Disruption of auxin transport is associated with aberrant leaf development in maize

Despite recent progress, the mechanisms governing shoot morphogenesis in higher plants are only partially understood. Classical physiological studies have suggested that gradients of the plant growth regulator auxin may play a role in controlling tissue differentiation in shoots. More recent molecular genetic studies have also identified knotted1 like homeobox (knox) genes as important regulators of shoot development. The maize (Zea mays L.) mutant rough sheath2 (rs2) displays ectopic expression of at least three knox genes and consequently conditions a range of shoot and leaf phenotypes, including aberrant vascular development, ligular displacements, and dwarfism (R. Schneeberger, M. Tsiantis, M. Freeling, J.A. Langdale [1998] Development 125: 2857-2865). In this report, we show that rs2 mutants also display decreased polar auxin transport in the shoot. We also demonstrate that germination of wild-type maize seedlings on agents known to inhibit polar auxin transport mimics aspects of the rs2 mutant phenotype. The phenotype elaborated in inhibitor-treated plants is not correlated with ectopic KNOX protein accumulation.

IL-6, DHEA and the ageing process.

The age-related increase in circulating IL-6 levels in humans which has been attributed to a decline in DHEA production by the adrenal gland is currently attracting attention because of its possible relevance to the aetiology and management of a number of age-related clinical disorders. The potential importance of these observations and suggestions has prompted us to perform more detailed studies on the relationship between IL-6 and DHEA. Using immunoassay techniques we have found in normal healthy individuals over the age of 40 an inverse relationship between plasma DHEA levels and the presence of detectable levels of IL-6 (more than 1 pg/ml). In vitro, studies also revealed that low dose (10(-6)-10(-8) M) of DHEA and DHEAS inhibited the production of IL-6 in unstimulated human spleen cell suspension cultures whilst enhancing its release by explant cultures of the same tissue. In contrast they had no effect on immunoglobulin production. These studies suggest that there is a real, but complex relationship between IL-6 production and DHEA levels which warrants further investigation.

Hepatocyte growth factor (HGF) protects c-met-expressing Burkitt's lymphoma cell lines from apoptotic death induced by DNA damaging agents.

The relative sensitivity of neoplastic cells to DNA damaging agents is a key factor in cancer therapy. In this paper, we show that pretreatment of Burkitt's lymphoma cell lines expressing the c-met protooncogene with hepatocyte growth factor (HGF) protects them from death induced by DNA damaging agents commonly used in tumour therapy. This protection was observed in assays based on morphological assessment of apoptotic cells and DNA fragmentation assays. The protection was dose- and time-dependent -- maximal protection requiring pre-incubation with 100 ng/ml HGF for 48 h. Western blotting analysis and flow cytometric studies revealed that HGF inhibited doxorubicin- and etoposide-induced decreases in the levels of the anti-apoptotic proteins Bcl-X(L), and to a lesser extent Bcl-2, without inducing changes in the pro-apoptotic Bax protein. Overall, these studies suggest that the accumulation of HGF within the microenvironment of neoplastic cells may contribute to the development of a chemoresistant phenotype.

Organ culture of human lymphoid tissue. I. Characteristics of the system.

The major aim of three-dimensional tissue culture is to preserve the natural architecture of the tissue and thereby allow the cells to retain their original functions during in vitro cultivation. Here we describe a method for the rapid preparation of three-dimensional tissue explants from human lymphoid organs. The precision-cut tissue slices are of uniform size and thickness and can be cryopreserved and stored in liquid nitrogen without substantial loss of viability or functionality of the cells. Upon in vitro culture, cells within the explants survived as well as their counterparts cultured in single cell suspension. However, spontaneous immunoglobulin (Ig) production in explants started more promptly and often reached considerably higher levels than that in suspension cultures run in parallel. Lymphocytes within the slices could be activated by polyclonal stimuli such as PHA, as shown by the upregulation of the activation markers CD23 and CD25 on B and T cells, respectively. However, approximately five-fold higher concentrations of mitogen than those used for suspension cultures were needed. Taken together, the system presented here constitutes a potent tool for the investigation of the complex interactions leading to activation and differentiation of B and T cells in lymphoid organs.

Organ culture of human lymphoid tissue. II. Marked differences in cytokine production and proliferation between slice and suspension cultures of human spleen.

