Primary and secondary autologous mixed-lymphocyte interactions in mice occur via antigens encoded by genes in the I region of the major histocompatibility complex

The antigens stimulating the autologous mixed-lymphocyte reactions (AMLR) were studied in experiments employing specific blocking antisera and through panels of secondary stimulating cells differing in the MHC. Antisera directed to the entire I region, I-A, I-EC, or A?Eα were all effective in blocking the primary AMLR. A principal role for those determinants encoded in I-A was indicated by secondary stimulation experiments. In secondary cultures, I-EC encoded determinants generated weaker responses in the absence of I-A homology.     

Potentiation of the in vitro cytotoxic response to syngeneic lymphoma cells by soluble products of tuberculin-sensitive lymphoid cells stimulated with PPD

Spleen cells from rats immunized with the syngeneic (C58NT)D Gross virus induced lymphoma have previously been shown to differentiate into cytotoxic effector cells following restimulation with tumor cells in vitro. Previous work has also demonstrated that the addition of PPD-primed syngeneic spleen cells and PPD to cultures of (C58NT)D-primed spleen cells will potentiate the in vitro cytotoxic response to tumor antigens. In the studies presented here, the potentiating effect was found to be mediated by a soluble factor(s) released by nonadherent cells from BCG-primed rats. The release of this immunopotentiating factor(IPF) required the presence of PPD and varied with the concentration of PPD added. IPF was produced by BCG-primed spleen, lymph node, and thymus cells. Maximal production of IPF in PPD-stimulated cultures was obtained after 6–12 hr of incubation. Supernatants obtained after 30 hr of incubation lacked apparent IPF activity when tested initially, but activity was recovered after mild heat treatment. Recovery of IPF activity after heat exposure is best explained by the presence of a heat-labile inhibitor. IPF itself is stable to heat treatment to 56 °C for 40 min. IPF was also shown to be capable of enhancing immune responses to histocompatibility antigens in vitro.     

Suppression of lymphokine production: I. Macrophage-mediated inhibition of MIF production

Macrophages have been found to suppress the in vitro production by stimulated T lymphocytes of a lymphokine, migration inhibitory factor. When macrophages isolated from primary MSV-induced tumors were added to antigen-stimulated MSV-immune spleen cells, a complete suppression of MIF production was observed. This suppression was nonspecific, since MIF production by antigen-stimulated alloimmune spleen cells and by PHA-stimulated normal spleen cells was also inhibited. Suppressor macrophages could also be induced by inoculation with Corynebacterium parvum, whereas light mineral oil-induced peritoneal macrophages had no detectable effect on MIF production. The failure to detect MIF in the supernatants of stimulated cultures containing activated macrophages appeared to be due to inhibition of lymphokine production rather than to absorption or inactivation of MIF or to interference with the assay for detection of MIF. Macrophages were able to suppress MIF production only when added during the first 4–5 hr of culture and they had no effect when added later. These data show that activated macrophages can nonspecifically suppress lymphokine production and that this appears to be due to inhibition of an early step in lymphocyte stimulation.     

The effects of corticosteroids on immunoregulation in sarcoidosis.

The effects of in vivo hydrocortisone administration on the kinetics and functional capabilities of cells involved in the immune response in sarcoidosis were examined. Untreated sarcoidosis patients have a decrease in the absolute numbers of circulating T lymphocytes (P < 0.05). However, with regard to the proportions of T lymphocyte subpopulations, there is an increase in the relative proportions of IgG Fc receptor positive T cells (T_G) (P < 0.01), which have suppressor capabilities in certain in vitro systems of mitogen-induced antibody production, and a relative decrease in IgM Fc receptor positive T lymphocytes (T^M) which have helper effects in this system (P < 0.05). Additionally, sarcoidosis patients have circulating “suppressor” monocytes capable of suppressing anti-sheep red blood cell (SRBC) plaque-forming cell (PFC) responses by pokeweed mitogen (PWM)-stimulated lymphocytes. The in vitro removal of this cell abrogated this depressed response (P < 0.01). Intravenous administration of hydrocortisone produced a transient absolute T lymphocytopenia (P < 0.01) accompanied by a relative increase in T^G cells (P < 0.01) and a relative decrease in T_M cells (P < 0.02). Four hours after hydrocortisone therapy, at the point of maximal hydrocortisone-induced monocytopenia (P < 0.01), the suppressed ability of sarcoidosis lymphocytes to synthesize and secrete in vitro anti-SRBC antibody after polyclonal activation was corrected (P < 0.01), and PFC responses comparable to those seen in untreated normal subjects were obtained. These studies demonstrate that corticosteroid administration has profound effects on certain in vitro demonstrable immunoregulatory abnormalities in sarcoidosis.     

