MRL/lpr Thy1.1小鼠中一个新的T细胞亚群的发现

目的研究ＭＲＬ／ｌｐｒＴｈｙ１．１小鼠中Ｔ细胞的异常、异常Ｔ细胞的起源及其与淋巴腺病理的关系。方法取出生后不同时期ＭＲＬ／ｌｐｒＴｈｙ１．１小鼠及正常Ｃ５７ＢＬ／６小鼠的造血淋巴组织，制备单细胞悬液并用流式细胞技术检测其细胞表面标记，然后以ｔ检验进行统计学处理。结果发现ＭＲＬ／ｌｐｒＴｈｙ１．１小鼠中存在着一个异常的、与淋巴腺病理密切相关的Ｔ细胞亚群，其表型为Ｔｈｙ１．１－ＴＣＲαβ＋Ｌｙ５＋，而且这一细胞亚群随着年龄增长而迅速增加，至１９周龄，其可以占淋巴结细胞总数的８０％。同时，淋巴结及脾脏中传统Ｔ细胞（Ｔｈｙ１．１＋ＴＣＲαβ＋Ｌｙ５－）随着Ｔｈｙ１．１－ＴＣＲαβ＋Ｌｙ５＋细胞的增加而逐渐减少。结果还表明，这一细胞亚群既不在胸腺也不在骨髓中分化成熟，但这一细胞亚群起源于某些异常骨髓造血细胞。结论ＭＲＬ／ｌｐｒＴｈｙ１．１小鼠中存在着一个起源于某些异常骨髓造血细胞的表型为Ｔｈｙ１．１－ＴＣＲαβ＋Ｌｙ５＋的随着年龄增长而迅速增加的Ｔ细胞亚群     

Quantitative and qualitative changes in the intraepithelial lymphocyte population in the uterus of nonpregnant and pregnant sheep.

PROBLEM: Previous studies have revealed the presence of a unique population of CD45R+ granulated cells in the sheep uterine epithelium. In the present study, dramatic changes in this cell population and in the nongranulated lymphocytes in the uterine and endometrial glandular epithelium of non-cycling, cycling, pregnant, and postparturient sheep are described. In noncycling and cycling sheep, the granules in the granulated intraepithelial cells were small. From days 55 to 134 of pregnancy, the granules in these cells were large, and there was a significant increase (P < 0.01) in the proportion of this cell population in the uterine epithelium but not in the endometrial glandular epithelium located in the deeper region of the stroma. The number of these cells declined dramatically (P < 0.01) from 2 to 15 days after parturition. Both the tissue distribution and the time of activation of these cells suggests they are different from the granulated lymphocytes described in placentae of mice and man. CONCLUSIONS: It is concluded that this unique population of granulated cells is derived from lymphocytes, and that these cells become metabolically active from mid- to late-pregnancy and may play a physiological role during pregnancy or birth. In contrast, the number of nongranulated intraepithelial lymphocytes were suppressed throughout pregnancy and they probably do not play a role in pregnancy.     

A human suppressor lymphocyte subpopulation identified by a heteroantiserum

A rabbit antiserum has been prepared using peripheral mononuclear cells from a patient with common variable hypogammaglobulinaemia (CVH) who had demonstrable suppressor cells in vitro. This antiserum (anti-Ck), after absorption with pooled normal glass-non-adherent cells and platelets, showed selective reactivity with subpopulations of normal human peripheral lymphocytes. Using indirect immunofluorescence, anti-Ck labelled 36 +/- 8% (mean +/- 1 s.d.) of normal human peripheral lymphocytes. Anti-Ck has the following pattern of reactivity with enriched cell populations: 18% of normal E-rosette-positive cells, 70% of normal E-rosette-negative cells, 60% of lymphocytes from CVH patients, 82% of T gamma lymphocytes, 82% of Con A-activated lymphocytes and 81% of Fc receptor-positive lymphocytes. Anti-Ck reacted neither with glass non-adherent cells (which had been used for absorption), nor with thymocytes, monocytes, granulocytes, membrane immunoglobulin-positive lymphocytes, lymphocytes from patients with chronic lymphocytic leukaemia, or Bristol 7 (B-cell line) cells. The in vitro proliferative response to concanavalin A, phytohaemagglutinin and pokeweed mitogen was significantly enhanced after complement-mediated lysis of normal peripheral lymphocytes using anti-Ck. Immunoglobulin production as measured by a reverse haemolytic plaque assay was also significantly increased by lysis of cell subpopulations with anti-Ck. These studies show that a heteroantiserum can be prepared which identifies antigens common to subsets of human lymphocytes which contain functional suppressor cells.     

