A new isolation method for rat intraepithelial lymphocytes.

Intraepithelial lymphocytes (IELs) play critical roles in gut immunity. In mice, gammadelta T cells are a large component of the IEL population. In the rat, gammadelta IELs are reportedly much less common, but technical issues suggest that previous analyses should be interpreted cautiously. The study of IELs in rats has been impeded by isolation procedures that are lengthy and complex, leading to small cell yields. For this reason, it is possible that rat IELs analyzed in previous studies have not been representative of the entire IEL compartment. We report a new method for the isolation of rat IELs that is based on the selective removal of intestinal epithelial cells under conditions that leave the basement membrane undisturbed. The method is rapid and requires neither enzymatic digestion, nor surgical removal of Peyer's patches, nor vigorous mechanical manipulation of the intestine. The yield of rat IELs using this method is 5- to 10-fold greater than that reported for other methods. Morphological and phenotypic analyses demonstrated that the purified cell population is comprised of IELs and is not contaminated with lamina propria or Peyer's patch lymphocytes. Phenotypic analysis revealed five major subsets of IELs based on differential cell surface expression of CD4, CD8, and alphabeta T cell receptor (TcR). Among the alphabetaTcR- cells was a population of gammadelta T cells present at levels not previously detected. The isolation of IEL sub-populations using this methodology should facilitate studies of the function of these cells in gut immunity.     

Antigen-binding thymus-derived lymphocytes: I. Rapid method for isolation of theta-positive antigen-stimulated cells

A rapid method for separating SRBC-stimulated populations of theta (θ)-positive thymus-derived lymphocytes from bone marrow-derived lymphocytes and plaque-forming cells has been developed using cotton wool columns. The column effluent was monitored for plaque-forming and `rosette'-forming cells. Stimulated θ-negative cells were found to adhere preferentially to cotton wool. The total recovery of small lymphocytes was 12±3 per cent and the yield of rosette-forming cells was 5.5±4 per cent of which 85±5 per cent were θ-positive.     

A simple method for the isolation of murine peripheral blood lymphocytes

A simple, rapid and reproducible semi-microtechnique was developed for the isolation of murine peripheral blood lymphocytes. Murine peripheral blood was collected from brachial vessels and centrifuged through a Ficoll--Hypaque gradient in a microfuge tube yielding 1--1.5 X 10(6) lymphocytes per mouse.     

A method for isolation and culture of lymphocytes from endoscopic biopsies

Human small and large intestinal lamina propria lymphocytes have been successfully prepared from endoscopic biopsies by a combined enzymatic and mechanical method which gives higher yields of viable mucosal lymphocytes than previously reported, despite the small size of the biopsy samples. Viability of the cells was demonstrated by dye exclusion and they could be satisfactorily maintained in short-term culture. Phytohaemagglutinin-P (PHA-P) transformation characteristics of intestinal lymphoid cells and those of peripheral blood were studied in 20 patients with Crohn's disease and 10 control subjects. Peripheral blood lymphocytes were separated according to this technique, no decrease in viability being observed when compared to a standard Ficoll-Hypaque gradient technique. Endoscopically abnormal (EA) and endoscopically normal (EN) Crohn's tissue showed significantly different responses to PHA-P (P<0.001), EA tissue lymphocytes giving lower blastogenic responses.     

A rapid method for the isolation of functional thymus-derived murine lymphocytes

A rapid method is described for effectively removing immunoglobulin-bearing cells from either primed or unprimed mouse spleen and lymph node cell suspensions. Incubation of cell suspensions in nylon wool columns for 45 min at 37 °C resulted in a 9 to 100-fold depletion of immunoglobulin-bearing cells and a complementary 1.5 to 2-fold enrichment of T cells in the column effluent populations. The effluent population, derived from passage of spleen cells through these columns, was virtually devoid of B precursor and memory cell activity, but contained all of the helper cell and cytotoxic effector cell precursor activity when compared to unfractionated spleen cells.     

