Identification of Immunogenic Epitopes of the 170-kDa Subunit Adhesin of Entamoeba histolytica in Patients with Invasive Amebiasis

ABSTRACT. Entamoeba histolytica causes amebic dysentery (AD) and liver abscess (ALA). Little is known about protective immunity to amebiasis, and studies in this area have been complicated by the paucity of defined ameba antigens. We examined the proliferative responses of peripheral blood mononuclear cells (PBMC) from patients with AD and ALA to a recombinant protein containing a portion of the 170 kDa adhesin of E. histolytica (170CR), and to two synthetic peptides (1 and 2) derived from the 170 kDa sequence that were predicted to contain T cell epitopes. A significant number of patients with AD and ALA had PBMC that proliferated to 170CR molecule, and several individuals with ALA and AD had T cells that recognized one or both peptides. Contrarily, individuals from a non-endemic region for amebiasis did not respond to 170CR protein, or to both peptides. In regard to antibody response, nine of fifteen patients with ALA showed antibodies to 170CR protein. These same patients had antibodies to peptide 2. We identified peptides from 170-kDa adhesin that may contain both T and B cell epitopes recognized by some patients with invasive amebiasis. These peptides may be valuable reagents in studies of the immune response to amebiasis.     

Complement-dependent induction of DNA synthesis and cell proliferation in human liver connective tissue cells in vitro

Summary Liver connective tissue cells (LCTC) isolated from patients with fibrotic livers have morphological and biochemical characteristics of myofibroblasts. We have examined the proliferation of LCTC derived from normal livers and from livers with fibrosis of different etiologies, as well as proliferation of skin fibroblasts. We have compared proliferation rates in the presence of fresh human serum and heat-inactivated serum. While skin fibroblast and LCTC from normal liver showed no difference, proliferation of LCTC from fibrotic livers was markedly decreased in the presence of heat-inactivated serum. We demonstrate that the native complement component C1 is a factor involved in the induction of DNA synthesis and proliferation of LCTC isolated from fibrotic livers. We propose that native C1, acting probably in cooperation with other growth factors, is involved in the expansion of connective tissue cells during the development of liver fibrosis.     

Modified single node culture method—a new micropropagation method for chickpea

Summary Chickpea was micropropagated by axillary shoot proliferation (ASP) and modified single node culture (MSNC) methods. Maximum propagule proliferation occurred on Murashige and Skoog (MS) medium enriched with 1–10 μM N⁶-benzyladenine and 0.01 μM α-naphthaleneacetic acid. The propagules were rooted on MS medium containing 1 μM 3-indolebutyric acid and B₅ vitamins. Regenerated plants were fertile and phenotypically similar to control plants grown from seed. The MSNC method was four times more efficient than the ASP method in terms of the number of plants produced per explant.     

Hydrogen Peroxide and the Proliferation of Bhk-21 Cells

Intracellular levels of H2O2 in BHK-21 cells are not static but decline progressively with cell growth. Exposure of cells to inhibitors of catalase, or glutathione peroxidase, not only diminishes this decline but also depresses rates of cell proliferation, suggesting important growth regulatory roles for those antioxidant enzymes. Other agents which also diminish the growth-associated decline in intracellular levels of H₂O₂, such as the superoxide dismutase mimic, copper II—(3,5-diisopropylsalicylate)₂, or docosahexaenoic acid, also reduced cell proliferation. In contrast, proliferation can be stimulated by the addition of 1 μM exogenous H₂O₂ to the culture medium. Under these conditions, however, intracellular levels of H₂O₂ are unaffected, whereas there is a reduction in intracellular levels of glutathione. It is argued that critical balances between intracellular levels of both H₂O₂ and glutathione are of significance in relation both to growth stimulation and inhibition. In addition growth stimulatory concentrations of H₂O₂, whilst initially leading to increased intracellular levels of lipid peroxidation breakdown products, appear to “trigger” their metabolism, possibly through aldehyde dehydrogenase, whose activity is also stimulated by H₂O₂     

‘Lymphocyte-like’cells in ascidians: Precursors for vertebrate lymphocytes?

There has been much speculation that the evolutionary precursors of vertebrate lymphocytes may exist in the ascidians (proto chordates), but conclusive evidence has remained elusive especially as membrane bound immunoglobulin has not been detected in any invertebrate. This paper reviews new evidence which indicates that the ‘lymphocyte-like’ cells in ascidians have functional, as well as morphological, similarities with vertebrate lymphocytes. Firstly, these cells have been linked with in vivo non-self recognition events such as allograft rejection in solitary ascidians and non-fusion reactions between colonial ascidians. Secondly, they have been shown to be cytotoxic towards xenogenic targets in vitro and to use cytolytic mechanisms similar to those of cytotoxic T-cells. Thirdly, they are able to proliferate in vitro in response to mitogens or allogeneic cells. It is therefore suggested that, apart from immunoglobulin production and clonal selection, there is persuasive evidence that the ‘lymphocyte-like’ cells of ascidians constitute a primordial form of vertebrate lymphocyte.     