Monoclonal antibody associated with a lymphocyte subpopulation in chronic lymphocytic leukemia

An IgG monoclonal antibody that detects a subpopulation of lymphocytes found in peripheral blood and bone marrow of patients with CLL and malignant lymphoma is described. The initial immunization used to achieve the resultant monoclonal antibody included the use of cells obtained by DNA transformation of mouse L-cells with the DNA obtained from a morphologically altered somatic cell hybrid between primary human CLL peripheral lymphocytes and a flat-revertant Chinese hamster ovary (CHO) cell line designated GRC+L-73. Hybridomas were thus selected as potentially recognizing antigens associated with the morphological transformation induced by hybridization of CHO cells with lymphocytes from lymphocytic malignancies. One such hybridoma, designated 37-28, was selected for further investigation. The monoclonal antibody produced was IgG (gamma G2a) and detects a subpopulation of lymphocytes present in hematological specimens of some of the lymphocytic malignancies.     

Studies on the lymphocyte subpopulation in systemic lupus erythematosus by monoclonal antibodies

T cells and T-cell subsets were studied with monoclonal antibodies in 15 patients with systemic lupus erythematosus (SLE) during a 6-wk period. During active states of disease, all investigated T-cell subsets decreased, but the reduction of suppressor cells seemed to be more pronounced, therefore the helper/suppressor ratio increased. Less suppressor cells could be detected during clinical impairments and more during improvements.     

Analysis of human lymphocyte protein expression. I. Identification of subpopulation markers by two-dimensional polyacrylamide gel electrophoresis

This study provides new knowledge on the changes in protein expression that differentiate the functionally and phenotypically different cells of the human immune system. Purification by flow cytometry of normal lymphocytes (both T and B cells), monocytes and granulocytes, combined with high-resolution two-dimensional polyacrylamide gel electrophoresis, revealed reproducible qualitative and quantitative changes between these cell populations. Characteristic profiles of marker proteins for each cell type were identified. Determination of markers for T lymphocyte subpopulations was achieved by the comparative analysis of normal T cells separated on the basis of CD4 and CD8 expression in combination with the analysis of cells from patients with T cell chronic lymphocyte leukemia. These results suggest that the modulation or regulation of proteins is very strictly controlled in lymphoid differentiation, and that several quantitative and a few qualitative differences can give rise to completely different phenotypes. Thus, instead of detecting numerous random differences among lymphocyte protein patterns, rather stringent regulation of protein expression in each subpopulation was found.     

Characterization of a lymphocyte subset displaying a unique regulatory activity in human decidua

One of the most intriguing mechanisms of early pregnancy is the maternal immune tolerance toward her semi-allogeneic fetus, specifically in face of the accumulation of lymphocytes to high numbers at implantation sites. Here, we propose that a regulatory decidual lymphocyte (dL) population prevent the activation of reactive T cells and by that may maintain immune tolerance in the decidua. dLs were isolated from first trimester decidua and were then co-cultured with PBMC that were stimulated with anti-CD3 mAbs. Cytokine secretion to the media as well as the proliferative response were tested. The data demonstrate that dLs inhibit the production of IFN-gamma, tumor necrosis factor-alpha (TNF-alpha) and IL-5 but not CD25 expression, IL-2 production or proliferation in the responder PBMC. Suppression is mediated by a cell contact-dependent mechanism, was not restricted by the MHC and was not reversed by the addition of exogenous IL-2 although the inhibitory sub-population was identified as CD3+CD4+CD25+Foxp3+ natural regulatory T cells (Treg). Interestingly, suppression can also be overcome by the addition the endotoxin LPS, suggesting a mechanism for preterm labor triggered by chorioamnionitis. While these characteristics are in contrast to known peripheral CD4+CD25+ Treg activity, we identified these cells as the cellular subset responsible for the regulatory activity, suggesting that in decidua a functionally unique regulatory lymphocyte subset exist. These findings suggest the existence of a dynamic regulatory system in human decidua that is highly responsive to environmental factors.     

