Activity of human erythrocyte gangliosides as a receptor to HVJ

Liposomes could bind and fuse efficiently to human erythrocytes in the presence of HVJ when they contained gangliosides isolated from human erythrocytes. Sialosylparagloboside, which has a terminal sequence of NeuAcα2?3Ga1β1?4GlcNac, has a much higher receptor activity to the virus than GD_1a, GD_1b, GT_1b, and GT_1a, all of which contain the terminal sequence of NeuAcα2?3Galβ1?3GalNAc or NeuAcα2?8NeuAcα2?3Galβ1?3GalNAc. The activity of sialosylparagloboside is comparable to that of glycophorin, a major sialoglycoprotein of human erythrocytes, when compared on the basis of the required amount (as sialic acid) of compounds. The high affinity of sialosylparagloboside to the viral HANA protein is also suggested by the finding that it showed high inhibitory activity against HVJ-mediated binding of glycophorin liposomes to erythrocytes. Sialosylparagloboside was also highly susceptible to the viral sialidase, the other biological function of HANA protein.     

The facilitating effect of gangliosides on the electrogenic (Na+/K+) pump and on the resistance of the membrane potential to hypoxia in neuromuscular preparation

The effects have been investigated of a mixture of gangliosides from beef brain cortex (GM₁, GD_1a, GD_1b and GT₁) either added to the bathing medium or injected intraperitoneally on muscle fibres and nerve terminals in mouse diaphragm. The electrogenic (Na⁺/K⁺) pump activity of muscle fibres enriched with sodium was increased by 38% after 2-h pretreatment with gangliosides (5×10^–8 mol ·l^–1). Muscles from animals treated with gangliosides did not show the substantial depolarization of the resting membrane potential (RMP) in K⁺-free solution (6 h) shown by control muscles. Further, treatment with gangliosides slowed the changes in muscle fibre RMP and frequency of the miniature end-plate potentials in oxygen deprived muscles.     

Evidence that the Thy-1 molecule is the target for T cell mitogenic antibody against brain-associated antigens

Rabbit anti-mouse brain (RaMBr) antiserum can induce Lyt-1⁺, Lyt-2^?, T cells to proliferate and stimulates the same T cell subset to induce B cell proliferation. The aim of this report is to demonstrate that the mitogenic determinant recognized on the T cell surface by RaMBr antiserum is located on the Thy-1 molecule expressing the products of the Thy-1^a and Thy-1^b alleles. Evidence is drawn from serological and genetic experiments. The brain T cell cross-reactive, mitogenic determinant is not expressed on Thy-1^? mutants of the BW5147 T cell lymphoma that fail to express the Thy-1 molecule but do express other T cell surface proteins such as T-200 and gp 69, 71. Monoclonal anti-Thy-1.1 and anti-Thy-1.2 antibodies block the binding to the appropriate T cells of the majority of the serum antibody from RaMBr antiserum. The absorption of mitogenic antibody was blocked in a similar fashion, thus demonstrating the close association of the determinant and the Thy-1 antigen defined by monoclonal alloantibodies. The mitogenic and Thy-1.1 determinants are probably located on the same molecule because of the data obtained with the BW5147 Thy-1^? mutants and the observation that Thy-1^a T cells, which express a lower level of surface Thy-1 than Thy-1^b T cells, also express lower levels of the determinant recognized by RaMBr antiserum. Furthermore, in (AKR × DBA/2)F₁ mice (Thy-1^a/b) which express less Thy-1.1 antigen than Thy-1.2 at the surface, the mitogenic determinant was found to be prefentially associated with Thy-1.2. The coordinated genetic control of the surface levels of the Thy-1 determinant and the mitogenic determinant suggests that both determinants are situated on the same molecule in the T cell membrane.     

