Murine spleen lymphocytes bearing receptors for peanut agglutinin: VII. Separation and functional characterization

Murine spleen lymphocytes having receptors for peanut agglutinin (PNA) were visualized by a specific rosetting technique. The number of lymphocytes forming PNA rosettes (PNA_R⁺) is dependent on the PNA concentration used. T lymphocytes are the primary PNA-rosetting lymphocytes for low PNA concentration (2.5 μg/ml), while both T and B lymphocytes are involved in high PNA concentration (10 μg/ml). Functional studies show that when spleen lymphocytes are separated at a low PNA concentration, the PNA_R⁺ do not respond to T mitogens and suppress antigen-specific immune responses. However, when spleen cells are separated at a high PNA concentration, the PNA_R⁺ do not exhibit differential functional properties as compared to non-PNA-rosetting lymphocytes. The implication of these findings is discussed.     

Relationship between the expression of class I antigen and reactivity of chicken thymocytes

The expression of major histocompatibility complex (MHC) class I antigens in ontogenesis and the distribution of B-F⁺ cells, defined by means of a monoclonal antibody, were studied by indirect membrane immunofluorescence tests on suspensions of thymus, bursa, spleen, peripheral blood lymphocytes (PBL) and red blood cells (RBC) from 18-day-old chicken embryos and chickens from 1–90 days after hatching. At 18 days of incubation and at the first day after hatching, RBC, PBL, and the cells from bursa and thymus are negative. The percentage of positive PBL and bursal cells increases up to 9 days after hatching. By 2 weeks after hatching almost 100 % of the RBC, PBL, bursa, and spleen cells were positive whereas the thymus showed only 20% positive cells. Analysis on 4-m-thick, frozen acetone-fixed tissue sections of thymus showed that medullary cells are positive, while the cortical area is negative. The graft-versus-host (GvH) competence of these thymus subpopulations was compared after sorting by the fluorescence-activated cell sorter and injection into MHC incompatible embryos. GvH reactivity was associated primarily with the B-F⁺ population. Double staining studies with peanut agglutinin (PNA)-fluorescein isothiocyanate and a rabbit-anti-Ig tetramethyl isothiocyanate-conjugate proved that the PNA^– thymocytes are identical with B-F⁺ thymocytes.Abbreviations used in this paper: FACS fluorescence-activated cell sorter - FCS fetal calf serum - FITC-Ig fluorescein isothiocyanate-conjugated immunoglobulin - GvH graft-versus-host - HAT hypoxanthineaminopterin-thymidine - HBSS Hanks' balanced salt solution - IIF indirect immunofluorescence - MCA monoclonal antibody - MHC major histocompatibility complex - NWL normal white Leghorn - OS Obese strain - PBL peripheral blood lymphocytes - PBS phosphate-buffered saline - PNA peanut agglutinin - RBC red blood cells - TRITC-Ig tetramethyl isothiocyanate-conjugated immunoglobulin     

Increased tumor-infiltrating CD8+Foxp3+ T lymphocytes are associated with tumor progression in human gastric cancer

Background

CD8⁺Foxp3⁺ T lymphocytes have been detected in tumors. However, the distribution, phenotypic features, and regulation of these cells in gastric cancer remain unknown.

Methods

The levels of CD8⁺Foxp3⁺ T lymphocytes in the peripheral blood, tumor-draining lymph nodes, non-tumor tissues, and tumor tissues of patients with gastric cancer were detected by flow cytometry. Foxp3 induction in CD8⁺Foxp3^? T cells was investigated in vitro. The suppressive function of CD8⁺Foxp3⁺ T lymphocytes was analyzed by their effect on CD4⁺ T-cell proliferation and IFN-γ production. The percentages of CD8⁺Foxp3⁺ T lymphocytes were evaluated for the association with tumor stage.