Identification of a unique epigenetic sub-microenvironment in prostate cancer

The glutathione S-transferase P1 (GSTP1) gene promoter is methylated in tumour cells in more than 90% of prostate carcinomas. Recently, GSTP1 promoter methylation was identified in tumour-associated stromal cells in addition to the tumour epithelium. To define the extent and location of stromal methylation, epigenetic mapping using pyrosequencing quantification of GSTP1 promoter methylation and an anatomical three-dimensional reconstruction of an entire human prostate specimen with cancer were performed. Normal epithelium and stroma, tumour epithelium, and tumour-associated stromal cells were laser capture-microdissected from multiple locations throughout the gland. As expected, the GSTP1 promoter in both normal epithelium and normal stromal cells distant from the tumour was not methylated and the tumour epithelium showed consistently high levels of promoter methylation throughout. However, tumour-associated stromal cells were found to be methylated only in a localized and distinct anatomical sub-field of the tumour, revealing the presence of an epigenetically unique microenvironment within the cancer. Morphologically, the sub-field consisted of typical, non-reactive stroma, representing a genomic alteration in cells that appeared otherwise histologically normal. Similar epigenetic anatomical mapping of a control prostate gland without cancer showed low background methylation levels in all cell types throughout the specimen. These data suggest that stromal cell methylation can occur in a distinct sub-region of prostate cancer and may have implications for understanding tumour biology and clinical intervention.     

Principal lymphocyte subpopulation in local host response to human oesophageal cancer

Summary We investigated what subpopulations of tumour-infiltrating lymphocytes (TIL) play a key role in in vivo function and what determines the degree of local host response represented by lymphocyte infiltration in human oesophageal cancer. We examined the increased subpopulation of TIL in good responders (GR) (patients with intensively TIL infiltrated tumours) when compared with poor responders (PR) (patients with weakly TIL infiltrated tumours). The frequency of each subpopulation was determined by quantitative flow-cytometric measurement on TIL separated from fresh tumours. Of TIL in GR, the frequency of CD3⁺ cells increased significantly (P<0.05) but the frequencies of CD16⁺, Leu7^–, and CD16⁺ Leu7^– cells were low and did not increase significantly compared with those in PR. With respect to T-cell subsets of TIL in GR, the frequency of CD8⁺ cells was significantly higher than that in PR (P<0.01), and CD4⁺/CD8⁺ ratio was lower than that in PR (P< 0.025). On two-colour analyses, most of CD8⁺ cells (cytotoxic/suppressor T-cells: Tc/s) did not co-express CD11b and the frequency of CD8⁺ CD11b^– cells (cytotoxic T-cell: Tc) increased significantly compared with that in PR. Clinicopathological and phenotypic analysis of peripheral blood lymphocytes revealed that there are no major differences in general immunocompetence between GR and PR. These results suggest that Tc/s, especially Tc, might play a key role in local host response. They also suggest that not only the general immune status of the host but also the identification of class I major histocompatibility complex antigens by the host at the tumour site may strongly affect the degree of host response in oesophageal cancer.     

Anti-inflammatory effect of ibuprofen-loaded embolization beads in sheep uterus

Embolization of blood vessels may result in a variety of side effects which can include pain and inflammation. The objective of this study was to assess the release and effect of ibuprofen (IBU) from Bead Block microspheres (BB) loaded with IBU (IBU-BB) on the foreign body inflammatory reaction in a sheep uterine artery model. Both uterine arteries of 12 hormonally cycled ewes were embolized with 0.5 mL of 500-700 microm BB (n = 6) or IBU-BB (n = 6). Animals were sacrificed at 1 week (1W) or 3 weeks (3W) (n = 3 each group). The gross examination of the organs was performed and distribution of the beads in the tissue was assessed. Inflammation was estimated histologically by quantitative and semiquantitative classification of inflammatory cells on HES and MGG stains and use of videoanalysis after immunohistolabeling with CD-antibodies to a variety of inflammatory cells. At 1W, a significant decrease of inflammatory response was observed for IBU-BB relative to BB in terms of number of lymphocytes and of immunohistochemical staining for CD172a, MHC-II, CD3, and CD4. At 3W, the inflammatory response for IBU-BB was similar to that for BB at 1W in terms of cell populations and moderate intensity. There was no or low amounts of staining for CD8 and CD45RA and none for CD21 in all four groups. Immunohistochemical detection of IBU showed that some drug was still present in the beads at 1W but none was detectable at 3W suggesting it had all eluted. These results signify that the inflammatory response is dampened by the action of IBU eluted from the beads and that IBU-BB can delay postembolization inflammatory reaction.     

