Avidity enhancement of L-selectin bonds by flow: shear-promoted rotation of leukocytes turn labile bonds into functional tethers

L-selectin is a key lectin essential for leukocyte capture and rolling on vessel walls. Functional adhesion of L-selectin requires a minimal threshold of hydrodynamic shear. Using high temporal resolution videomicroscopy, we now report that L-selectin engages its ligands through exceptionally labile adhesive bonds (tethers) even below this shear threshold. These tethers share a lifetime of 4 ms on distinct physiological ligands, two orders of magnitude shorter than the lifetime of the P-selectin-PSGL-1 bond. Below threshold shear, tether duration is not shortened by elevated shear stresses. However, above the shear threshold, selectin tethers undergo 14-fold stabilization by shear-driven leukocyte transport. Notably, the cytoplasmic tail of L-selectin contributes to this stabilization only above the shear threshold. These properties are not shared by P-selectin- or VLA-4-mediated tethers. L-selectin tethers appear adapted to undergo rapid avidity enhancement by cellular transport, a specialized mechanism not used by any other known adhesion receptor.     

Myelin-associated glycoprotein and myelin galactolipids stabilize developing axo-glial interactions

We have analyzed mice that lack both the myelin-associated glycoprotein (MAG) and the myelin galactolipids, two glial components implicated in mediating axo-glial interactions during the myelination process. The single-mutant mice produce abnormal myelin containing similar ultrastructural abnormalities, suggesting that these molecules may play an overlapping role in myelin formation. Furthermore, the absence of the galactolipids results in a disruption in paranodal axo-glial interactions, and we show here that similar, albeit less severe, abnormalities exist in the developing MAG mutant. In the double-mutant mice, maintenance of axo-glial adhesion is significantly more affected than in the single mutants, supporting the overlapping function hypothesis. We also show that independently of MAG, galactolipids, and paranodal junctional components, immature nodes of Ranvier form normally, but rapidly destabilize in their absence. These data indicate that distinct molecular mechanisms are responsible for the formation and maintenance of axo-glial interactions.     

Light spectral quality effects on the growth of potato (Solanum tuberosum L.) nodal cuttings in vitro

Summary The effects of light spectral quality on the growth of in vitro nodal cuttings of potato (Solanum tuberosum L.) cultivars Norland, Superior, Kennebec, and Denali were examined. The different light spectra were provided by Vita-Lite fluorescent (VF) (a white light control), blue fluorescent (BF), red fluorescent (RF), low-pressure sodium (LPS), and a combination of low-pressure sodium plus cool-white fluorescent lamps (LPS/CWF). For all cultivars, stem lengths after 4 wk were longest under LPS, followed by RF, LPS/CWF, VF, and BF (in descending order). Microscopic studies revealed that cells were shortest when cultured in BF or VF environments, and were longest in RF or LPS lamp environments. The highest number of axillary branches occurred on plantlets grown with LPS or LPS/CWF, whereas the lowest number occurred with BF. No leaf or stem edema (callus or gall-like growths) occurred with LPS or LPS/CWF lighting, and no edema occurred on cv. Norland plantlets, regardless of lighting. Results suggest that shoot morphologic development of in vitro grown potato plants can be controlled by controlling irradiant spectral quality.     

Retroviral transduction of interferon-γ cDNA into a nonimmunogenic murinefibrosarcoma: generation of T cells in draining lymph nodes capable of treating established parental metastatic tumor