Results

The frequency of CD8⁺Foxp3⁺ T lymphocytes in tumor tissues was significantly higher than that in non-tumor tissues, and similar results were also observed in tumor-draining lymph nodes compared with peripheral blood. Most intratumoral CD8⁺Foxp3⁺ T lymphocytes were activated effector cells (CD45RA^?CD27^?). TGF-β1 levels were positively correlated with the frequency of CD8⁺Foxp3⁺ T lymphocytes in tumor tissues, and in vitro TGF-β1 could induce the generation of CD8⁺Foxp3⁺ T lymphocytes in a dose-dependent manner. Furthermore, intratumoral CD8⁺Foxp3⁺ T lymphocytes suppressed the proliferation and IFN-γ production of CD4⁺ T cells. Finally, intratumoral CD8⁺Foxp3⁺ T lymphocytes were significantly increased with tumor progression in terms of tumor-node-metastasis (TNM) stage.

Conclusions

Our data have shown that increased intratumoral CD8⁺Foxp3⁺ T lymphocytes are associated with tumor stage and potentially influence CD4⁺ T-cell functions, which may provide insights for developing novel immunotherapy protocols against gastric cancer.     

Increased expression and functionality of the gap junction in peripheral blood lymphocytes is associated with hypertension-mediated inflammation in spontaneously hypertensive rats

Background

Imbalances in circulating T lymphocytes play critical roles in the pathogenesis of hypertension-mediated inflammation. Connexins (Cxs) in immune cells are involved in the maintenance of homeostasis of T lymphocytes. However, the association between Cxs in peripheral blood T lymphocytes and hypertension-mediated inflammation remains unknown. This study was designed to investigate the role of Cxs in T lymphocytes in hypertension-mediated inflammation in spontaneously hypertensive rats (SHRs).

Methods

The systolic blood pressure (SBP) in Wistar-Kyoto (WKY) rats and SHRs was monitored using the tail-cuff method. The serum cytokine level was determined using ELISA. The proportions of different T-lymphocyte subtypes in the peripheral blood, the expressions of Cx40/Cx43 in the T-cell subtypes, and the gap junctional intracellular communication (GJIC) of peripheral blood lymphocytes were measured using flow cytometry (FC). The accumulations of Cx40/Cx43 at the plasma membrane and/or in the cytoplasm were determined using immunofluorescence staining. The in vitro mRNA levels of cytokines and GJIC in the peripheral blood lymphocytes were respectively examined using real-time PCR and FC after treatment with Gap27 and/or concanavalin A (Con A).

Results

The percentage of CD4⁺ T cells and the CD4⁺/CD8⁺ ratio were high, and the accumulation or expressions of Cx40/Cx43 in the peripheral blood lymphocytes in SHRs were higher than in those of WKY rats. The percentage of CD8⁺ and CD4⁺CD25⁺ T cells was lower in SHRs. The serum levels of IL-2, IL-4 and IL-6 from SHRs were higher than those from WKY rats, and the serum levels of IL-2 and IL-6 positively correlated with the expression of Cx40/Cx43 in the peripheral blood T lymphocytes from SHRs. The peripheral blood lymphocytes of SHRs exhibited enhanced GJIC. Cx43-based channel inhibition, which was mediated by Gap27, remarkably reduced GJIC in lymphocytes, and suppressed IL-2 and IL-6 mRNA expressions in Con A stimulated peripheral blood lymphocytes.

Conclusions

Our data suggest that Cxs may be involved in the regulation of T-lymphocyte homeostasis and the production of cytokines. A clear association was found between alterations in Cxs expression or in Cx43-based GJIC and hypertension-mediated inflammation.

     

Polyclonal activation of human lymphocytes and induction of cytotoxic lymphocytes by streptococcal preparations

Polyclonal activation of human peripheral blood lymphocytes (PBLs)in vitro by preparations ofStreptococcus pyogenes Su strain (OK-432) and other heat-killed strains was investigated. The streptococcal preparations tested induce a proliferative response of PBLs via interleukin-2 (IL-2)-independent pathways. The proliferative response is accompanied by the generation of lymphoblastic cells (LBCs), which consist of heterologous lymphocyte populations: CD4⁺ helper type of T cells, and CD4^–CD8^– double-negative (DN) lymphocytes, including both CD3⁺ TcR ⁺ T cells and CD2⁺CD3^– immature type of T or non-T cell type of lymphocytes. Almost all the LBCs express Leu19, TfR (transferrin receptor), LFA-1 and CD38 (OKT10) antigens, which are expressed on activated T cells, NK cells and some other lymphocytes. The proliferative response of human PBLs is also accompanied by the generation of potent cytotoxic activity against NK-sensitive and -resistant targets. C-dependent cytolysis and cell sorting experiments of OK-432-activated LBCs revealed that both CD3⁺ and CD3^– types of CD4^–CD8^– DN lymphocytes, but not CD4⁺ helper T cells, may be major populations responsible for the cytotoxicity induced. On the other hand, CD4^–CD8 T cells may be required for the proliferation of PBLs and generation of cytotoxic effector cells. These results suggest that the OK-432 and other streptococcal preparations stimulate the human PBLsin vitro to induce the proliferation/activation of CD4⁺ T cells, mediating the following generation of DN cytotoxic effector lymphocytes.     