The stimulating effect of ganglioside injections on the recovery of choline acetyltransferase and acetylcholinesterase activities in the hippocampus of the rat after septal lesions

The effect of intramuscular administration of a mixture of gangliosides (21% GM₁, 39.7% GD_1a,, 16% GD_1b, 19% GT₁ in a daily dose of 50 mg per kg upon the time course of changes in hippocampal acetylcholinesterase and choline acetyltransferase activities after extensive medioventral septal lesions in the rat was checked on days 3, 5, 18 and 50 after the operation. Following the early decrease in the enzyme activities to about 25% of control due to degeneration, a gradual recovery up to about 50% of control activity at the 50th day was found. When gangliosides were administered, the recovery in the activity of both enzymes was more pronounced. The ratio of the enzyme activities from the animals injected with gangliosides to that from uninjected animals was 1.45 and 1.48 on the 18th day and 1.62 and 1.50 on the 50th day after the operation, for choline acetyltransferase and acetylcholinesterase activity, respectively. Since no significant effect of ganglioside injection was seen at early postoperative times i.e. on days 3 and 5, the effects seen on days 18 and 50 seem to be specifically due to facilitation of the recovery processes and not to retardation of the degeneration processes.Assuming that the spontaneous recovery of cholinergic enzyme activity reflects reinnervation of the hippocampus through collateral sprouting, gangliosides would seem to facilitate the regrowth of new cholinergic nerve terminals.     

Exogenously applied gangliosides (GM1, GD1a and Gmix) fail to facilitate the induction of long-term potentiation (LTP) in the slices of hippocampus and superior colliculus of the guinea pig

To confirm the effect of gangliosides on the facilitation of the induction of long-term potentiation (LTP), slices of hippocampus and superior colliculus from guinea pig were prepared. One group of slices was incubated in the standard medium containing gangliosides (GM₁, GD_1a, or a mixture of gangliosides from bovine brain, each at a concentration of 70 μM) for 2 h. The other group of slices was incubated in the standard medium only. In both groups of slices, tetanic stimulation induced LTP in a similar manner. In low Ca²⁺ (1 mM) medium, LTP was not induced in either group of slices. Thus, the present experiments could not confirm previous reports indicating that exogenously applied gangliosides facilitate the induction of LTP.     

Biochemical purification of detergent-solubilized H-2 alloantigens

A multistep Chromatographic fractionation scheme is described for purifying the H-2K^b and H-2D^b major histocompatibility antigens as isolated from the murine lymphoblastoid cell line EL4 (H-2^b haplotype). The membrane-integrated antigen molecules were solubilized with the non-ionic detergent NP-40 and were purified by gel filtration chromatography, ion exchange chromatography and affinity chromatography with lentil lectin conjugated to Sepharose. During the latter procedure, use of a linear gradient monosaccharide elution effected partial separation of the H-2K^b and H-2D^b antigens. At this stage the H-2 glycoproteins were highly purified based on several criteria. Upon polyacrylamide gel electrophoresis in SDS the major band migrates with a mol. wt of approximately 45,000 daltons corresponding to the mol. wt of antigens obtained by immunoprecipitation. Moreover, near identity of the profiles of the arginine-containing tryptic peptides from chromatographically-purified and immunoprecipitated H-2K^b preparations suggests that a high degree of homogeneity has been achieved in the Chromatographic purification. As is demonstrated, mg quantities of the H-2K^b and the H-2D^b antigens can be isolated and partially separated from each other by this purification scheme thereby opening the way for structural studies of the H-2K and H-2D molecules by a variety of biochemical methods.     

Ganglioside syndrome,a new autoimmune neurologic disorder,experimentally induced with brain gangliosides

Rabbits intensively immunized with total bovine brain gangliosides or a certain molecular species of gangliosides, such as GD_1a and GM₁, respectively, in complete Freud's adjuvant developed a variety of neurologic symptoms and signs which closely resembled experimental allergic encephalomyelitis, i.e., a sluggish righting reflex, muscular weakness, hindleg paralysis (flaccid in the case of GD_1a and rigid in GM₁) and body weight loss, frequently followed by death. Histological examinations of GD_1a-immunized rabbits showed extensive degeneration of the peripheral nerves, including breakdown of myelin and fragmentation of axis cylinders. In central nervous tissues, however, only mild damage was observed in some cases. Similar neurologic symptoms and signs were also induced in guinea pigs. The results suggest that this syndrome might be induced by a cell-mediated autoimmune response to gangliosides.     

