Protective effects of ginsenoside Rg1 on aging Sca-1⁺ hematopoietic cells.

In adults, bone hematopoietic cells are responsible for the lifelong production of all blood cells. It is affected in aging, with progressive loss of physiological integrity leading to impaired function by cellular intrinsic and extrinsic factors. However, intervention measures, which directly inhibit the aging of hematopoietic cells, remain to be investigated. In the present study, 10 µmol/l ginsenoside Rg1 (Rg1) markedly alleviated the aging phenotypes of Sca­1+ hematopoietic cells following in vitro exposure. In addition, the protective effects of ginsenoside Rg1 on the aging of Sca­1+ hematopoietic cells was confirmed using a serial transplantation assay in C57BL/6 mice. The mechanistic investigations revealed that Rg1­mediated Sca­1+ hematopoietic cell aging alleviation was linked to a series of characteristic events, including telomere end attrition compensation, telomerase activity reconstitution and the activation of genes involved in p16­Rb signaling pathways. Based on the above results, it was concluded that ginsenoside Rg1 is a potent agent, which acts on hematopoietic cells to protect them from aging, which has implications for therapeutic approaches in hemopoietic diseases.

[Effect of SIRT6/NF-κB signal axis in delaying hematopoietic stem/progenitor cell senescence with ginsenoside Rg1].

OBJECTIVE: To investigate the effect of SIRT6/NF-κB signal axis in delaying hematopoietic stem/progenitor cell senescence with ginsenoside Rg1, in order to provide theatrical and experimental basis for looking for methods for delaying HSC senescence. METHOD: Sca-1 + HSC/HPC was isolated by magnetic cell sorting (MACS) and divided into five groups: the normal control group, the aging group, the positive control group, the Rg1 anti-senescence group, and the Rg1-treated group. Senescence-associated ß-galactosidase (SA-ß-Gal) staining, cell cycle analysis and hemopoietic progenitor cell mix (CFU-Mix) were adopted to determine the effect Rg1 in delaying or treating Sca-1 + HSC/HPC senescence biology. The mRNA and protein of senescence regulation molecules SIRT6 and NF-KB were examined by realtime fluorescence quantitative PCR (FQ-PCR) and western blotting. RESULT: Compared with the senescence group, the Rg1 anti-senescence group and the Rg1-treated group showed lower percentage in SA-ß-Gal-stained positive cells, decreased cell proportion in G1 phase, increased number of CFU-Mix, up-regulated in SIRT6 mRNA and protein expression, down-regulation in NF-KB mRNA and protein expression. The Rg1 anti-senescence group showed more evident changes in indexes than the Rg1-treated group. CONCLUSION: Rg, may inhibit Sca-1 + HSC/HPC senescence induced by t-BHP by regulating SIRT6/NF-KB signal path.

Cardiac stem cells with electrical stimulation improve ischaemic heart function through regulation of connective tissue growth factor and miR-378.

AIMS: In this study, we investigated whether pre-conditioning (PC) by electrical stimulation (EleS) induces cytoprotective effect on cardiac stem cells (CSCs) and determined its underlying molecular mechanisms. METHODS AND RESULTS: Sca-1(+) CSCs were isolated from male C57BL6 mice (12 weeks) hearts. PC of CSCs with EleS ((EleS)CSCs) was carried out for 3 h at 1.5 V followed by exposure to 300 µM H2O2 for 5 h. Cytoprotective effects and cell adhesion ability were significantly increased by EleS as evaluated by transferase-mediated dUTP nick-end labelling (TUNEL), lactate dehydrogenase (LDH) release assay, and adhesion assay. EleS increased phosphorylation of AKT, focal adhesion kinase (FAK), and glycogen synthase kinase (GSK3ß), as well as decreased caspase-3 cleavage. Interestingly, inhibition of AKT or FAK abolished the pro-survival effects of EleS. We found that connective tissue growth factor (Ctgf) was responsible for EleS-induced CSC survival and adhesion.The survival rate of (EleS)CSCs after transplantation in the infarcted myocardium was significantly increased together with improvement in cardiac function. Importantly, knockdown of Ctgf abolished EleS-induced cytoprotective effects and recovery of cardiac function. Furthermore, we identified miR-378 as a potential Ctgf regulator in (EleS)CSCs. CONCLUSION: EleS enhanced CSC survival in vitro and in vivo as well as functional recovery of the ischaemic heart through an AKT/FAK/CTGF signalling pathway. It is suggested that Ctgf and miR-378 are novel therapeutic targets for stem cell-based therapy.