Recently, we have established a method for the culture of human spleen slices in vitro. The procedure allows thin slices (200-350 microns) of human spleen to be cultured for up to 7 days. Using this method, we have previously established that unstimulated spleen slices spontaneously synthesize and secrete considerably higher levels of immunoglobulin than suspension cultures of the same tissue run in parallel. In this study, we report that there are also marked differences in the cytokine secretion profile between slices and suspensions and in their proliferative response. In brief, control and PHA-stimulated spleen slices secrete high levels of IL-1 beta, IL-6, IL-8 and IL-11 while the levels found in suspension supernatants are appreciably lower. By way of contrast, high levels of IL-2, IL-4, IL-10 and TNF alpha are found in suspension culture supernatants following PHA stimulation while the response in slice cultures is extremely low. These differences are also reflected in the results obtained at the cellular (intracellular cytokine) level. Additional studies reveal that spontaneous immunoglobulin production observed in spleen slices can be inhibited by the addition of specific antibodies to IL-1 beta, IL-6 and TNF alpha and that the bulk of the IL-6 and IL-1 beta detected in culture supernatants represents de novo synthesis. Finally, the background and mitogen-stimulated proliferative response of tissue slices is meagre compared with that observed in spleen suspensions suggesting that proliferation in the former is held under strict control. Collectively, we believe that the tissue slice procedure described provides us with a system for studying integrated events in lymphoid tissues in vitro and evaluating immunomodulatory substances of potential clinical importance.

The follow-up study of skin reactivity to recall antigens and E- and EAC-RFC profiles in blood in asbestos workers.

We have determined cutaneous DTH reactions to SK-SD and PPD and peripheral blood lymphocyte profiles in a group of asbestos workers in two consecutive surveys. It was found that asbestosis and, to a lesser extent, the presence of ANA are significantly correlated with the lack of response to the above antigens. 83% of asbestos workers when tested at a 4 year interval fell into the same two categories of responsiveness (lack of response or response at least to one antigen). The asbestosis cases had lower total lymphocyte count as well as proportions and absolute number of E-RFC as compared to asbestos workers without asbestosis and/or ANA. Furthermore, the latter group showed the lower percentages and absolute number of E-RFC than the matched controls. The presence of ANA is also correlated with lower proportions of E-RFC. However, this is related at least in part to asbestosis.

The effects of human seminal plasma and PGE2 on mitogen induced proliferation and cytokine production of human splenic lymphocytes and peripheral blood mononuclear cells.

The effects of human seminal plasma (HSP) and prostaglandin E2 (PGE2) on the proliferative responses of human splenic lymphocytes and peripheral blood mononuclear cells (PBMCs) to phytohaemagglutinin (PHA), anti-CD3 and anti-CD3 plus anti-CD28 mAb have been studied. Th1 and Th2 cytokines were also measured in the supernatants of selected cultures. Both HSP and PGE2 reproducibly inhibit the proliferative response to PHA and anti-CD3 mAb in a dose dependent manner. These effects were observed with both fresh and frozen human PBMCs and splenic lymphocytes. HSP and PGE2 however were less effective in inhibiting the co-stimulatory response induced by anti-CD3 plus anti-CD28 mAb. In addition, the HSP and PGE2 treatment used inhibited the production of the Th1 cytokines IL-2 and IFNg but had a differential modulatory effect on Th2 cytokine production, namely enhancing the production of IL-6 whilst simultaneously impairing the synthesis of IL-4 and IL-10.

The immunosuppressive contribution of prostaglandin components of human semen and their ability to elevate cyclic adenosine monophosphate levels in peripheral blood mononuclear cells.

We have compared the ability of fractions from seminal plasma to suppress lymphocyte proliferation and examined the effects of these fractions in raising intracellular cyclic adenosine monophosphate (cAMP) in the same preparations of peripheral blood mononuclear cells. Human seminal plasma is very effective at raising cAMP but seminal plasma stripped by C-18 reverse phase columns is inactive. Both prostaglandin E (PGE) and 19-hydroxy PGE contribute to the elevation of cAMP and a combination of these two prostaglandins is as effective as whole seminal plasma in raising cAMP but not as effective in inhibiting lymphoproliferation. These results suggest that human seminal plasma prostaglandins act through the EP2 receptor to inhibit T cell and NK cell function and thus attenuate both the cellular and humoral actions of the female's immune system.

Relative immunosuppressive activity of human seminal prostaglandins.