Perforin, granzyme B, and FasL expression by peripheral blood T lymphocytes in emphysema

Background

It is generally accepted that emphysematous lungs are characterized by an increase in the numbers of neutrophils, macrophages, and CD8⁺ T lymphocytes, the lasts having increased cytotoxic activity. Because systemic inflammation is also a component of emphysema, we hypothesize that peripheral CD8⁺ T lymphocytes of emphysematous smokers who show evidence of systemic inflammation will have higher expression of cytotoxic molecules.

Methods

We assessed parameters of systemic inflammation in normal individuals (smokers or non-smokers) and in emphysematous subjects with an active smoking history by measuring serum interleukine-6, C-reactive protein, and tumor necrosis factor. Expression of perforin, granzyme B, and FasL protein by CD8⁺ T lymphocytes, CD4⁺ T lymphocytes, and natural killer cells were assessed by flow cytometry while perforin, granzyme B, and FasL mRNA expression were measured on purified systemic CD8⁺ T lymphocytes by real-time PCR.

Results

Emphysematous smokers had higher levels of serum interleukine-6 than normal subjects. Even with the presence of systemic inflammation in emphysematous smokers, the percentage of peripheral CD8⁺ T lymphocytes, CD4⁺ T lymphocytes, and NK cells expressing perforin and granzyme B protein was not different between the three groups.

Conclusion

Despite evidence of systemic inflammation, peripheral T lymphocytes of emphysematous smokers did not show higher levels of cytotoxic markers, suggesting that increase of activated T lymphocytes in the emphysematous lung may be due to either activation in the lung or specific peripheral recruitment.     

外周血Treg细胞、T淋巴细胞及其亚群与早期宫颈癌的关系及对淋巴结转移的预测价值研究

摘要 目的：分析外周血Treg细胞、T淋巴细胞及其亚群与早期宫颈癌的关系及对淋巴结转移的预测价值。方法：选择我院自2017年1月至2020年12月接诊的60例接受子宫颈癌根治术及盆腔淋巴清扫术的早期宫颈癌患者作为观察组,另选同期的60例健康体检者作为对照组。比较两组外周血Treg细胞、T淋巴细胞及其亚群水平,使用受试者工作特征曲线（ROC）下面积（AUC）评价外周血Treg细胞、T淋巴细胞及其亚群对淋巴结转移的预测效能。结果：观察组外周血Treg细胞、CD8⁺T细胞水平高于对照组,CD3⁺T细胞、CD4⁺T细胞、CD4⁺/CD8⁺比值均低于对照组（P＜0.05）;观察组术后外周血Treg细胞、CD8⁺T细胞水平较术前降低,CD3⁺T细胞、CD4⁺T细胞、CD4⁺/CD8⁺比值均较术前升高（P＜0.05）;在60例早期宫颈癌患者中,发生淋巴结转移12例;淋巴结转移组术前外周血Treg细胞水平、CD8⁺T细胞高于非淋巴结转移组,CD3⁺T细胞、CD4⁺T细胞、CD4⁺/CD8⁺比值均低于非淋巴结转移组（P＜0.05）;经多因素Logistic回归分析,外周血Treg细胞、CD3⁺T细胞、CD4⁺/CD8⁺比值均是早期宫颈癌患者发生淋巴结转移的独立预测因素（P＜0.05）;经ROC曲线分析,外周血Treg细胞、CD3⁺T细胞联合CD4⁺/CD8⁺比值预测早期宫颈癌患者发生淋巴结转移的AUC为0.910。结论：外周血Treg细胞、T淋巴细胞及其亚群水平与早期宫颈癌的病情演变有关,其中外周血Treg细胞、CD3⁺T细胞联合CD4⁺/CD8⁺比值预测淋巴结转移的效能较好,值得进一步研究应用。     