Specific antigens of chicken thymus

Purified plasma membranes from chicken thymus and bursa cells were prepared and solubilized with sarkosyl (sodium salt of N-methyl-N-(1-oxodecyl)-glycine). Antisera to solubilized thymus plasma membrane (TPM) were produced in rabbits and the globulin fraction obtained by ammonium sulfate precipitation. Four precipitating antigens were detected in solubilized TPM by immunoelectrophoresis. Following absorption with chicken serum and bursa plasma membrane (BPM) immunosorbents, three antigens, designated T₁, T₂, T₃, were specific for the TPM fraction, and one antigen, T₁, was found in soluble extracts of thymus tissue. Absorption with isolated plasma membrane and whole cells indicated that the T₁ and T₂ antigens in solubilized TPM are associated with the plasma membrane but not expressed on the surface of the cell. A common antigen, designated BT, was detected in BPM and TPM fractions and in membrane preparations of spleen. The antigens were not detected in any other tissues or cells including brain, circulating lymphocytes and erythrocytes.     

Functional activities of antibodies against brain-associated T cell antigens II. Stimulation of T cell-induced B cell proliferation

B cells could be stimulated to proliferate by mitomycin C-treated normal T cells that were activated by rabbit anti-mouse brain antiserum (RaMBr). The B cell proliferation peaked 48 h after the initiation of the cultures. The T cell subset involved was Lyt-1⁺, 2^?. The B and T cells participating in the interaction did not need to be matched at the H-2 locus. A long-term T cell line (C.1.a), which was genetically restricted by the H-2 complex in its antigen (ovalbumin)-specific interaction with B cells, could also be induced by RaMBr to stimulate B cell proliferation. However, the RaMBr-mediated interaction of C.1.a with B cells was genetically unrestricted. This suggests that T cell activation by RaMBr can bypass the requirement for the recognition of I region antigens, which is a feature of antigen-specific interactions between T and B cells. In fact, RaMBr antibody appeared to function by bringing T cells into contact with B cells by the interaction of the B cell surface membrane Fc receptors with the Fc portion of RaMBr antibody bound to the T cell surface. Brain-associated antigen(s) appeared to play a unique role in T-B cell interactions because antibodies against other T cell surface antigens were inactive, e.g. those which remained in rabbit anti-mouse thymocyte antiserum at a high titer after absorption with mouse brain tissue. Furthermore, the brain-associated T cell antigen involved in the cellular interaction was differentiated from the Thy-1 antigen by the failure of xeno-anti-Thy-1.1 antisera to induce the interaction of B6.PL.Thy-1² T cells with B cells, and the presence of activity in rat anti-AKR mouse brain serum which did not contain antibodies against Thy-1.1 or Thy-1.2 determinants. The data appear to support the hypothesis that the brain-associated T cell antigen, as defined serologically in these studies, may represent a molecule which plays a role in the immunological functions of the Lyt-1⁺, Lyt-2^? T cell subset.     

Radioautographic Analysis of Surface Markers on Decidual Cells Shared by Cells of the Lymphomyeloid Tissues

ABSTRACT: Stromal type decidual cells recovered from the murine decidua by a mild collagenase dispersion procedure contain immunoregulatory cells whose ultimate precursors may originate from the bone marrow. To explore the familial relationship of these cells with other cells of the immune system, a battery of cell surface markers recognized on lymphomyeloid cells were examined and quantitated at the morphological level on typical stromal type decidual cells of the dispersed CBA mouse decidua at 8–14 days of syngeneic pregnancy, using a sensitive radioautographic technique. Cells were either labeled directly by exposure to ¹²⁵I-labeled monoclonal antibodies against Thy-1, Mac-1, or Lyt antigens or indirectly by a sequential exposure to monoclonal anti-I-A^k (Ia. 17) or monospecific anti-I-J^k antibodies and ¹²⁵I-labeled Protein A. Decidual cells were found to be Thy-1± (13–73% positive, the incidence rising with advancing gestation in the decidua basalis), Mac-1 ± (present on 6–11% on day 8 and 17–32% on day 12), I-A^?, 1-J^?, and Lyt^?. Macrophages within the decidua were Thy-1^?, Lyt^?, Mac-1 ⁺, and I-A± (present on 5–61% of cells, the incidence rising with advancing gestation). These surface properties of decidual cells along with the presence of FcR, an absence of C₃R, and the presence of a unique marker Dec-1 reported by us earlier, distinguish them from most other stromal type immunoregulatory cells such as Langerhans cells (I-A⁺, FcR⁺, C₃R⁺, Mac-1^?, Thy-1^?), follicular dendritic cells (I-A⁺, FcR⁺, Thy-1^?, Mac-1^?, Dec-1^?), and dendritic reticular cells (I-A⁺, FcR^?, C₃R^?, Thy-1^?, Lyt^?, Mac-1). However, a substantial subpopulation of decidual cells share a few properties (1-A^?,Thy-1⁺) with another marrow derived stromal type cell in the epidermis termed as epidermal dendritic cells that remain to be investigated for other surface markers such as Mac-1, Dec-1, and FcR.     