Induction of a cell-transferable suppression of alloreactivity by photodamaged lymphocytes.

We have reported previously that splenocytes from BALB/c mice acutely rejecting CBA/j skin allografts were exposed to 8-methoxypsoralen (8-MOP) and ultraviolet A light and infused several times intravenously into naive BALB/c recipients; the recipients were hyporesponsive to CBA/j alloantigens in skin graft and delayed-type hypersensitivity assays, as well as in mixed leukocyte culture and cytotoxicity assays. We currently expand on this work by showing that donor-specific tolerance can be transferred adoptively to naive syngeneic animals via unfractionated splenocytes from mice rendered tolerant by the previous protocol. This suppressed response to alloantigen was transferred optimally with splenocytes taken from mice on the sixth day after the final treatment with PET cells. We further demonstrate that the cells that are adoptively transferring suppression are radiosensitive, Thy-1+, Lyt-2+, L3T4- T lymphocytes.

Histological and immunohistochemical characterization of joint inflammation and flare-up reactions induced by cloned MT4,Lyt-2-T cells.

This report describes the histological and immunohistochemical characterization of joint inflammations and flare-up reactions in mice induced by cloned MT4+,Lyt-2-T cells. The T-cell clone used was specific for the antigen methylated bovine serum albumin (mBSA) and was inoculated locally into a joint together with the antigen. The histological examination was performed in methylmethacrylate sections, and the various cell types were quantified in distinct regions of the knee joint. The infiltrates consisted predominantly of granulocytes admixed with small numbers of histiocytes. Few lymphocytes were present, while plasma cells were not found. Fibrosis was prominent in the later stages of the inflammation. Immunohistochemical analysis of total unfixed, non-decalcified sections using monoclonal antibodies revealed the presence of T cells which were predominantly of the helper phenotype, sporadic B cells, and a considerable number of Ia-positive cells. Macrophages were scattered throughout the infiltrate. The synovial lining was shown to express Ia antigens and to contain cells that stained with macrophage markers. Cell clusters were found including helper T (Th) cells, some B cells, and Ia-positive cells. These results are in line with immunohistological examinations in other arthritis models and resemble the early events in human rheumatoid arthritis. The data indicate that activated helper T cells are required and sufficient to give rise to the inflammatory infiltrates that are characteristic of the inflammations and exacerbations in human rheumatoid arthritis.

The peritoneal Ly-1 (CD5) B cell repertoire is unique among murine B cell repertoires.

Ly-1 (CD5) B cells and conventional B cells represent two distinct lineages of murine B cells which are distinguishable by expression of surface molecules, organ location, ontogeny and development and antibody production in vivo. In order to assess whether the different developmental pathways of Ly-1 B cells and conventional B cells result in different antibody repertoires, we have used limiting dilution analyses to determine frequencies of B cells making antibodies capable of binding to a range of antigens including haptens, proteins, bacterial polysaccharides and bromelain-treated mouse red blood cells. Starting populations of B cells were purified from spleen, peritoneum and bone marrow of adult BALB/c mice or from spleens of newborn mice by use of the fluorescence-activated cell sorter. The peritoneal Ly-1 B cell repertoire was found to be different from that of conventional B cells, with between 5- and 100-fold higher frequencies of clones producing IgM antibodies capable of binding to the antigens tested. However, when tested, the majority of Ly-1 B cell anti-haptenic antibodies did not show the high affinity binding or fine specificity characteristics of specific antibodies elicited in immune responses in vivo. The high frequencies of antigen-reactive antibodies within the Ly-1 B repertoire are most likely explained by the presence of clones secreting low-affinity or multireactive antibodies. The Ly-1 B cell repertoire is not mirrored in repertoires from either newborn B cells or virgin B cells in adult bone marrow. Therefore, either Ly-1 B cells develop from distinct precursors with intrinsically different mechanisms of V gene usage and recombination, or newly formed Ly-1 B are heavily selected on specificity for entry into this peritoneal lineage. If the second alternative is true, bacterial antigens in the gut are not required for selection of this unique repertoire, as Ly-1 B cells in germ-free mice also show the multireactive repertoire characteristic of this B cell lineage in normal mice.