Allelic allotype inclusion and exclusion among rabbit Ig-bearing lymphocytes of peripheral blood and lymphoid organs

Mononuclear cells isolated from peripheral blood, appendix, sacculus rotundus, mesenteric lymph nodes, and spleen of b⁴b⁵ heterozygous rabbits were examined for surface Ig allotypes of the b locus. Ig allotype-bearing cells were detected as cells binding erythrocytes or bacteria coated with monospecific anti-b4 or anti-b5 antibody (Ab). Rosetting the cells with Ab-coated erythrocytes indicated that many peripheral blood lymphocytes, but relatively few appendix cells, bore both the b4 and b5 allotypes. Lymphocytes bearing both the b4 and b5 allotypes were also detected by incubating the cells with a mixture of Escherichia coli coated with anti-b4 Ab and Gaffkya tetragena coated with anti-b5 Ab. The percentage of Ig-positive lymphocytes binding both bacteria was 22–31% in the peripheral blood, 4–6% in the appendix, 3–5% in the sacculus rotundus, 4–10% in the mesenteric lymph nodes, and 5% in the spleen. Thus, the percentage of double-bearing lymphocytes was higher in the blood than in the appendix, sacculus rotundus, mesenteric lymph nodes, or spleen. The b4b5-bearing cells in the blood were not cells with adsorbed cytophilic Ab, since these cells still bore both the b4 and b5 allotypes after pronase digestion and Ig regeneration. These double-bearing lymphocytes, i.e., cells exhibiting allelic allotype inclusion, are probably less differentiated cells.     

Expansion of regulatory GITR+CD25low/-CD4+ T cells in systemic lupus erythematosus patients

Introduction

CD4⁺CD25^low/-GITR⁺ T lymphocytes expressing forkhead box protein P3 (FoxP3) and showing regulatory activity have been recently described in healthy donors. The objective of the study was to evaluate the proportion of CD4⁺CD25^low/-GITR⁺ T lymphocytes within CD4⁺ T cells and compare their phenotypic and functional profile with that of CD4⁺CD25^highGITR⁻ T lymphocytes in systemic lupus erythematosus (SLE) patients.

Methods

The percentage of CD4⁺CD25^low/-GITR⁺ cells circulating in the peripheral blood (PB) of 32 patients with SLE and 25 healthy controls was evaluated with flow cytometry. CD4⁺CD25^low/-GITR⁺ cells were isolated with magnetic separation, and their phenotype was compared with that of CD4⁺CD25^highGITR⁻ cells. Regulatory activity of both cell subsets was tested in autologous and heterologous co-cultures after purification through a negative sorting strategy.

Results

Results indicated that CD4⁺CD25^low/-GITR⁺ cells are expanded in the PB of 50% of SLE patients. Expansion was observed only in patients with inactive disease. Phenotypic analysis demonstrated that CD4⁺CD25^low/-GITR⁺ cells display regulatory T-cell (Treg) markers, including FoxP3, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), transforming growth factor-beta (TGF-β), and interleukin (IL)-10. In contrast, CD4⁺CD25^highGITR⁻ cells appear to be activated and express low levels of Treg markers. Functional experiments demonstrated that CD4⁺CD25^low/-GITR⁺ cells exert a higher inhibitory activity against both autologous and heterologous cells as compared with CD4⁺CD25^highGITR⁻ cells. Suppression is independent of cell contact and is mediated by IL-10 and TGF-β.

Conclusions

Phenotypic and functional data demonstrate that in SLE patients, CD4⁺CD25^low/-GITR⁺ cells are fully active Treg cells, possibly representing peripheral Treg (pTreg) that are expanded in patients with inactive disease. These data may suggest a key role of this T-cell subset in the modulation of the abnormal immune response in SLE. Strategies aimed at expanding this Treg subset for therapeutic purpose deserve to be investigated.     

