for long-term effects on reproduction and survival. By detecting sub-lethal concentrations that may impair recruitment to the population and the continuation of the life cycle, chemical control of invasive bivalves could be targeted to early life stages and to reduce potential environmental impacts resulting from the higher concentrations required to control adults.     

EA rosette forming lymphoid cells: distribution as to cell types and organs from chickens of different developmental stages.

The interaction of chicken spleen cells with sheep erythrocytes coated with chicken antibody (EA complexes) was studied using a rosette assay. The results reported in this paper indicate that subpopulations of chicken lymphocytes, monocytes, and heterophils have a receptor for EA. The formation of rosettes between chicken lymphoid cells and sheep erythrocytes (SRBC) was dependent upon the concentration of antibody used to sensitize the SRBC. In a developmental study of rosette-forming lymphocytes (RFL), the bursa was the first site of appearance of large numbers of RFL. The percentage of RFL in the bursa reached a peak at 17 days of embryonic life, and declined to a low by hatching. The percentage of RFL in the spleen, however, began to increase at the time of hatching and by 6 weeks of age the spleen far surpassed the bursa in percentage RFL. At no age were significant numbers of RFL detected in the thymus.     

Functional interactions of human and murine lymphoid cells. I. Analysis of primary and secondary responses

In this study human T-cell responses against murine alloantigens were analyzed. The results show that optimal primary responses are obtained from peripheral blood mononuclear cells only when murine splenic adherent cells (SAC) were used as antigen. Further analysis revealed that human T cells were able to respond directly to murine cells without the need for antigen reprocessing; however, human interleukin 1 (IL-1) was required for optimal stimulation. In contrast, secondary proliferative responses to murine cellular antigens could be induced from primed T cells even in the absence of SAC and/or IL-1. These proliferative responses, and in addition, cytotoxic T-cell responses, were specific for the priming antigen. Long-term human T-cell lines specific for murine alloantigens were found to replace the need for murine T cells in antigen-specific murine B-cell responses to sheep red blood cells. The mechanism of help delivered by the human T cells appeared to be by the release of nonspecific helper-T-cell factors. The evidence presented for this is the inability of these cells to stimulate cells from mice that express the X chromosome B-cell defect xid.     

Intermediate filament protein expression in early developmental stages of the mouse

Expression patterns of intermediate filament proteins have been studied during early mouse embryo development. For this purpose, pre-implantation embryos at different stages of development after in vitro fertilization were studied using antibodies to cytokeratins, vimentin and lamins, using the indirect immunofluorescence assay. The levels of expression were quantitated and localization of the protein constituents was assessed by means of confocal scanning laser microscopy. Our studies showed that, although the embryos grew in culture, vimentin could not be detected in a filamentous organization. Immunofluorescence for cytokeratins was only positive from the 8-cell stage onwards. In the morula stage an increased level of cytokeratin expression was observed with a transitional staining pattern, combining a filamentous and a diffuse occurrence. In the blastocyst stages profound cytokeratin filaments were seen in trophoblast cells but not in the inner cell mass. When the cytokeratin subtypes were analysed separately, it became apparent that expression levels of cytokeratins 8 and 18 increased gradually up to a filamentous pattern in the blastocyst stage. Cytokeratins 7 and 19, although elevated in the latter stage and showing a filamentous distribution, were not found as prominently as cytokeratins 8 and 18. A-type as well as B-type lamins could be detected in all developmental stages examined, as a faintly reactive nuclear lamina. In blastocysts both lamin types were detected in trophoblast as well as in inner cell mass.