Gene modification of tumor cells with the cDNA for interferon (IFN) has been shown to increase the immunogenicity of some tumor cells. In order to explore further the possible therapeutic relevance of these previous findings, two clones of the nonimmunogenic MCA-102 fibrosarcoma of C57BL/6 origin were retrovirally transduced with the cDNA encoding murine IFN: 102.4JK (4JK), a clone with relatively high major histocompatibility complex (MHC) class I expression, and 102.24JK (24JK), a clone with low expression of surface MHC class I molecules. Retroviral transduction of tumor cells with the cDNA encoding for IFN resulted in a substantial up-regulation of MHC class I surface expression in the 24JK clone but little change of class I in the 4JK clone. In an attempt to generate antitumor lymphocytes, these gene-modified cells were inoculated into mouse footpads and draining lymph nodes (DLN) were removed, dispersed, and cultured in vitro for 10 days with irradiated tumor cells and interleukin-2. DLN from mice bearing either unmodified tumor or tumor transduced with cDNA encoding neomycin resistance (Neo ^R) or IFN, were used to treat recipients harboring 3-day pulmonary metastases induced by the parental, unmodified tumor. Treatment with DLN cells obtained following the injection of 24JK tumor cells modified with the gene for IFN significantly reduced the number of pulmonary metastases in four separate experiments, compared to groups treated by DLN cells generated from inoculation of either the unmodified, parental 24JK clone or the same clone transduced with theNeo ^R gene only. In contrast, DLN cells induced either by IFN-transduced 4JK (high expression of MHC class I) or an unmodified 4JK tumor (moderate expression of MHC class I) had significant but equal therapeutic efficacy. Although the in vitro growth rate of tumor cell lines was unaffected by the insertion of the mouse IFN cDNA, their in vivo (s.c.) growth rates were significantly slower than those of the nontransduced tumors. Thus, after retroviral transduction of the murine IFN cDNA into a nonimmunogenic tumor with a very low level of surface expression of MHC class I, modified tumor cells could elicit therapeutic T cells from DLN capable of successfully treating established pulmonary metastases upon adoptive transfer. This strategy significantly confirms previous observations on the potential therapeutic effects of gene modification of tumor cells with IFN and extends the realm of therapeutic possibilities to include the use of DLN cells for the development of T-cell based immunotherapies against nonimmunogenic human tumors.     

B lymphocyte differentiation in lethally irradiated and reconstituted mice

The recovery of the B lymphocyte compartments was investigated in lethally irradiated mice reconstituted with fetal liver cells. This was done by means of immunofluorescence on frozen sections of spleen, lymph nodes and Peyer's patches. The first B lymphocyte recovery in the spleen was observed on day 8, a few days earlier than in lymph nodes and Peyer's patches (day 13). These early B cells in the spleen were found in the central part of the periarteriolar lymphatic sheath (PALS). Later on, while increasing in number, the B cells formed growing follicles at the periphery of the PALS. Subsequently, brightly fluorescent B cells appeared in the marginal zone, which surrounded the follicles. Another two weeks later, around day 30, also germinal center formation was observed in the follicles of the spleen. B cell development in lymph nodes and Peyer's patches started somewhat later than in the spleen, but once started, the recovery of the different compartments was completed very fast. Germinal center reactions were found in lymph nodes and Peyer's patches already on day 25, and thus earlier than in the spleen, but later than the first occurrence of the strongly fluorescent cells in the marginal zone. Apparently, germinalcenter formation is not essential for the recovery of the population of brightly fluorescent B cells in the marginal zone after irradiation and reconstitution.     

Effect of cerebrocrast on the lymphocyte blast transformation activity in normal and streptozotocin‐induced diabetic rats

Both IDDM and NIDDM are characterized by deviations in peripheral T and B lymphocyte count, T_helper:T_suppressor ratio, as well as by impaired T_suppressor function. These abnormalities may promote insulin antibody and other antibody production, contributing to overt diabetes mellitus development in early stage of the disease. In the present study we explored the effects of cerebrocrast (1,4‐dihydropyridine derivative) administration on Con A‐ and IL‐2‐stimulated tissue lymphocyte blast transformation activity and on the thymus and lymph node mass in normal and streptozotocin (STZ)‐induced diabetic rats. It was established that cerebrocrast, administered four times at the doses of 0·05 and 0·5 mg kg⁻¹, has long‐term (up to 14 days) effects on the immune system and protects against the toxic effect of STZ in STZ‐induced diabetic rats, preventing thymus and lymph node mass loss. We conclude that cerebrocrast administration leads to the increase in number and activity of T_helper and T_suppressor lymphocytes. Glycolysis and DNA synthesis in these cells is augmented under the influence of cerebrocrast administration. We propose that the increase in lymphocyte suppressive activity caused by cerebrocrast administration may prevent the development of IDDM and NIDDM in patients with pre‐diabetes, but in patients with early and overt diabetes mellitus the drug administration may prevent the overexpression of insulin antibodies and other antibodies. The effect of cerebrocrast on the de novo production of insulin and IL‐2 receptors may be beneficial for IDDM and NIDDM patients. Copyright © 1999 John Wiley & Sons, Ltd.     