Human seminal plasma contains uniquely high concentrations of prostaglandins of the E series which are believed to contribute to its immunosuppressive effects in vivo. In order to obtain further insight into their activity we have compared the immunosuppressive properties in vitro of PGE1, PGE2 and 19-OH PGE using three immunological systems known to be modulated by prostaglandins, namely, mitogen induced lymphocyte proliferation, IL-2 and transferrin receptor expression and NK-cell mediated cytotoxicity. These studies revealed that PGE1 and PGE2 exerted a greater immunosuppressive effect than 19-OH PGE, but considerably higher levels of 19-OH PGE in semen might contribute the majority of immunosuppressive activity in vivo. Our studies also show that the lower stability of 19-OH PGE in culture media may be responsible for its lower immunosuppressive effect observed in vitro.

Expression of a common secretory granule specific protein as a marker for the extracellular organelles (prostasomes) in human semen.

OBJECTIVE: To further characterize prostasomes, trilamellar to multilamellar vesicles that are thought to originate from acinar cells of the human prostate and present in appreciable amounts in normal human semen. Purified prostasomes were shown to have immunosuppressive activity in vitro as measured by inhibition of mitogen-induced lymphocyte proliferation and inhibition of superoxide generation by neutrophils. A granule membrane protein, called granulophysin, has recently been identified in the membranes of platelet dense granules. Antibodies that recognize granulophysin also stain granules in different cell types including leukocytes, melanocytes, neurones, endothelial cells, and epithelial cells. DESIGN: The presence of epitopes recognized by antigranulophysin monoclonal antibody in prostasomes was investigated using indirect immunofluorescence and subsequent cytofluorimetric analysis. The protein was also analyzed by Western blotting. Reactivity of antigranulophysin antibody with the prostate tissue was studied by immunoperoxidase staining. RESULTS: A majority of prostasome particles specifically reacted with antigranulophysin antibody. In lysates prepared from prostasomes, a broad band of 32 to 37 kd was detected by Western blotting. CONCLUSION: This report defines granulophysin as a constituent membrane molecule of prostasomes that may serve as a useful marker in elucidation of prostasome function.

Generation and preliminary characterization of three mouse monoclonal antibodies reacting with human B lymphocytes.

The paper describes generation and characterization of three monoclonal antibodies (BR3, BR16, BR31) specific for human B lymphocytes. BR3 and BR31 stain highly purified population of B lymphocytes but fail to react with granulocytes, monocytes and E+ lymphocytes. BR16 in addition to B cells also stains 10% peripheral T cells and 70% granulocytes. BR3 staining pattern closely correlates that obtained with antihuman Ig both in peripheral blood and in lymphoid tissues. BR31 recognizes a subpopulation of B lymphocytes (about 50%).

Mouse T-T hybridomas expressing receptors for syngeneic erythrocytes.

The report describes generation of mouse T-T hybridomas expressing receptors for murine erythrocytes. The obtained cell lines form 30-85% rosettes with mouse red cells without any preference for H-2 compatible erythrocytes. They do not bind xenogeneic erythrocytes. The role of Thy 1 antigen in constitution of receptors for erythrocytes was excluded since anti-Thy 1 monoclonal antibodies had no influence on rosetting activity. Rosette formation is inhibited by certain sugars showing the same pattern of inhibition as in the case of autologous rosette formation by CBA mouse thymocytes.

Specificity of anti-T cell antibodies from BCG infected mice.

BCG infected mice were found to produce anti-T cell cytotoxic autoantibodies (CAA)6. Absorption studies with intact or desialyzed thymocytes and splenic T cells showed that CAA consisted of two kinds of antibody with different target cell specificities, one (CAA-2) able to recognize determinants on desialyzed T cells, and another (CAA-1) able to bind to both intact and desialyzed thymocytes. Normal thymocytes did not remove the antibodies specific to desialyzed lymphocytes. These two antibodies were separated by affinity chromatography on agarose. Cytotoxicity inhibition, using various sugars as inhibitors, indicated that lactose and lactosamine were potent inhibitors of CAA-2. In contrast CAA-1 was not inhibited by any of the sugars tested. Functional experiments revealed that CAA-2 inhibited the secondary anti-SRBC IgG antibody response without effecting the IgM response. The anti-SRBC antibody production (both IgM and IgG) was not affected by CAA-1. The possible interference of these antibodies with carbohydrate specific cell interactions are discussed.