Biased immunoglobulin variable region gene expression by Ly-1 B cells due to clonal selection.

Most, if not all, autoantibodies specific for bromelain-treated mouse erythrocytes recognize the common membrane phospholipid, phosphatidyl choline (PtC). Anti-PtC antibodies are produced by 5%-15% of CD5+ Ly-1 B cells of normal unimmunized mice, but not by detectable numbers of conventional CD5- B cells. At 1 week of age PtC-specific B cells are undetectable but then increase dramatically over the next 3 to 4 weeks to reach adult numbers. We report here that PtC-specific Ly-1 B cells in B10.H-2aH-4bp/Wts mice predominantly express either of two heavy and kappa chain variable (V) region gene combinations. In addition, the sequence and length of DH genes are conserved among cells expressing the same V gene combination, and the V kappa-J kappa junctions of one group involve unusual splice sites. Preferential V gene rearrangement models are insufficient to explain the DH and V kappa-J kappa junctional sequences or the delayed appearance of this specificity, and so they cannot solely account for the high frequency of PtC-specific cells. These characteristics are more consistent with antigen selection. We therefore attribute the frequent use of the two V region gene combinations to selection for cells that express them and conclude that the expressed V gene repertoire of Ly-1 B cells in adult mice is influenced by antigen selection. Apparently, there is no selection for mutant anti-PtC antibodies of higher affinity during the formation of the Ly-1 B repertoire because the V region genes expressed by PtC-specific cells are unmutated. Our findings are consistent with an important, germ line-encoded function for the immunoglobulin products of these gene combinations.

The relationship between induced and spontaneous autoantibodies in MRL mice: the role of Ly-1 B cells?

The murine response to bromelain-treated mouse red blood cells (BrMRBC) is derived from Ly-1 B cells. It has been proposed that this B-cell subset produces a variety of other autoantibodies and is elevated in autoimmune mouse strains. We have studied the ability of MRL lpr/lpr and the non-autoimmune congenic MRL +/+ mice to make autoantibodies to BrMRBC and immunoglobulin (rheumatoid factors, RF). Following lipopolysaccharide (LPS) stimulation we found the numbers of autologous plaque-forming cells (PFC) to be low in both lpr and MRL +/+ mice, suggesting low Ly-1 B-cell numbers. This observation is consistent with the view that Ly-1 B cells in the mouse may not give rise to pathologically relevant RF.

The interaction of the xid and me genes.

The murine "motheaten" (me) mutation has been bred onto the NFS background and combined with the X-linked immunodeficiency (xid) mutation to investigate the effect of the xid-induced B cell maturational block on the widespread immune dysfunction, high levels of autoantibodies, and early mortality found in the motheaten mice. The xid markedly reduced spontaneous IgM secretion by spleen cells, serum IgM, anti-ssDNA antibodies, anti-bromelain-treated-erythrocyte antibodies, and T cell binding (but not thymocytotoxic) antibodies; however, neither phenotype nor mortality was affected, suggesting that other factors are responsible for early death. Marked expansion of the Ly-1+ B cell pool was prevented by xid in the motheaten mouse leaving only a very small population of sIgM-positive B cells. This failure of non-Ly-1+ B cell development in me/me X xid mice suggests that me/me leads to inhibition of non-Ly-1+ B cells and preferential expansion of Ly-1+ B cells in motheaten mice, perhaps as a result of their high levels of maturation and activation factors.

