The Investigation on the Value of Repeat and Combination Test of ACA and Anti‐β2‐GPI Antibody in Women with Recurrent Spontaneous Abortion

Problem In order to investigate the value of anticardiolipin antibodies (ACA) and anti‐β₂‐GPI antibodies detection in screening autoimmune type recurrent spontaneous abortion and its clinic application in antiphospholipid syndrome diagnosis, we adopt repeat combined ACA and anti‐β₂‐GPI antibodies detection in this study. Method of study Sera were collected from patients and work‐up was done for detection of ACA and anti‐β₂‐GPI antibodies by enzyme‐linked immunosorbent assay (ELISA). The work‐up was done for detection of antibodies once in every 6 weeks for 14 times consecutively. Results The repeated and combined detection of ACA and anti‐β₂‐GPI antibodies detection could raise the positivity rate up to 21.8% (P < 0.05) in comparison with positive for ACA alone (14.1%), positive for anti‐β₂‐GPI alone (3.1%), and concurrently positive for both ACA and anti‐β₂‐GPI antibodies (4.6%). In 91 confirmed positive antiphospholipid antibodies (APA) patients, with more frequent screening for ACA and anti‐β₂‐GPI antibodies, more patients with APA were found. The positive rate of five and more screenings was over 81.32%, which was statistically significant (P < 0.05), in comparison with that of four or less screenings (68.13%). Conclusion Our data implied that it would be appropriate to take over five or more screenings of combined ACA and anti‐β₂‐GPI antibodies detection in suspect patients to facilitate the positive diagnostic rate for autoimmune type RSA.     

Correlation Between β2-Glycoprotein Antibodies and Antiphospholipid Antibodies in Patients with Reproductive Failure

PROBLEM: Antiphospholipid antibodies (APAs) are important in the etiology of reproductive failure. Studies have shown that binding proteins are necessary for the detection of APAs. One of these, β₂-glycoprotein, has been shown to be necessary for detection of anticardiolipin antibodies. It is felt that some APAs may be directed to the binding protein itself, or to a combination of the binding protein and phospholipid. METHOD OF STUDY: In this study, a comparison of APAs vs. anti β₂-glycoprotein antibodies was performed on the sera of 123 women younger than 40 years of age with a history of reproductive failure. Antibodies to six phospholipid epitopes, cardiolipin, phosphatidyle-thanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, and phosphatidylserine, were measured. RESULTS: Of the 123 women tested, 33 had one or more positive immunoglobulin (Ig)G antibodies to phospholipids, of which 9 were to cardiolipin. However, only 1 of 123 women had IgG antibodies to β₂-glycoprotein and she was APA negative. Thirty-eight of 123 women had one or more IgM antibodies to phospholipids, with none directed to cardiolipin IgM. In contrast, only 8 of the 123 women had IgM antibodies to β₂-glycoprotein. Five of the eight patients had IgM APA; 4 of 5 had IgM antibodies to PE, 1 to PI. CONCLUSIONS: There is no correlation between β₂-glycoprotein antibodies and APA status in this population. To date, our most sensitive test for detecting phospholipid autoimmune-mediated in vitro fertilization failure still appears to be the ELISA assay for APA.     

αβ Lineage-committed thymocytes can be rescued by the γδ T cell receptor (TCR) in the absence of TCR β chain

Commitment of the αβ and γδ T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCRγδ-transgenic or TCRβ knockout mice, both of which are unable to generate TCRαβ-positive T cells, develop phenotypically αβ-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCRβ protein, the γδTCR can promote the development of αβ-like thymocytes, which, however, do not expand significantly and do not mature into γδ T cells. These results show that commitment to the αβ lineage can be determined independently of the isotype of the TCR, and suggest that αβ versus γδ T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCRγ and δ gene rearrangements on αβ T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.     