Identification of a unique double-negative regulatory T-cell population

Regulatory T (Treg) cells represent one of the main mechanisms of regulating self-reactive immune cells. Treg cells are thought to play a role in down-regulating immune responses to self or allogeneic antigens in the periphery. Although the function of Treg cells has been demonstrated in many experimental settings, the precise mechanisms and antigen specificity often remain unclear. In a hepatitis B e antigen-T-cell receptor (HBeAg-TCR) double transgenic mouse model, we observed a phenotypically unique (TCR+) CD4- /CD8- CD25(+/-) GITR(high) PD-1(high) FoxP3-) HBeAg-specific population that demonstrates immune regulatory function. This HBeAg-specific double-negative regulatory cell population proliferates vigorously in vitro, in contrast to any other known regulatory population, in an interleukin-2-independent manner.     

Presence of a distinct CD8+ and CD5- leucocyte subpopulation in the sheep liver.

The relative proportions of CD5, CD4, CD8, SBU-T19 and surface immunoglobulin (sIg)-positive lymphocytes in normal sheep livers were determined by flow cytometry analysis of isolated liver leucocytes and compared to lymphocyte subpopulations found in peripheral blood and hepatic lymph nodes. In contrast to the blood, very low numbers of SBU-T19+ cells were found in both the liver and hepatic lymph nodes. The ratio of helper versus suppressor/cytotoxic T cells in the liver was less than 1, which was much lower than in blood or hepatic lymph nodes. A large proportion of liver lymphocytes were characterized by a low intensity staining for the CD8 antigen (CD8L). Two-colour flow cytometry analysis revealed that the CD8L cells were negative for the CD5 pan T-cell marker. It is suggested that these CD8LCD5- cells are natural killer (NK) cells similar to those found in the gut. An influx of CD8LCD5- cells was observed in leucocyte-infiltrated liver lesions at later stages of a rejection response to the cestode Taenia hydatigena.     

Phenotypic and functional characteristics of a T lymphocyte regulator subpopulation involved in stimulation of hematopoiesis during stress

Research Institute of Pharmacology, Tomsk Scientific Center, Academy of Medical Sciences of the USSR. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 107, No. 5, pp. 590–593, May, 1989.     

A unique human B lymphocyte antigen defined by a monoclonal antibody

We produced a hybridoma designated 4G7 from a mouse immunized with chronic lymphocytic leukemia cells. The 4G7 hybridoma secretes an IgG1 antibody that is specific for normal and malignant B lymphocytes. Using dual color immunofluorescence staining, this antibody reacted with all immunoglobulin-positive cells but no T cells in normal peripheral blood. There was no detectable 4G7 antigen on monocytes, platelets, red cells, granulocytes, or phytohemagglutinin-activated T cells. When PBL were depleted of 4G7 positive cells and stimulated with pokeweed mitogen, secreted immunoglobulin levels fell to less than 10% of control values on Day 5 and less than 1% of control on Day 7. This antibody was reactive with 155 of 176 B lineage neoplasms on which it was screened. Thirty-five cases of myeloid or T-lymphoid malignancy were negative. Our studies show that the 4G7 antigen modulates in the presence of excess antibody. Free 4G7 antigen was not found circulating in human serum. The cell surface antigen identified by 4G7 was sensitive to pronase proteolysis but resistant to trypsin and chymotrypsin digestion. A comparison of 4G7 with other known B-cell antibodies indicates that the 4G7 antigen has not been previously identified. This antibody is of use for the identification of normal B lymphocytes, the study of B-cell differentiation, and the characterization of lymphoid malignancies.     

Identification of a novel astrovirus in domestic sheep in Hungary

The family Astroviridae consists of two genera, Avastrovirus and Mamastrovirus, whose members are associated with gastroenteritis in avian and mammalian hosts, respectively. We serendipitously identified a novel ovine astrovirus in a fecal specimen from a domestic sheep (Ovis aries) in Hungary by viral metagenomic analysis. Sequencing of the fragment indicated that it was an ORF1b/ORF2/3′UTR sequence, and it has been submitted to the GenBank database as ovine astrovirus type 2 (OAstV-2/Hungary/2009) with accession number JN592482. The unique sequence characteristics and the phylogenetic position of OAstV-2 suggest that genetically divergent lineages of astroviruses exist in sheep.