The type of dietary fat affects the severity of autoimmune disease in NZB/NZW mice.

The type of dietary fat dramatically affects the onset of autoimmune disease in lupus-prone female New Zealand Black/New Zealand White F1 (B/W) mice. Disease development was strikingly slowed in mice fed a diet containing quantities of omega-3 fatty acids (fish oil, FO). By 10 months of age, 94% of the FO mice were still living, whereas all the mice fed a saturated fat diet (lard,L) were dead. Those mice fed a corn oil (CO) diet were intermediate with 35% alive at the 10-month time evaluation. Long after the L and CO groups had succumbed to glomerulonephritis, the FO group had negligible proteinuria. Both B and T cell function, particularly antibody production and resultant circulating immune complex (CIC) levels, were modified by the type of dietary fat. FO mice exhibited lower levels of anti-ds-DNA and lower levels of CICs than L or CO mice. B/W antibody response to a T-independent antigen (DNP-dextran) was enhanced at 8 months of age in FO mice, whereas it was suppressed in L mice. T-dependent (sheep red blood cell) responses at that time period were reduced in all the diet groups, a reflection of the reduced numbers of accessory T cells as determined by FACS analysis. The natural killer (NK) response to YAC-1 cells decreased in the L group from 5 to 9 months of age but remained unchanged in the CO and FO groups. Severe glomerulonephritis was the most common histopathologic finding in the L and CO groups. Arteritis was found in the spleens of nearly all the L and CO mice. Arteritis of the heart, colon and intestine, stomach, kidney, and liver were also seen principally in the L mice. In contrast, most FO mice had minimal to mild glomerulonephritis and no or minimal arteritis in the spleen. It is likely omega-3 fatty acids of fish oil reduce immune-complex-induced glomerulonephritis through production of prostaglandin metabolites with attenuated activity and/or through altering cell membrane structure and fluidity, which may, in turn, affect the responsiveness of immune cells.

Altered natural killer and natural cytotoxic cellular activities in lpr mice.

Three strains of mice bearing the autosomal recessive lpr gene (MRL, C57BL/6, and C3H) that had spontaneously developed a lupus-like disease were studied sequentially for functional natural killer (NK) and natural cytotoxic (NC) cell activity. Natural killing was impaired in spleen and bone marrow cells from all the lpr strains, as well as from the congenic strain MRL--+/+, which develops a late onset lupus-like disease. The NK cell activity was found to be depleted as early as 2 months of age in all lpr strains, and decreased further with age. NK activity was augmentable by Poly I:C and interleukin 2 (IL-2), suggesting that the residual cells can respond to NK modulators. In contrast with NK cell activity, NC activity was not decreased in lpr mice but could be augmented by IL-3-rich supernatants. The spontaneous decrease in NK cell activity was associated with an increased autologous plaque-forming cell (APFC) response to bromelin-treated mouse red blood cells, which is produced primarily by B cells possessing the Ly-1 phenotype (Lyt-1+ B). When NK cell activity was increased by exogenous administration of Poly I:C, the APFC response diminished. Treatment of spleen cells with anti-asialo GM1 prior to Poly I:C treatment resulted in a decreased NK response but increased both APFC and Lyt-1+ B cells. The possible regulation of autoreactivity by NK cells is discussed.

Phosphatidyl choline is recognized by a series of Ly-1+ murine B cell lymphomas specific for erythrocyte membranes.