Tissue-specific expression of LeY antigen in high endothelial venules of human lymphoid tissues

In this study, we demonstrated that the anti-Le^Yantibody (BM-1) especially reacted with high endothelial venules (HEVs) in peripheral lymph nodes of blood group O individuals. The Le^Yexpression on HEVs showed a unique tissue-specific pattern, i.e., a large amount of the Le^Yexpression in peripheral lymph nodes and no or small amounts in mesenteric lymph node. Statistical analysis showed that there was the significant difference between the percentage of Le^Y-positive HEVs in peripheral lymph nodes and mesenteric lymph nodes. No expression of Le^Ywas observed in vessels of Payer's patch, thymus, spleen and other non-lymphoid organs. In blood group A or B individuals, the reactivity between HEVs and anti-Le^Yantibody increased after enzyme digestion with -N-acetylgalactosaminidase or -galactosidase. These findings show that the expression of difucosylated blood group ABH antigens are especially expressed on HEVs in peripheral lymph nodes. Furthermore, the tissue-specific pattern suggests that these antigens may be related to intercellular adhesion between lymphocytes and HEVs.     

Lectin binding sites on CD34+ human haematopoietic stem cells and lymphocytes from peripheral blood: an ultrastructural post-embedding study

This study was performed to obtain a better insight into the glycosylation pattern of human CD34⁺ haematopoietic stem cells and lymphocytes from peripheral blood using an ultrastructural post-embedding technique. Lectins applied were derived from Canavalia ensiformis (Con A), Triticum vulgare (WGA), Lycopersicon esculentum (LEA), Limulus polyphemus (LPA), Ulex europaeus-I (UEA-I), Bauhinia purpurea (BPA), Glycine max (SBA), Helix pomatia (HPA), Arachis hypogaea (PNA) and Erythrina cristagalli (ECA). Our results showed almost identical staining patterns with both CD34⁺ cells and mature lymphocytes from peripheral blood. Con A displayed a prominent reactivity with the nuclear envelope and a weak staining of the plasma membrane. As demonstrated by an elaborate lectin double-labelling technique, WGA revealed an opposite staining pattern. Following neuraminidase treatment of sections, BPA, PNA and SBA exhibited a prominent staining of the plasma membrane in CD34⁺ cells and lymphocytes as well. Membrane reactivity with HPA was restricted to the majority of lymphocytes, presumably T-lymphocytes. Infrequently occurring dense cytoplasmic (lysosomal) bodies were reactive with a variety of lectins, and a weak diffuse nuclear labelling was observable with LPA, UEA-I, WGA and Con A. It is tempting to speculate that carbohydrate moieties on plasma membranes may be involved in the complex mechanisms characterizing cell-to-cell interactions (adhesion) and particularly in the so-called phenomenon of homing. This revised version was published online in November 2006 with corrections to the Cover Date.     

Interaction of peanut agglutinin,a lectin specific for nonreducing terminal d-galactosyl residues,with embryonal carcinoma cells

Peanut agglutinin (PNA), a lectin specific for terminal d-galactosyl residues, was found to react with embryonal carcinoma cells, but not with their differentiated derivatives. Receptors for PNA were detectable at the surface of all cells of the quasinullipotent F9 line and on only 50% of the multipotent PCC3/A/1 line. The fraction of the PCC3/A/1 population which expresses the F9 antigen was found to be included in the subpopulation carrying the PNA receptors. PNA⁺ and PNA^? subpopulations of PCC3/A/1 were separated by a PNA-mediated reversible agglutination of PNA⁺ cells with rabbit erythrocytes. These subpopulations were essentially F9⁺ and F9^?, respectively.     

Early postnatal development of the immune system in piglets: the redistribution of T lymphocyte subsets

In this study, we have characterised postnatal changes in T lymphocyte subsets, especially γδ T lymphocytes, in blood, spleen and lymph nodes. Detection was carried out using two-colour flow cytometry and three-colour immunohistochemistry. During ontogeny, there was a significant increase in the total percentage of γδ T cells in the spleen and blood. In the lymph nodes, there were no age-dependent changes in the total percentage of γδ T cells, but the percentage of the γδTCR⁺CD8⁺ subpopulation significantly increased. The tissue distribution of γδTCR⁺CD8⁺ and γδTCR⁺CD8⁻ cells in the lymph nodes is random and not collocated with a particular area of the organ. Furthermore, postnatal development was characterised by an increasing frequency of CD8⁺CD3⁺CD4⁻γδTCR⁻, which was compensated by a decreasing proportion of CD4⁺ lymphocytes. Double positive CD4⁺CD8⁺ lymphocytes were rare during the first month of life and a significant age-dependent increase of these cells was found in all the compartments monitored.     