Concentration-dependent modulations of potassium and calcium currents of rat osteoblastic cells by arachidonic acid

We show that the voltage-gated K⁺ and Ca²⁺ currents of rat osteoblastic cells are strongly modulated by arachidonic acid (AA), and that these modulations are very sensitive to the AA concentration. At 2 or 3 μm, AA reduces the amplitude and accelerates the inactivation of the K⁺ current activated by depolarization; at higher concentrations (≥5 μm), AA still blocks this K⁺ current, but also induces a very large noninactivating K⁺ current. At 2 or 3 μm, AA enhances the T-type Ca²⁺ current, close to its threshold of activation, whereas at 10 μm, it blocks that current. AA (1–10 μm) also blocks the dihydropyridine-sensitive L-type Ca²⁺ current. Thus, the effect of AA on Ca²⁺ entry through voltage-gated Ca²⁺ channels can change qualitatively with the AA concentration: at 2 or 3 μm, AA will favor Ca²⁺ entry through T channels, both by lowering the voltage-gated K⁺ conductance and by increasing the T current, whereas at 10 μm, AA will prevent Ca²⁺ entry through voltage-gated Ca²⁺ channels, both by inducing a K⁺ conductance and by blocking Ca²⁺ channels.     

Efficient generation of chimaeric mice using embryonic stem cells after long-term culture in the presence of ciliary neurotrophic factor

The aim of our study was to evaluate whether ciliary neurotrophic factor (CNTF) can substitute for leukaemia inhibitory factor (LIF) in maintaining pluripotential embryonic stem (ES) cells in culture. Two subclones of D3 ES cells were used to assess cell proliferation and differentiation in the presence of CNTF, LIF or Buffalo rat liver (BRL) cell-conditioned medium, or in the absence of exogenous differentiation inhibiting factors. ES cells maintained in medium supplemented with CNTF for up to four weeks were injected into blastocysts to investigate theirin vivo pluripotency in terms of chimaera formation. CNTF inhibited ES cell differentiation in a dose-dependent manner. The most effective concentration was 10 ng CNTF per ml of medium. The effects of CNTF on ES cell differentiation and proliferation were comparable to those of LIF at the same concentration. BRL cell-conditioned medium was less effective at preventing ES cell differentiation but induced their proliferation very markedly. Both ES cell clones efficiently formed chimaeras after long-term culture with CNTF as the only differentiation inhibiting agent. The ability of these ES cells to colonize the germ-line is the ultimate proof that CNTF can preserve the pluripotency of ES cells.     

Effect of essential fatty acids on circulating T cell subsets in patients with colorectal cancer

The effect of essential fatty acids (EFA), given orally as dietary supplements, on the responsiveness in vitro of peripheral blood lymphocytes (PBL), to the mitogen concanavalin A have been studied in 10 patients with localized and 14 patients with advanced colorectal cancer. The degree of lymphocyte activation was assessed by measuring the amount of tritiated [³H]thymidine incorporated into newly synthesised lymphocyte DNA. The results were expressed as stimulation indices. T cell responses to concanavalin A stimulation showed a significant reduction of stimulation indices following EFA supplementation, in both the localized (P=0.026) and advanced (P=0.016) tumour groups, when compared with pretreatment activity in vitro. Mixing experiments, using EFA-supplemented and non-EFA-supplemented lymphocytes with concanavalin A, suggest no enhancement of T suppressor cell activity. Cell surface marker analysis (fluorescence-activated cell sorting for CD phenotyping) revealed a reduction of absolute numbers of CD4⁺ and CD8⁺ lymphocytes following EFA supplementation. The stimulation indices returned to presupplementation values 3 months following cessation of EFA intake. There was no significant change of these indices in the control (no EFA supplementation) advanced tumour group tested. This study suggests that EFA supplementation in patients with colorectal cancer selectively reduces circulating PBL. and T cell subset (including suppressor cells) numbers and/or activity. Such effects may have an important outcome in patients with malignant disease.This work was supported by grants from the Grampian Health Broad, the Royal College of Surgeons of Edinburgh, and Scottish Hospital Endownment Research Trust     

Inhibition of the proliferation of cultured immortalized schwann cells by forskolin with a decreased basal level of diacylglycerol

The repetitive passages of a Schwann cell culture results in the appearance of immortalized cells. In order to investigate the direct effects of cyclic AMP (cAMP) on Schwann cell proliferation, we used the immortalized Schwann cells because the responses of a short-term Schwann cell culture to agents increasing the intracellular cAMP are more complicated and it does not seem that all of them are due to the direct effects of cAMP. By adding up to 200 M of forskolin, an adenylate cyclase activator, to the culture medium, Schwann cell proliferation was inhibited and the intracellular 1,2-diacylglycerol (DG) level was decreased in a dose-dependent manner to 44 and 53% of the control values, respectively. The protein phosphorylation activity in the cytosol from the cell treated with 100 M forskolin, assayed with myelin basic protein as the acceptor, decreased to 78% and this inhibition was then reversed by the addition of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeable DG, to the assay mixture. The cell proliferation inhibited by forskolin was also restored by the addition of OAG. These data suggest that cAMP inhibits both the activity of protein kinase C (PKC) and consequently cell proliferation through suppression of intracellular DG level, an activator of PKC. Since the inositol 1,4,5-triphosphate level and the hydrolysis of phosphatidylcholine to DG and phosphorylcholine were not affected, forskolin therefore appears to suppress the de novo synthesis of DG.