Cells from 6 of 14 different Ly-1+ murine B cell lymphomas bound to synthetic liposomes encapsulating fluorescein. The liposomes were made from distearoyl phosphatidyl choline (DSPC), distearoyl phosphatidyl glycerol (DSPG), and cholesterol. In all cases, liposome binding was due to recognition of phosphatidyl choline by the surface IgM on the tumor cells. Liposome binding could be inhibited by DSPC but not by DSPG, and the number of liposomes bound per cell was directly related to the cell surface concentration of IgM. The IgM secreted by a hybridoma derived from one of the lymphomas, CH12, was shown to agglutinate liposomes, and was used in a solid-phase immunoassay to study inhibition of liposome binding by pure phospholipids; DSPC and sphingomyelin both inhibited, whereas DSPG did not. The Ig borne by the six lymphomas that bind phosphatidylcholine also bind to both SRBC and bromelain-treated mouse erythrocytes. The idiotypic of CH12 IgM is similar to that expressed by Ly-1+ normal splenic B cells of the same specificity. The significance of these data in relation to other commonly studied autoantigens, and to the restricted specificity of normal Ly-1+ B cells is discussed.

Cross-reactive idiotypes and common antigen binding specificities expressed by a series of murine B-cell lymphomas: etiological implications.

A series of 27 B-cell lymphomas (designated the CH series), induced in B10.H-2aH-4b p/Wts mice by intense adoptive immunization with sheep erythrocytes, was found to represent a subset of the total B-cell repertoire. This subset was characterized by expression of a limited number of Ig heavy chain variable regions, as evidenced by the presence of cross-reactive idiotypes and common antigen binding specificities. Twenty-one of the 27 CH lymphomas studied were classified into five groups, defined by a particular cross-reactive idiotype; four of these groups were linked in a single network. Seven of 16 idiotypes defined by absorption analysis were present on lymphomas bearing either kappa or lambda light chains and so were localized to the heavy chain variable region. The surface Ig on 14 CH lymphomas was found to be specific for epitopes on certain erythrocytes (bromelain-treated autologous erythrocytes, sheep, and chicken erythrocytes) or E. coli. We propose that the CH lymphomas represent the malignant counterparts of a subset of idiotypically related, normal B cells in B10.H-2aH-4b p/Wts mice. Perturbation of this idiotype network, by hyperimmunization with an antigen for which some of the members are specific (sheep erythrocytes), increases the risk for neoplasia. Possible mechanisms for this are discussed.

Regulation of naturally occurring autoantibody secretion by a radiosensitive lymphocyte: initial characterization and ontogeny.

Low level irradiation (400-500 R) of normal mice or of murine spleen cells resulted in the detection of an enhanced number of plaque-forming cells against bromelain-treated autologous red cells (Br MRBC) 1 day later. The mechanism responsible for the increased numbers of plaques is apparently the elimination of a suppressor T cell since the addition of thymocytes or of Lyt 1+2+ splenic cells to cultures of irradiated cells reversed the radiation-induced increase. Studies on the ontogeny of the phenomenon indicate that anti-Br MRBC plaques can be formed by spleen cells taken from mice shortly after birth although adult levels are not reached until after 3 weeks of age. Radiation-induced increases in the number of plaques were not seen until 3 weeks of age, thus, suggesting a temporal developmental sequence of the ability to produce autoantibodies and to regulate such production.

T-cell-mediated enhancement of host-versus-graft reactivity in mice fed a diet enriched in vitamin A acetate.

Retinol (vitamin A) and some of its derivatives have an important role in: (1) regulating growth, proliferation and differentiation of various tissues and (2) maintaining reproduction and visual function in man and higher animals. Vitamin A and retinoids are also known as potent immunoregulatory and antineoplastic agents. Their ability to increase reactivity to histoincompatible tissues is well documented but the mechanism of this action is unclear. Here we report that mice fed on an otherwise conventional diet supplemented with vitamin A acetate (VAA) respond to 10(5) semiallogeneic cells (a suboptimal dose) in a host-versus-graft (HvG) reaction, whereas mice on a conventional diet do not. It is possible to transfer this enhanced immune reactivity by injecting lymphoid cells from VAA-fed animals into those syngeneic mice maintained on the conventional diet. Using a positive selection technique, we demonstrate that the phenotype of the cell probably responsible for this phenomenon is Lyt 1+ 2-.