T cell recruitment from the thymus to the spleen in tumor-bearing mice

Summary After inoculation of tumor cells (methylcholanthrene-induced sarcoma), the number of Thy 1⁺ cells and PNA (peanut agglutinin) binding cells, which were shown to be different subpopulations were increased in the spleen of thymus-intact mice, in contrast this increase was not observed in adult thymectomized mice. In experiments performed concurrently with splenic cell analysis, we found that the plasma PGE₂ levels declined in parallel with the tumor growth. Prevention of such a decline of plasma PGE₂ level by replenishment with exogenous PGE₂ inhibited the splenic cell increase in tumor bearers. In the tumorbearing mice, cell traffic systems from the thymus to the periphery was ascertained by injecting fluorescein diacetate (FDA) into the thymus and observing fluorescein positive cells in the periphery. We suggest that increased recruitment of thymic cells to the periphery may be mediated by PGE₂ in the presence of a tumor.Abbreviations PNA⁺ cells, peanut agglutinin binding cells - Ig⁺ cells, surface IgM positive cells - Thy 1⁺ Thy 1.2 antigen positive cells - PGE₂ prostaglandin E₂     

Post-natal ontogenesis of graft-versus-host reactivity of peanut agglutinin lectin-negative thymocytes in the chicken

Chicken thymocyte fractionation by peanut agglutinin lectin yields two cell subpopulations which differ in their GvH competence when injected into major histocompatibility complex (MHC)-(B locus) incompatible embryos. The nonagglutinated PNA^? fraction displayed a degree of alloreactivity similar to that of peripheral blood lymphocytes while the PNA⁺ cells were nonreactive. Analysis of chickens at the age of 4 to 24 weeks showed that the development of GvH-reactive PBL preceded by 12 weeks the maturation of GvH-reactive PNA^? thymocytes. Possible interpretations for this difference between the time of appearance of GvH reactivity of PNA^? thymocytes and PBL are discussed.     

Tumor-infiltrating CD4<Superscript>+</Superscript> T cells in patients with gastric cancer

Background

T lymphocytes play an indispensably important role in clearing virus and tumor antigen. There is little knowledge about impacts of inhibitory molecules with cytokine on tumor-infiltrating CD4⁺ T-cells in the presence of gastric cancer (GC). This study investigated the distribution of tumor-infiltrating T-cells subset and the differentiation as well as inhibitory phenotype of T-cells from blood and tissues of GC patients.

Materials and methods

Patients with GC diagnosed on the basis of pre-operative staging and laparotomy findings were approached for enrollment between 2014 and 2015 at the Affiliated Cancer Hospital of Zhengzhou University, China. Phenotypic analysis based on isolation of tumor-infiltrating lymphocytes and intracellular IFN-γ staining assay is conducted. Statistical analysis is performed to show significance.

Results

The results showed that the percentage of CD4⁺ T-cells among CD3⁺ cells in tumors was significantly higher than that in the matched paraneoplastic tissue. CD4⁺ CD25^high CD127^low regulatory T-cells (Tregs), PD-1⁺, Tim-3⁺, and PD-1⁺ Tim-3⁺ cells were up-regulated on tumor infiltrating T-cells from patients with GC compared to their expressions on corresponding peripheral blood and peritumoral T-cells. Blockades of PD-1⁺ and Tim-3⁺ were effective in restoring tumor infiltrating T-cells’ production of interferon-gamma (IFN-γ). Combined PD-1⁺ and Tim-3⁺ inhibition had a synergistic effect on IFN-γ secretion by CD4⁺ T-cells.

Conclusion

The results suggested that the composition, inhibitors, and location of the immune infiltrate should be considered when evaluating antitumor immunotherapy. A new insight into the mechanisms underlying T cell dysfunction is provided.