不同分离介质对小鼠原代肝星状细胞分离纯化效果的影响

目的 比较不同的分离介质对小鼠原代肝星状细胞（hepatic stellate cells,HSCs）分离纯化效率的差异.方法 肝门静脉原位灌注,预灌注不含钙镁离子的D-Hanks液及含有Ⅳ型胶原酶的灌注液,取消化后的肝脏,分别采用Ficoll-Paque PLUS、Percoll、Nycodenz作为分层介质,密度梯度离心纯化原代HSCs.显微镜下观察细胞纯度并计算细胞得率.锥虫蓝染色判断细胞活率.激光共聚焦显微镜观察原代HSCs的固有荧光情况.油红O染色观察获得原代HSCs的纯度情况.结果 采用Ficoll-Paque PLUS、Percoll、Nycodenz分离细胞得率分别为（1＆#177;0.5）＆#215;106,（1＆#177;0.5）＆#215;105,（0.8＆#177;0.5）＆#215;106;纯度分别为75％ ～ 85％,91％ ～95％,91％ ～95％;活率均高于90％.结论 用Nycodenz作为分离介质分离肝星状细胞的方案最优.     

改良法分离大鼠肝星状细胞

目的　探讨一种高效、经济、稳定的肝星状细胞的分离方法 ,为探索肝纤维化的发生机制提供细胞模型。方法　采用链霉蛋白酶、胶原酶原位灌流 ,以Nycodenz为分离介质 ,单层一步密度梯度离心法 ,分离大鼠的肝星状细胞。结果　肝星状细胞的获得率为 3 .95× 10 7 只、纯度为 97% ,存活率为 98%。结论　本法是一种较理想的分离肝星状细胞的方法。     

A method for the isolation of t lymphocytes from normal and neoplastic ocular tissue

Summary We have employed a simple organ culture method to isolate in vitro the small numbers of T lymphocytes present in choroidal melanomas, as well as normal choroid. Subsequent growth and expansion of the isolated T cells was accomplished in the presence of Interleukin-2 (IL-2; T Cell Growth Factor).     

Adhesion of lymphocytes to hepatic endothelium.

Chronic inflammation occurs when factors that regulate the process of leucocyte recruitment are disrupted, and it is dependent on recruitment, activation, and retention of lymphocytes within tissue microenvironments. The molecular mechanisms that mediate lymphocyte adhesion to vascular endothelial cells have been described by several groups, but the signals involved in the recruitment of lymphocytes via the hepatic circulation have yet to be elucidated fully. This article considers the liver as a model of organ specific lymphocyte recruitment. In this context, the roles of leucocyte and endothelial adhesion molecules and chemokines in lymphocyte recruitment are discussed. The article also reviews the mechanisms that regulate lymphocyte recirculation to the liver under both physiological and pathological conditions and draws parallels with other organs such as the gut and skin.     

Adhesion of lymphocytes to hepatic endothelium.

Chronic inflammation occurs when factors that regulate the process of leucocyte recruitment are disrupted, and it is dependent on recruitment, activation, and retention of lymphocytes within tissue microenvironments. The molecular mechanisms that mediate lymphocyte adhesion to vascular endothelial cells have been described by several groups, but the signals involved in the recruitment of lymphocytes via the hepatic circulation have yet to be elucidated fully. This article considers the liver as a model of organ specific lymphocyte recruitment. In this context, the roles of leucocyte and endothelial adhesion molecules and chemokines in lymphocyte recruitment are discussed. The article also reviews the mechanisms that regulate lymphocyte recirculation to the liver under both physiological and pathological conditions and draws parallels with other organs such as the gut and skin.     

A rapid method for the isolation of functional human T lymphocytes using hydroxyapatite column fractionation

Passage of peripheral blood lymphocytes through a column of hydroxyapatite resulted in a 7-20-fold depletion of immunoglobulin-bearing cells, a 20-fold depletion of monocytes, and a 1.3-fold enrichment of T cells. The effluent population was virtually devoid of B lymphocyte precursors and monocytes, whereas helper cell and suppressor cell populations remained intact. This method will facilitate the rapid preparation of T-enriched cell populations.     