Deletion of the α2A/α2C‐adrenoceptors accelerates cutaneous wound healing in mice

The α2‐adrenoceptors regulate the sympathetic nervous system, controlling presynaptic catecholamine release. However, the role of the α2‐adrenoceptors in cutaneous wound healing is poorly understood. Mice lacking both the α2A/α2C‐adrenoceptors were used to evaluate the participation of the α2‐adrenoceptor during cutaneous wound healing. A full‐thickness excisional lesion was performed on the dorsal skin of the α2A/α2C‐adrenoceptor knockout and wild‐type mice. Seven or fourteen days later, the animals were euthanized and the lesions were formalin‐fixed and paraffin‐embedded or frozen. Murine skin fibroblasts were also isolated from α2A/α2C‐adrenoceptor knockout and wild‐type mice, and fibroblast activity was evaluated. The in vivo study demonstrated that α2A/α2C‐adrenoceptor depletion accelerated wound contraction and re‐epithelialization. A reduction in the number of neutrophils and macrophages was observed in the α2A/α2C‐adrenoceptor knockout mice compared with wild‐type mice. In addition, α2A/α2C‐adrenoceptor depletion enhanced the levels of nitrite and hydroxyproline, and the protein expression of transforming growth factor‐β and vascular endothelial growth factor. Furthermore, α2A/α2C‐adrenoceptor depletion accelerated blood vessel formation and myofibroblast differentiation. The in vitro study demonstrated that skin fibroblasts isolated from α2A/α2C‐adrenoceptor knockout mice exhibited enhanced cell migration, α‐smooth muscle actin _protein expression and collagen deposition compared with wild‐type skin fibroblasts. In conclusion, α2A/α2C‐adrenoceptor deletion accelerates cutaneous wound healing in mice.     

Murine T Cell Determination of Pregnancy Outcome: I. Effects of Strain, αβ T Cell Receptor, γδ T Cell Receptor,and γδ T Cell Subsets

PROBLEM: T cells bearing αβ T cell receptor (TcR) and γδ TcR are present at the fetomaternal interface, and the latter, which express surface activation markers, can react with fetal trophoblast cell antigens. What is the role of these cells? METHOD: Using stress-abortion-prone DBA/2-mated CBA/J and abortion-resistant C57/B16 mice, αβ, γδ, and CD8^+/- T cell subsets were measured in spleen and uterine decidua. The effect of immunization against abortion and administration of anti-TcR antibody in vivo was examined. Cytokine synthesis was measured by intracellular staining of Brefeldin A-treated cells. RESULTS: Abortion-prone matings showed an unexpected accumulation of γδ T cells beginning in the peri-implantation period and this was suppressed by immunization against abortion. The immunization deleted γδ T cells producing the abortogenic cytokines, TNF-α and γ-interferon, and increased production of the anti-abortive cytokines, IL-10 and transforming growth factor-β2 (TGF-β2). Immunization also boosted the number of αβ T cells which were present in the decidua as early as 2 days after implantation. In vivo injection of GL4 (anti-δ) depleted γδ T cells producing Th1 cytokines in the peri-implantation period, and prevented abortions, whereas H57 (anti-β) decreased the number of αβ T cells and led to 100% abortions. CD8⁺ T cells present in peri-implant decidua before onset of abortions were mostly αβ TcR⁺, although some were γδ⁺. Changes in γδ and αβ T cells in pregnancy were most dramatic in uterine tissue. CONCLUSION: Although decidual γδ T cells after formation of a distinct placenta and fetus produce anti-abortive TGF-β2-like molecules and IL-10, prior events can lead to abortion. High local production of TNF-α and γ-interferon develop during the peri-implantation phase because of an excessive increase in the Th1 cytokine⁺ subset of γδ cells; these cytokines may be contributed by other tissues in decidua, and the contribution of bioactive factors by γδ T cells may augment the cytokine pool. In contrast, αβ T cells (which may be inactivated by stress that causes abortions) may mediate the anti-abortive effect of alloimmunization. Alloimmunization involves a shift from a Th1 to a Th2 pattern in the γδ T cells in decidua.     

In vivo application of mAb directed against the γδ TCR does not deplete but generates “invisible” γδ T cells

mAb targeting the γδ TCR have been used for γδ T‐cell depletion with varying success. Although the depletion‐capacity of the anti‐γδ TCR mAb clone GL3 has been disputed repeatedly, many groups continue to use γδ T‐cell depletion protocols involving the mAb clone UC7‐13D5 and find significant biological effects. We show here that treatment with both GL3 and UC7‐13D5 antibodies does not deplete γδ T cells in vivo, but rather leads to TCR internalization and thereby generates “invisible” γδ T cells. We addressed this issue using anti‐γδ TCR mAb injections into WT mice as well as into reporter TCR delta locus‐histone 2B enhanced GFP knock‐in mice, in which γδ T cells can be detected based on an intrinsic green fluorescence. Importantly, the use of TCR delta locus‐histone 2B enhanced GFP mice provided here for the first time direct evidence that the “depleted” γδ T cells were actually still present. Our results show further that GL3 and UC7‐13D5 mAb are in part cross‐competing for the same epitope. Assessed by activation markers, we observed in vitro and in vivo activation of γδ T cells through mAb. We conclude that γδ T‐cell depletion experiments must be evaluated with caution and discuss the implications for future studies on the physiological functions of γδ T cells.     