Interferon action: role of membrane gangliosides.

The antiviral activity of human (fibroblast + leukocyte) and mouse fibroblast interferon was neutralized by preincubation with ganglioside before application to cells. Ganglioside mixtures were as effective as pure gangliosides, with no particular ganglioside specificity observed for neutralization of human interferon. Ganglioside-agarose derivatives removed human interferon from solution; interferon was eluted with buffers containing urea, sodium dodecyl sulfate, and mercaptoethanol. Ganglioside-deficient transformed mouse cell lines were relatively insensitive to interferon action. Treatment of such cells with ganglioside led to an increase in membrane ganglioside content and in two of three cell lines to an increase in cell sensitivity to mouse interferon; in mouse SVS AL/N and TAL/N cells, GM₂, GT₁, and a mixture of crude gangliosides were effective whereas GM₁ and GD_1a were without effect. The interferon sensitivity of untransformed cells, which failed to take up appreciable ganglioside, was not increased by treatment with exogenous ganglioside; the sensitivity of one GM₁-deficient transformed cell line ($\text{K-Balb}\text{C-3T3}$) was not increased by pretreatment with ganglioside. These results indicate an interaction between gangliosides and several types of interferon and suggest a role for membrane gangliosides in the antiviral action of interferon.     

Intact and subcellular form of thymocytes of rats are exceptionally immunogenic in mice for inducing anti-Thy-1 T cell-independent class 2 antibody responses

Results of the present study show that the primary anti-Thy-1.1 antibody response to rat antigen in Thy-1.2 mice is induced exclusively by thymocyte antigen. Thy-1 antigens of brain and bone marrow, which expressed much Thy-1 antigen, were poorly immunogenic if at all. Brain Thy-1 antigen considerably inhibited the immunogenicity of thymocyte Thy-1. Moreover, we found that subcellular form of thymocytes induce as high antibody responses as intact thymocytes do. The subcellular thymocyte Thy-1 antigen behaved as TI-2 antigen, inducing a good response in athymic nude mice but not in CBA/N mice with a B cell defect. The significance of these findings is discussed in relation to the possible physiological activity of Thy-1 or Thy-1-linked molecules on thymocytes specifically mediating lymphocyte differentiation.     

COMPARATIVE IMMUNOGENICITY OF THY-1.1 AND THY-1.2 ANTIGENS

The immunogenicity of Thy-1 antigens was examined using the Thy-1 congenic mouse strains A and A/Thy-1.1. A disequilibrium of response was found between these strains. A strain mice rejected skin grafts and produced high titres of antibody to lymphoid cells from A/Thy-1.1 strain mice. However, in the reverse combination grafts were not rejected nor was there any significant antibody response. A/Thy-1.1 mice did not respond to Thy-1.2 antigen when it was presented on an H-2 allogeneic background (A·SW strain) but did respond to it when non-H-2 incompatibilities were involved (B10·A strain donor). We postulate the existence of Ir gene(s) for the response to Thy-1.2 antigens which are linked to H-2. The failure of A/Thy-1.1 strain mice to respond to Thy-1.2 antigens in the absence of carrier determinants is the result of a deletion (in the germ line) of the appropriate Ir gene itself resulting from the long association of the H-2^a haplotype of the A mouse (donor of the genetic background of A/Thy-1.1 strain) with the Thy-1.2 gene.     

Type-1 chain histo-blood group antigens (Lea,monosialosyl-Lea,disialosyl-Lea,Leb, and H) in normal and malignant human endometrium