An improved and rapid method for the isolation of rat lymph node or spleen T lymphocytes for T cell proliferation assays.

To detect and compare the capacity of antigen presenting cells to present antigen in a T cell proliferation assay, it is necessary to obtain a pure population of antigen-primed T cells that gives low background proliferative responses. Therefore in this paper we present a newly developed isolation method of antigen-primed T lymphocytes from rat spleen or lymph nodes. This method uses a nylon wool column to deplete most of the adherent cells and B cells, followed by an indirect elimination method with magnetic beads to remove contaminating Ia-positive cells. We compared this method with two commonly used isolation methods, namely a 1.5 h adherence step, followed by a nylon wool column and a Sephadex G-10 column and a 1.5 h adherence step followed by a passage through two consecutive columns of Sephadex G-10. The best T cell enrichment (98% OX-19/52-positive cells) was achieved with the newly developed method, in which the contamination of Ia-positive cells, predominantly B cells and dendritic cells (DC), was diminished to less than 2%. The background response of this population was low and differed significantly with the common methods. Antigen-specific T cell responses induced by splenic DC, expressed as stimulation index, gave very specific responses and showed a steep rise with increasing DC concentrations compared to the common methods. Therefore we conclude that we developed an improved, rapid and reproducible method for the isolation of rat spleen or lymph node T lymphocytes suitable for T cell proliferation assays.     

Xenogenic transfer of human lymphocytes in tolerized mice.

Female nu/+ or BALB/c mice were immunized with human peripheral blood lymphocytes (PBL) before and during pregnancy. Pups born to these mothers were inoculated with human PBL or human fetal bone marrow and thymus cells. Tolerization of the pups to human PBL was observed without graft-versus-host reaction. Presence of human immunoglobulins was observed in the pups for 3-4 weeks. Human T cells also could be detected for a period of 3-4 months in these mice.     

Age-decrease of cells sensitive to an autoantibody-specific for thymocytes and thymus-dependent lymphocytes in NZB mice

NZB mice naturally produce an autoantibody which in the presence of complement is specifically cytotoxic for thymocytes and thymus-dependent lymphocytes (T-cells) in the peripheral lymphoid tissues (lymph nodes and spleen) and the circulation of mice. Using a direct cytotoxicity test with a NZB mouse serum pool which contained the high titred autoantibody, a progressive decrease was observed with age in the proportion of the autoantibody-sensitive cells in mesenteric lymph node, spleen, and blood of NZB mice in comparison with mice of other strains (C57BL/6J and NZW). The numerical decrease in the population of autoantibody-sensitive cells was evident at younger age and greater degree in the peripheral blood than in the lymph node and spleen. The age-decrease in the number of autoantibody-sensitive cells in lymph node and spleen contrasted with the numerical increase in nucleated cells in these organs. The age-decrease in the proportion and number of the autoantibody-sensitive cells in the blood exceeded the decrease in the blood lymphocyte count. This finding indicated that T-cells in the blood are selectively depleted with the ageing of NZB mice. A similar observation was made on the blood lymphocytes of (NZB × NZW)F₁ hybrid mice. The depletion of T-cells in the blood in association with the production of natural thymocytotoxic autoantibody is termed autoimmune thymus-dependent lymphocytopenia.     

Purification and quantification of T and B lymphocytes by an affinity method.

Affinity surfaces were produced by coupling human immunoglobulin (HGG) to the surface of tissue culture grade plastic-ware with a water-soluble carbodiimide followed by treatment with anti-HGG antisera. Surface immunoglobulin SIg) bearing human B lymphocytes attach to these surfaces when centrifuged on to them and unattached cells could be recovered by inverting the trays or dishes. Optimal cell attachment conditions could be rapidly evaluated by counting cells attached to representative areas of multi-well trays and percentage of SIg-bearing cells quantified. Evidence was obtained for cell attachment through Fc receptors as well as SIg using unrelated antigen--antibody-coated trays. This could be prevented by using the F (ab')2 fragments of the antisera. Under these conditions specific attachment through K and lambda light chains could be achieved with normal and chronic lymphocytic leukaemic lymphocytes. Using tissue culture plastic Petri dishes and relatively small quantities of antiserum, larger numbers of lymphocytes could be processed to produce T lymphocytes containing less than 1 per cent of contaminating SIg-positive cells.     