Antigen detected on normal human B lymphocytes by BR31 monoclonal antibody--the nature of the antigen and its functional properties.

Monoclonal BR31 antibody reacts with a subpopulation of B lymphocytes from normal human blood and tissue. The paper shows that the antigen recognized by BR31 is a protein or at least depends on protein synthesis for its expression. BR31 mAb does not cause spontaneous proliferation of B cells. However, it greatly enhances proliferation of normal tonsil B cells in response to SAC and PMA. No costimulation was observed when the cells were cultured in the presence of BR31 mAb and anti-Ig or BCGF. It was also found that there is a subpopulation of B cells which is able to proliferate with SAC or PMA only in the presence of BR31 mAb.

Reactivation of antibody genes in Epstein-Barr virus transformed human B cell lines. A preliminary report.

Three immortalised human B cell lines, which had either last their capacity to secrete specific antibody or secreted low levels of antibody were studied in an attempt to reactivate or enhance antibody synthesis. A variety of different stimuli known to cause activation, proliferation and differentiation of normal B cells were used including polyclonal activators and recombinant interferon. Four of them, (namely LPS, anti-IgM, IL2 and IL6) effectively increased specific antibody synthesis after three days of culture. These preliminary experiments show that the loss or decline in immunoglobulin production by immortalised human B cells is, at least in some cases, reversible.

The use of tissue slices in immunological investigations.

Our knowledge of cellular and molecular events taking place during various immune responses comes mainly from in vitro studies where isolated cells are exposed to defined stimuli. This reductionist approach has greatly advanced our understanding of the immune system. However, these studies do not truly reflect the complexity of the integrative events which take place in both primary and secondary lymphoid tissue in vivo. In order to address this problem we have developed a tissue culture procedure which involves the cultivating of precision cut human spleen slices at gas/liquid interface. We have shown, that cells in this culture system show marked differences in cytokine and immunoglobulin production in comparison with conventional single cell suspension cultures, obtained from the same spleen and run in parallel. In this review article we describe the basic technique, results which we have obtained in this system and discuss the possible basis of the observed differences.

IgM of mice infected with Mycobacterium bovis interacts selectively with T cells.

In the course of experimental BCG infection in mice, cytotoxic autoantibodies (CAA) of IgM class reacting with thymocytes and T lymphocytes were found. CAA were not cytotoxic for bone marrow cells and B lymphocytes. CAA reacts with PNA+, glass wool-adherent, hydrocortisone-sensitive thymocytes. The reaction of thymocytes with CAA and complement, prevented suppressor cell induction from such treated cells. The importance of CAA in mycobacterial infection is discussed.

Characterization of lymphocyte E rosette inhibitory factor in sera from tuberculous patients.

Rosette inhibitory factor (RIF) in serum of patients suffering from active pulmonary tuberculosis, after in vitro contact with lymphocytes from healthy subjects, inhibits spontaneous rosette formation of these lymphocytes with sheep red blood cells (SRBC). RIF is not a C-reactive protein. By the use of chromatography on Sephadex G-200 and single analytic flotation, RIF was found to be contained in the beta-lipoprotein fraction. The presumable mechanism of formation of this factor and suggestions concerning its action on T cells are discussed.

Partial characterization of a factor present in normal human and animal serum which stimulate E rosette formation by lymphocytes from tuberculous patients.

The influence of sera from healthy human beings on ability of lymphocytes from patients with active pulmonary tuberculosis to form rosettes with SRBC was studied. Lymphocytes incubated for 2 hours with sera from healthy subjects recovered ability to form rosettes spontaneously with SRBC. The factor responsible for stimulation of the lymphocytes is contaned in the albumin fraction. Sera from mice, rabbits, guinea pigs and fetal calf serum had a similar effect to that of sera from healthy human beings. The character of this factor and the mechanism of its action are discussed. The stimulating factor is accompanied by an inhibitor.

T and B lymphocytes in patients with pulmonary tuberculosis.

Counts of T and B lymphocytes were made in the peripheral blood from patients with active and inactive pulmonary tuberculosis and in healthy subjects. In patients with active pulmonary tuberculosis the numbers of T cells were significantly lower than in the controls. After treatment for two months, if the patient's condition improved the percentage of T cells returned to nearly normal levels. If the patient failed to improve, the numbers of T cells remained low. The mechanism of these phenomena is discussed.