Type-1 chain histo-blood group antigens such as the Lewis (Le)^a, monosialosyl-Le^a, Le^b and H antigens show an increased expression in endometrial carcinomas. However, the possibility that these antigens are expressed under genetic or hormonal influence in endometrial carcinomas has not been considered. In the present study, the expression of type-1 chain carbohydrate antigens in normal and malignant endometrium was evaluated by immunohistochemistry and related to both genetic and hormonal factors. The glands of normal, non-secretory endometria expressed, in contrast with surface epithelial cells, Le^a, Le^b, disialosyl-Le^a, and H determinants infrequently. Adenomatous hyperplasias and endometrial carcinomas showed an increased expression of type-1 chain carbohydrates that was qualitatively influenced by the erythrocyte Lewis phenotype and the secretor status. Whereas Le^a+b– non-secretors mainly accumulated Le^a antigen, and only limited amounts of Le^b antigen, Le^a–b+ secretors expressed H, Le^b and Le^a antigens. The expression of type-1 chain antigens showed no association with the serum-oestrogen level or to the hormone-receptor status. Thus the Lewis secretor status has a qualitative influence on the increased expression of type-1 chain antigens, which, however, seem to be unrelated to hormonal factors. Our findings suggest an increased activity of the Se-gene-defined or a closely related fucosyl-transferase in neoplastic endometrial epithelial cells.     

Nicotinamide riboside restores cognition through an upregulation of proliferator-activated receptor-γ coactivator 1α regulated β-secretase 1 degradation and mitochondrial gene expression in Alzheimer's mouse models

Nicotinamide adenine dinucleotide (NAD)⁺, a coenzyme involved in redox activities in the mitochondrial electron transport chain, has been identified as a key regulator of the lifespan-extending effects, and the activation of NAD⁺ expression has been linked with a decrease in beta-amyloid (Aβ) toxicity in Alzheimer's disease (AD). Nicotinamide riboside (NR) is a NAD⁺ precursor, it promotes peroxisome proliferator-activated receptor-γ coactivator 1 (PGC)-1α expression in the brain. Evidence has shown that PGC-1α is a crucial regulator of Aβ generation because it affects β-secretase (BACE1) degradation. In this study we tested the hypothesis that NR treatment in an AD mouse model could attenuate Aβ toxicity through the activation of PGC-1α-mediated BACE1 degradation. Using the Tg2576 AD mouse model, using in vivo behavioral analyses, biochemistry assays, small hairpin RNA (shRNA) gene silencing and electrophysiological recording, we found (1) dietary treatment of Tg2576 mice with 250 mg/kg/day of NR for 3 months significantly attenuates cognitive deterioration in Tg2576 mice and coincides with an increase in the steady-state levels of NAD⁺ in the cerebral cortex; (2) application of NR to hippocampal slices (10 μM) for 4 hours abolishes the deficits in long-term potentiation recorded in the CA1 region of Tg2576 mice; (3) NR treatment promotes PGC-1α expression in the brain coinciding with enhanced degradation of BACE1 and the reduction of Aβ production in Tg2576 mice. Further in vitro studies confirmed that BACE1 protein content is decreased by NR treatment in primary neuronal cultures derived from Tg2576 embryos, in which BACE1 degradation was prevented by PGC-1α-shRNA gene silencing; and (4) NR treatment and PGC-1α overexpression enhance BACE1 ubiquitination and proteasomal degradation. Our studies suggest that dietary treatment with NR might benefit AD cognitive function and synaptic plasticity, in part by promoting PGC-1α-mediated BACE1 ubiquitination and degradation, thus preventing Aβ production in the brain.     

Spontaneous production of interleukin 3 by T lymphocytes from autoimmune MRL/MP-lpr/lpr mice

MRL/MP-lpr/lpr (MRL/lpr) mice develop a lupus-like autoimmune disease and a massive generalized lymphadenopathy associated with proliferation of nonmalignant Thy-1⁺ Lyt-1⁺ cells. The mechanism(s) leading to outgrowth of these cells is unknown. We report here that Thy-1⁺, Lyt-1⁺, Lyt-2^? lymphocytes from spleens of MRL/lpr mice, but not from several strains of normal mice, spontaneously secrete IL3. The presence of IL3 is shown by: (a) the ability of the supernatants from unstimulated spleen cells of MRL/lpr (MRL/lpr SUP) to support growth of IL3 but not IL2 addicted cells and (b) the growth-promoting activity in MRL/lpr SUP was absorbed with IL3-dependent cells but not with IL2-dependent cells. Spontaneous release of IL3 was detected in supernatants from spleen cells of 6-week-old MRL/lpr mice and the titers of IL3 activity increased with age. Nylon wool-enriched cells from spleens of MRL/lpr mice proliferated in response to purified IL3 and IL3 secreted by MRL/lpr T cells, in a manner similar to nylon wool-passed cells from normal mice. The cells responding to both sources of IL3 were Thy-1⁺, Lyt-1⁺, Lyt-2^?. Thus, Thy-1⁺, Lyt-1⁺,2^? cells from spleen of MRL/lpr mice spontaneously secrete IL3 and respond normally to this lymphokine. Four Thy-1⁺, Lyt-1⁺,2^? cell lines derived from unstimulated spleen cells of MRL/lpr mice were established in culture with IL3. These IL3-sensitive T cell lines help syngeneic and H-2-compatible normal small “resting” B cells to mature into plasma cells secreting predominantly IgG₁, IgG₂ and IgA. Taken together, these data and previous findings that T cells from MRL/lpr mice have an impaired production of and response to IL2, strongly suggest that abnormal production of IL3 may account for the outgrowth of Thy-1⁺, Lyt-1⁺,2^? cells in the MRL/lpr mouse. Finally, a mechanism linking abnormal production of IL3 and B cell hyperactivity in these animals is proposed.     