     

Interaction of HMG proteins and H1 with hybrid PNA–DNA junctions

The objective of this study was to evaluate the effects of inserting peptide nucleic acid (PNA) sequences into the protein‐binding surface of an immobilized four‐way junction (4WJ). Here we compare the classic immobile DNA junction, J1, with two PNA containing hybrid junctions (4WJ‐PNA₁ and 4WJ‐PNA₃). The protein interactions of each 4WJ were evaluated using recombinant high mobility group proteins from rat (HMGB1b and HMGB1b/R26A) and human histone H1. In vitro studies show that both HMG and H1 proteins display high binding affinity toward 4WJ's. A 4WJ can access different conformations depending on ionic environment, most simply interpreted by a two‐state equilibrium between: (i) an open‐x state favored by absence of Mg²⁺, low salt, and protein binding, and (ii) a compact stacked‐x state favored by Mg²⁺. 4WJ‐PNA₃, like J1, shifts readily from an open to stacked conformation in the presence of Mg⁺², while 4WJ‐PNA₁ does not. Circular dichroism spectra indicate that HMGB1b recognizes each of the hybrid junctions. H1, however, displays a strong preference for J1 relative to the hybrids. More extensive binding analysis revealed that HMGB1b binds J1 and 4WJ‐PNA₃ with nearly identical affinity (K_Ds) and 4WJ‐PNA₁ with two‐fold lower affinity. Thus both the sequence/location of the PNA sequence and the protein determine the structural and protein recognition properties of 4WJs.     

Splenic lymphocytopoiesis and migration pattern of splenic lymphocytes

The spleens of young pigs were selectively labeled with tritiated thymidine ([³H]-TdR) and the relative and absolute numbers of labeled lymphocytes found 24 hr later in different lymphoid and nonlymphoid organs were determined autoradiographically. It was deduced that about 4.6 × 10⁹ lymphocytes (that is, about 15% of all splenic lymphocytes) are produced by the spleen per day and about 17% of the newly formed lymphocytes leave the spleen within the first day of labeling. Spleen-derived lymphocytes could be found in relatively high numbers in the lymph nodes, blood, gut-associated lymphoid tissues, and, surprisingly, in the bone marrow, whereas the concentration in the thymus was very low. In a second series, pigs were labeled with [³H]TdR and only the spleen was excluded from labeling. The labeling index of splenic small lymphocytes was about 10% 1 day later, indicating a high rate of influx of newly formed lymphocytes into the pig spleen. The spleen of the young pig is an important lymphocytopoietic organ and exports and imports newly formed lymphocytes at high rates.     

Effects of dietary conjugated linoleic acids on cellular immune response of piglets after cyclosporin A injection

The present study investigated the effects of dietary conjugated linoleic acid (CLA) on the cellular immune response of piglets after cyclosporin A (CsA) treatment. The experimental study had a 2×2 factorial design, and the main factors consisted of diets (0% or 2% CLA) and immunosuppression treatments (CsA or saline injection). CsA injection significantly increased feed : gain (F : G) of piglets (P<0.05); however, dietary CLA significantly decreased F : G of piglets (P<0.05). Dietary CLA partly ameliorated the deterioration of the feed conversion rate caused by CsA treatment (P<0.01). CsA treatment significantly decreased the percentages of CD4⁺ and CD8⁺ T lymphocytes in the thymus (P<0.01). Dietary CLA increased the percentages of CD4⁺ CD8⁺ double-positive and CD8⁺ single-positive T lymphocytes in the thymus (P<0.05), and had the trend to inhibit the decrease of CD4⁺ T lymphocytes in the thymus after CsA injection (P=0.07). CsA treatment significantly depleted the peripheral blood CD3⁺, CD4⁺ and CD8⁺ T lymphocytes (P<0.01). Dietary CLA significantly increased the number of peripheral blood CD8⁺ T lymphocytes and interleukin-2 (IL-2) production (P<0.05), and inhibited the decreases of peripheral blood CD3⁺, CD4⁺ and CD8⁺ T lymphocytes counts (P<0.01) as well as IL-2 production (P<0.05) after CsA treatment. Dietary CLA partly rescued the decrease of lymphocyte proliferation after CsA injection (P<0.05). In summary, dietary CLA effectively ameliorated CsA-induced cellular immunosuppression in piglets.