A simple method for the propagation of cervical lymphocytes.

Local immune function is most likely a key influence in the establishment of human papillomavirus infections and its subsequent disease. Unfortunately, little information is known about local cervical immunity, and even less is known about human papillomavirus immunoreactivity. In addition, studies of local immunoreactivity have been hampered by the technical difficulty in obtaining cervical lymphocytes. The objective of the present study was to develop a simple method for the propagation of cervical lymphocytes from biopsy-size specimens. Cervical tissue was obtained from women undergoing a hysterectomy. Cervical samples measuring approximately 3 by 5 by 2 mm were minced and divided into two portions. One portion was digested by standard digestion methods and density gradient lymphocyte separation. The sample was then immunocharacterized for CD4 and CD8 cells by flow cytometry. The other portion was minced into 1-mm3 sections, and each section was placed into a separate well with tissue culture medium and interleukin-2. Lymphocyte counts and immunophenotypic analysis were performed after 18 to 20 days in culture. After 18 to 20 days in culture, the analysis demonstrated that this method of direct lymphocyte culture from a biopsy specimen yielded approximately 1 x 10(6) to 5 x 10(6) lymphocytes. Immunophenotypic studies of the digested sample at day 0 revealed CD4-to-CD8 ratios of between 0.7:1 and 3.5:1, and at days 18 to 20 they revealed ratios of between 2.3:1 and 98:1. In summary, we developed a simple technique for propagating cervical lymphocytes from small tissue samples for the study of the local immune response. Studies are under way to optimize lymphocyte growth and to preserve CD8 populations.     

Infectious lymphocytes in lymphocytic choriomeningitis virus carrier mice.

The infectivity of blood and lymphoid organs of mice persistently infected with lymphocytic choriomeningitis virus was found to be predominantly associated with lymphocytes and both T and B cells were infectious. A hypothesis is presented in which it is assumed that lymphocytes in carrier mice are infected via their LCM virus-specific antigen receptors, thereby leading to their antigen-triggered clonal expansion followed by infection and functional inactivation.     

Genetic selection of mice for quantitative responsiveness of lymphocytes to phytohemagglutinin.

A two-way selection was performed in mice according to the quantitative in vitro response of lymph node lymphocytes to the mitogenic activity of phytohemagglutinin (PHA). The foundation population was composed of outbred mice produced by reciprocal mating of equal numbers of mice from four different colonies. The selective breeding was carried out by mating of mice at each generation giving the best or the lowest response, respectively. The progressive interline separation produced by 6 generations of selective breeding demonstrates that responsiveness to PHA is submitted to polygenic regulation. The heritability of the character investigated is 0.28 +/- 0.08. The interline separation is also found with another T mitogen, concanavalin A (Con A). In spleen cells PHA and Con A produce a similar interline difference. In contrast, the purified protein derivative of tuberculin (PPD) stimulated both lines equally, and E. coli lipopolysaccharide gave only a slightly higher response in high line. This finding implies that our selection based upon response to PHA did not influence B cell function.     

The radiosensitivity of T and B lymphocytes in mice.

The radiosensitivity of T and B lymphocytes in spleens of specific pathogen-free C3Hf/HeMs male mice was studied by the direct and indirect immunofluorescence technique. It was found that the radiobiological parameters characterizing the survival curve of Bpsi lymphocytes were DO = 200 R and n = 1-00. The T lymphocytes, on the other hand, were shown to consist of two distinct subpopulations with respect to their radiosensitivity. The radiobiological parameters of the radiosensitive fraction of T lymphocytes were Dq = 185 R, DO =195 R and n = 2-50. The DO value of the radioresistant T lymphocyte subpopulation was practically unmeasurable. It was estimated that approximately 8 per cent of the T lymphocytes present in the spleen of normal C3Hf mice belonged to this radioresistant subpopulation.