Serological and preliminary biochemical characteristics of a T lymphocyte differentiation antigen detected by rabbit antiserum to rat thymocyte membranes.

A rabbit antiserum against rat thymocyte membrane was studied using quantitative absorption analysis, and shown to contain a large amount of antibody against antigen(s) on lymphocytes but not liver, erythrocytes, kidney, heart, lung or brain tissue. Among lymphocytes the antigen(s) was at least 20-fold more abundant on thymocytes or T lymphocytes than on bone marrow or B lymphocytes, and is thus referred to as a T lymphocyte antigen. In saturating binding studies with absorbed antiserum 120 000 molecules of antibody were bound per thymocyte. The T lymphocyte antigenic activity was effectively solubilized from thymocyte membrane by deoxycholate, and its hydrodynamic properties suggested that only one molecule (or a group of very similar molecules) express the activity. The S₂₀,_w and v? values of the antigen were 5.7 S and 0.73 ml/g, and the Stoke's radius was 6.2 nm; a molecular weight (including any bound deoxycholate) of 1 50 000 was calculated. All antigenic activity in deoxycholate extracts bound to a lentil lectin affinity column suggesting the antigen may be a glycoprotein. On serological and biochemical grounds the antigen was distinct from Thy-1. In an appendix a method for determining the maximum binding of antibody per cell by absorption analysis is described.     

Suppression of inflammation response by a novel A₃ adenosine receptor agonist thio-Cl-IB-MECA through inhibition of Akt and NF-κB signaling

Adenosine, a purine nucleoside, is released from metabolically active cells into extracellular space and plays an important role in various pathophysiological processes. Adenosine regulates many biological responses including inflammation by the interaction with their receptors such as A₁, A_2A, A_2B, and A₃. Especially, A₃ adenosine receptor (A₃AR) is considered to be expressed in macrophage cells. To the end, A₃AR agonists have been reported to have an anti-inflammatory activity. In our continuous efforts to develop new anti-inflammatory agents, we found a novel adenosine analog, 2-chloro-N⁶-(3-iodobenzyl)-4′-thioadenosine-5′-N-methyluronamide (thio-Cl-IB-MECA), was a potent human A₃AR agonist. The study was designed to investigate whether thio-Cl-IB-MECA has an anti-inflammatory potential in mouse macrophage RAW 264.7 cells and mouse sepsis model in vivo. Thio-Cl-IB-MECA exhibited an effective anti-inflammatory activity. The expression of pro-inflammatory biomarkers including inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), and tumor necrosis factor (TNF-α) was suppressed by the treatment of thio-Cl-IB-MECA in the protein and mRNA levels in lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7 cells. Further examination revealed that thio-Cl-IB-MECA inhibited LPS-induced phosphatidylinositol 3-kinase (PI3 kinase)/Akt activation, NF-kB binding activity, and β-catenin expression. In addition, in in vivo LPS-induced mouse endotoxemia model, thio-Cl-IB-MECA exerted the increase of survival rate compared to vehicle-treated mouse. The analysis of the protein levels of iNOS, IL-1β, and TNF-α was also suppressed by the compound-treated groups in lung tissues. These results suggest that thio-Cl-IB-MECA might have an anti-inflammatory activity through the inhibition of pro-inflammatory cytokine expression by modulating PI3K/Akt and NF-κB signaling pathways.     