Effect of Elevated CO2 Concentration on the Starch Grain Accumulation in Chloroplasts from Soybean Leaves at Different Nodes

Soybean ( Glycine max (L.) Merr. ) plants were grown under ambient and elevated CO2 (plus 350 μL/L) concentration in cylindrical open-top chamber to examine their effects on the ultra- structure of chloroplasts. The upper, lower and mid-node leaves were harvested after 7 days full expansion under different CO2 concentrations and ultrathin section were prepared for transmission electron microscopy. In general, the average content of starch grains and thylakoid membranes in the chloroplasts under the elevated CO2 concentration were always higher than the control. Under higher CO2 concentration, there were smaller and less starch grains in the chloroplasts from upper-node leaves than those from mid-node leaves. The shape of their starch grains changed from elliptical to oval,and their thylakoid membranes and grana remained normal. At lower-node leaves, one or two oval, or three timer starch grains accumulated in the chloroplasts. In the mid-node leaves,however, some chloroplasts under higher CO2 concentration had rather large tim elliptical starch grains which could consequently cause disruption of grana and stroama thylakoids in the chloroplasts, whereas in other chloroplasts, the thylakoid membranes and grana were not deformed as the starch grains were smaller and elliptical. On the other hand, under higher CO2 concentration, the stacking degree of thylakoid membranes and starch grains accumulation in the mid-node leaves were significantly higher than those in the lower-node leaves,and slightly higher than the upper-node leaves. These results, in agreement with the chlorophyll contents and photosynthetic rate which reported by other authors in the past, indicated that the ultrastmcture response of the chloroplasts from different leaf nodes of soybeen under elevated CO2 coneentration were different. The seed yield of soybean at different nodes was decreased gradually from mid-nodes towards both upper- and lower-nodes. The greatest effect of elevated CO2 eoneentrafion on seed yeild was at the mid-node leaves. The variation of seed yields of soybean at different nodes under elevated CO2 concentration was in eoneert with the change in the ultrastmcture of chloroplasts and in turn the change in their photosynthetic rates of leaves at different nodes.     

Dendritic cells are dysfunctional in patients with operable breast cancer

Background: Dendritic cells (DCs) play a crucial role in presenting antigens to T lymphocytes and inducing cytotoxic T cells. DCs have been studied in patients with breast cancer to define the factors leading to failure of an effective systemic and locoregional anticancer host response. Methods: Purified DCs were obtained from peripheral blood (PB) and lymph nodes (LNs) of women with operable breast cancer, using immunomagnetic bead selection. The stimulatory capacity of DCs in the allogeneic mixed leukocyte reaction (MLR) and autologous T cell proliferation test (purified protein derivative (PPD) as stimulator), the expression of surface markers on DCs and the production of cytokines in vitro by DCs from patients with operable breast cancer and from healthy donors (controls) were studied. Results: 70–75% purified DCs were isolated from PB and LNs. PBDCs and LNDCs from patients with operable breast cancer demonstrated a reduced capacity to stimulate in an MLR, compared with PBDCs from normal donors (p<0.01). Autologous T cell proliferation in patients had a decreased ability to respond to PPD, when compared with controls (p<0.01). However, T cells from patients responded as well as control T lymphocytes in the presence of control DCs. PBDCs and LNDCs from patients expressed low levels of HLA-DR and CD86, and induced decreased interleukin-12 (IL-12) secretion in vitro, compared with DCs from normal donors (p<0.01). Conclusion: These data suggest a defective DC function in patients with operable breast cancer. Switched-off DCs in patients with early breast cancer and decreased IL-12 production may be important factors for progressive tumour growth.     

Toll-like receptor 2 is expressed on the intestinal M cells in swine

The Toll-like receptor (TLR) 2 binds a wide variety of microbial cell wall components. In this study, we investigated the expression pattern of TLR2 in adult swine gut-associated lymphoid tissues using real-time quantitative PCR, Western blotting, immunohistochemistry, and flow cytometric analysis. The mRNA for TLR2 was preferentially expressed in the mesenteric lymph nodes (MLNs) and Peyer's patches (Pps) of adult swine. Expression in these two tissues was approximately 15- and 9-fold higher than that of spleen, respectively. Western blotting further confirmed that the TLR2 protein was highly expressed in the MLNs and Pps. Interestingly, TLR2-expressing cells were found not only in immune cells, such as T cells and B cells, but also in membranous (M) cells. In addition, double immunostaining for TLR2 and cytokeratin 18 revealed that TLR2 was strongly expressed not only in the cytoplasm but also in the apical membrane of the pocket-like M cells. These results indicate that TLR2 on the MLNs and Pps enable the host defense to respond to a variety of cell wall components. Furthermore, the potential function of TLR2 as a pattern recognition receptor and its cellular distribution suggest that TLR2 plays an important role in ligand-specific transcytosis and transport in M cells.