The influence upon mitogenic and cellular immunologic reactive systems in vitro by poly(I:C) and BCG murine interferons induced in vivo.

We have previously reported that lymphocytes from W/Fu rats immunized with syngeneic (C58NT)D tumor cells were cytotoxic against these cells in a 4-hr ⁵¹Cr release assay. We have investigated the feasibility of cryopreserving lymphocytes and target cells and have selected freezing conditions which provide good yields of viable cells and functional activity. Lymphocytes from different animals had a recovery of 60–80% viability which resulted in a corresponding 55–75% recovery of cytotoxic activity. Repeated testing of lymphocyte cytotoxicity from a pool of frozen spleen cells against either fresh or frozen (C58NT)D cells gave reproducible cytotoxicity. In addition, recovery of high levels of lymphocyte function was also demonstrated when cryopreserved cells were employed in long-term cytotoxic assays, i.e., ³H-proline and ¹²⁵IUdR release assays, in the lymphoproliferative response to mitogens (PHA and Con A)³ or tumor cells (MLTI) as measured by ³H-thymidine incorporation, and in the in vitro generation of secondary cytotoxicity.By employing these cryoprotective techniques it is possible to have: 1) a population of lymphoid cells with known functional activity and 2) a pool of target cells with known susceptibility to lysis and antigenic content. Furthermore, the use of frozen cells as internal standards in each test also permits the analysis of assay variation as well as the study of variation in various cell types.     

不同影响因素对热原实验中兔筛选的影响

目的分析不同影响因素对新西兰兔的初次筛选合格率、二次筛选合格率的影响。方法根据2005版《中国药典》进行测定。结果初次筛选结果中,不同季节、体重值、性别、湿度新西兰兔的筛选率都有显著性差异,筛选时间在7～9月、体重值为1.7～2.0 kg、雄性的新西兰兔、在湿度为61～70%的条件下初次筛选率较高;在二次筛选结果中,不同季节、室温、湿度条件下新西兰兔的筛选率都有显著性差异,筛选时间在1～3月、在室温为22.1～23.0℃、湿度为61～70%的条件下新西兰兔的二次筛选率较高。结论在不同影响因素的条件下,新西兰兔的初次筛选合格率、二次筛选合格率均受到影响。     

Post-translational modifications in MeHg-induced neurotoxicity

Mercury (Hg) exposure remains a major public health concern due to its widespread distribution in the environment. Organic mercurials, such as MeHg, have been extensively investigated especially because of their congenital effects. In this context, studies on the molecular mechanism of MeHg-induced neurotoxicity are pivotal to the understanding of its toxic effects and the development of preventive measures. Post-translational modifications (PTMs) of proteins, such as phosphorylation, ubiquitination, and acetylation are essential for the proper function of proteins and play important roles in the regulation of cellular homeostasis. The rapid and transient nature of many PTMs allows efficient signal transduction in response to stress. This review summarizes the current knowledge of PTMs in MeHg-induced neurotoxicity, including the most commonly PTMs, as well as PTMs induced by oxidative stress and PTMs of antioxidant proteins. Though PTMs represent an important molecular mechanism for maintaining cellular homeostasis and are involved in the neurotoxic effects of MeHg, we are far from understanding the complete picture on their role, and further research is warranted to increase our knowledge of PTMs in MeHg-induced neurotoxicity.     

miR‐126 reduces trastuzumab resistance by targeting PIK3R2 and regulating AKT/mTOR pathway in breast cancer cells