Transgenic 4-1BBL-engineered vaccine stimulates potent Gag-specific therapeutic and long-term immunity via increased priming of CD44+CD62Lhigh IL-7R+ CTLs with up- and downregulation of anti- and pro-apoptosis genes

Human immunodeficiency virus type-1 (HIV-1)-specific dendritic cell (DC) vaccines have been used in clinical trials. However, they have been found to only induce some degree of immune responses in these studies. We previously demonstrated that the HIV-1 Gag-specific Gag-Texo vaccine stimulated Gag-specific effector CD8⁺ cytotoxic T lymphocyte (CTL) responses, leading to completely protective, but very limited, therapeutic immunity. In this study, we constructed a recombinant adenoviral vector, adenovirus (AdV)_4-1BBL, which expressed mouse 4-1BB ligand (4-1BBL), and generated transgenic 4-1BBL-engineered OVA-Texo_/4-1BBL and Gag-Texo_/4-1BBL vaccines by transfecting ovalbumin (OVA)-Texo and Gag-Texo cells with AdV_4-1BBL, respectively. We demonstrate that the OVA-specific OVA-Texo_/4-1BBL vaccine stimulates more efficient OVA-specific CTL responses (3.26%) compared to OVA-Texo-activated responses (1.98%) in wild-type C57BL/6 mice and the control OVA-Texo_/Null vaccine without transgenic 4-1BBL expression, leading to enhanced therapeutic immunity against 6-day established OVA-expressing B16 melanoma BL6-10_OVA cells. OVA-Texo_/4-1BBL-stimulated CTLs, which have a CD44⁺CD62L^high IL-7R⁺ phenotype, are likely memory CTL precursors, demonstrating prolonged survival and enhanced differentiation into memory CTLs with functional recall responses and long-term immunity against BL6-10_OVA melanoma. In addition, we demonstrate that OVA-Texo_/4-1BBL-stimulated CTLs up- and downregulate the expression of anti-apoptosis (Bcl2l10, Naip1, Nol3, Pak7 and Tnfrsf11b) and pro-apoptosis (Casp12, Trp63 and Trp73) genes, respectively, by RT² Profiler PCR array analysis. Importantly, the Gag-specific Gag-Texo_/4-1BBL vaccine also stimulates more efficient Gag-specific therapeutic and long-term immunity against HLA-A2/Gag-expressing B16 melanoma BL6-10_Gag/A2 cells than the control Gag-Texo_/Null vaccine in transgenic HLA-A2 mice. Taken together, our novel Gag-Texo_/4-1BBL vaccine, which is capable of stimulating potent Gag-specific therapeutic and long-term immunity, may represent a new immunotherapeutic vaccine for controlling HIV-1 infection.     

Developmental relationship between cytotoxic α/β T cell receptor-positive intraepithelial lymphocytes and Peyer's patch lymphocytes

Following intraduodenal priming of mice with reovirus, precursor cytotoxic T lymphocytes (pCTL) rapidly appear in intraepithelial lymphocytes (IEL) and Peyer's patches. These cells express CTL activity after secondary in vitro stimulation with reovirus-infected cells. Adoptive transfer of Peyer's patch lymphocytes from normal BALB/c mice into reovirus-infected CB.17 severe combined immunodeficiency mice results in the infection-dependent appearance of large numbers of both CD8⁺Thy-1⁺ and CD8^?Thy-1⁺, IEL that express the α/β T cell receptor (TcR). Phenotypic and functional characterization of IEL derived from conventionally reared, reovirus-infected mice also points to extensive similarities in the pCTL derived from Peyer's patches and IEL. As in the Peyer's patches, pCTL are persistent in the IEL compartment for up to 4 weeks after infection. A large percentage of IEL that are recovered from reovirus-primed mice after in vitro culture are CD8⁺Thy-1⁺ cells that express α/β TcR. Furthermore, depletion experiments demonstrate that the CD8⁺Thy-1⁺ population mediates the virus-specific CTL activity. Using limiting dilution analyses, it was estimated that 7 days after intraduodenal infection the average frequency of virus-specific pCTL was 197/10⁶ CD8⁺Thy-1+ IEL and 190/10⁶ CD8⁺Thy-1⁺ Peyer's patch lymphocytes. Taken together, these observations provide evidence that specific cellular immunity to reovirus in IEL is mediated, at least in part, by conventional cytotoxic T lymphocytes and that these cells are functionally and phenotypically similar to the pCTL derived from the Peyer's patches.