MicroRNAs (miRNAs) have been found to play a key role in drug resistance. In the current study, we aimed to explore the potential role of miR‐126 in trastuzumab resistance in breast cancer cells. We found that the trastuzumab‐resistant cell lines SKBR3/TR and BT474/TR had low expression of miR‐126 and increased ability to migrate and invade. The resistance, invasion and mobilization abilities of the cells resistant to trastuzumab were reduced by ectopic expression of miR‐126 mimics. In comparison, inhibition of miR‐126 in SKBR3 parental cells had the opposite effect of an increased resistance to trastuzumab as well as invasion and migration. It was also found that miR‐126 directly targets PIK3R2 in breast cancer cells. PIK3R2‐knockdown cells showed decreased resistance to trastuzumab, while overexpression of PIK3R2 increased trastuzumab resistance. In addition, our finding showed that overexpression of miR‐126 reduced resistance to trastuzumab in the trastuzumab‐resistant cells and that inhibition of the PIK3R2/PI3K/AKT/mTOR signalling pathway was involved in this effect. SKBR3/TR cells also showed increased sensitivity to trastuzumab mediated by miR‐126 in vivo. In conclusion, the above findings demonstrated that overexpression of miR‐126 or down‐regulation of its target gene may be a potential approach to overcome trastuzumab resistance in breast cancer cells.     

Epigenetic down-regulation of microRNA-126 in scleroderma endothelial cells is associated with impaired responses to VEGF and defective angiogenesis

Impaired angiogenesis in scleroderma (SSc) is a critical component of SSc pathology. MicroRNA-126 (miR-126) is expressed in endothelial cells (MVECs) where it regulates VEGF responses by repressing the negative regulators of VEGF, including the sprouty-related protein-1 (SPRED1), and phosphoinositide-3 kinase regulatory subunit 2 (PIK3R2). MVECs were isolated from SSc skin and matched subjects (n = 6). MiR-126 expression was measured by qPCR and in situ hybridization. Matrigel-based tube assembly was used to test angiogenesis. MiR-126 expression was inhibited by hsa-miR-126 inhibitor and enhanced by hsa-miR-126 Mimic. Epigenetic regulation of miR-126 expression was examined by the addition of epigenetic inhibitors (Aza and TSA) to MVECs and by bisulphite genomic sequencing of DNA methylation of the miR-126 promoter region. MiR-126 expression, as well as EGFL7 (miR-126 host gene), in SSc-MVECs and skin, was significantly down-regulated in association with increased expression of SPRED1 and PIK3R2 and diminished response to VEGF. Inhibition of miR-126 in NL-MVECs resulted in reduced angiogenic capacity, whereas overexpression of miR-126 in SSc-MVECs resulted in enhanced tube assembly. Addition of Aza and TSA normalized miR-126 and EGFL7 expression levels in SSc-MVECs. Heavy methylation in miR-126/EGFL7 gene was noted. In conclusion, these results demonstrate that the down-regulation of miR-126 results in impaired VEGF responses.     

Unique monoclonal antibodies specifically bind surface structures on human fetal erythroid blood cells

Background

Continuing efforts in development of non-invasive prenatal genetic tests have focused on the isolation of fetal nucleated red blood cells (NRBCs) from maternal blood for decades. Because no fetal cell-specific antibody has been described so far, the present study focused on the development of monoclonal antibodies (mAbs) to antigens that are expressed exclusively on fetal NRBCs.Methods: Mice were immunized with fetal erythroid cell membranes and hybridomas screened for Abs using a multi-parameter fluorescence-activated cell sorting (FACS). Selected mAbs were evaluated by comparative FACS analysis involving Abs known to bind erythroid cell surface markers (CD71, CD36, CD34), antigen-i, galactose, or glycophorin-A (GPA). Specificity was further confirmed by extensive immunohistological and immunocytological analyses of NRBCs from umbilical cord blood and fetal and adult cells from liver, bone marrow, peripheral blood, and lymphoid tissues.Results: Screening of 690 hybridomas yielded three clones of which Abs from 4B8 and 4B9 clones demonstrated the desired specificity for a novel antigenic structure expressed on fetal erythroblast cell membranes. The antigenic structure identified is different from known surface markers (CD36, CD71, GPA, antigen-i, and galactose), and is not present on circulating adult erythroid cells, except for occasional detectability in adult bone marrow cells.Conclusions:The new mAbs specifically bind the same or highly overlapping epitopes of a surface antigen that is almost exclusively expressed on fetal erythroid cells. The high specificity of the mAbs should facilitate development of simple methods for reliable isolation of fetal NRBCs and their use in non-invasive prenatal diagnosis of fetal genetic status.