Human γδ T Cells Express a Higher TCR/CD3 Complex Density Than αβ T Cells

The aim of our study was to compare CD3 expression on γδ T cells and αβ T cells in human patients. The antigen density of TCR and CD3 on both subsets was assessed by a quantitative method in eight patients. In parallel, we developed and validated a reliable direct tricolor staining protocol that we tested on samples from hospitalized and healthy individuals (n = 60). Our results demonstrate that human γδ T cells constitutively express approximately twofold more of the TCR/CD3 complex than αβ T cells. We suggest that this enhanced expression of the TCR/CD3 complex could contribute to the higher reactivity of γδ T cells compared to αβ T cells. These clinical laboratory results confirm the fundamental data described elsewhere. γδ T cells deserve further clinical investigations to understand their precise role in human immunity.     

γδ T cells are secondary participants in acute graft-versus-host reactions initiated by CD4+ αβT cells

To examine the role of T cell subpopulations in an acute graft-versus-host (GVH) reaction, γδ T cells and αβ T cells expressing one of the two prototypic Vβ gene families were negatively isolated from adult blood samples and injected into allogeneic chick embryos. CD4⁺ αβ T cells expressing either Vβ1 or Vβ2 receptors were equally capable of inducing acute GVH reactions, consistent with the idea that αβ T cell alloreactivity is determined by CDR3 variability. By themselves, the γδ T cells were incapable of inducing GVH reactions. However, host γδ T cells were recruited into the donor αβ T cell-initiated lesions, where they were activated and induced to proliferate. The data suggest that γβ T cells may play a secondary role in GVH reactions.     

Expression of pro-inflammatory cytokines by TCRαβ+ T and TCRγδ+ T cells in an experimental model of colitis

An inflammatory bowel disease (IBD) comparable to human ulcerative colitis is induced upon transfer of T cell-depleted wild-type (F1) bone marrow into syngeneic T cell-deficient (tgε26) mice (F1 → tgε26). Previously we have shown that activated CD4⁺ T cells predominate in transplanted tgε26 mice, and adoptive transfer experiments verified the potential of these cells to cause disease in immunodeficient recipient mice. Using flow cytometry for the detection of intracellular cytokine expression, we demonstrate in the present study that large numbers of CD4⁺ and CD8⁺ TCRαβ⁺ T cells from the intraepithelial region and lamina propria of the colon of diseased, but not from disease-free mice, produced interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Large numbers of T cells from peripheral lymphoid tissues of these animals also expressed IFN-α and TNF-α, but few expressed interleukin-4, demonstrating g strong bias towards Th1-type T cell responses in these animals. TCRγδ⁺ T cells, typically minor constituents of the inflammatory infiltrate of the colon in F1 → tgε26 mice, also expressed IFN-γ at a high frequency upon CD3 stimulation. In light of these findings we examined the potential involvement of TCRγδ⁺ T cells by testing their ability to induce colitis in tgε26 mice. We report here that tgε26 mice transplanted with T cell-depleted bone marrow from TCRα^null and TCRβ^null animals developed IBD. Furthermore, disease in these mice correlated with the development of peripheral and colonic TCRαδ⁺ T cells capable of IFN-γ production. These results suggest that IFN-γ may be a common mediator of IBD utilized by pathogenic T cells of distinct phenotype.     

Phosphoantigen activation induces surface translocation of intracellular CD94/NKG2A class I receptor on CD94− peripheral Vγ9 Vδ2 T cells but not on CD94− thymic or mature γ δ T cell clones

Most adult peripheral blood γ δ T cells express Vγ9/Vδ2-encoded TCR that recognize a restricted set of nonpeptidic phosphorylated compounds, referred to as phosphoantigens. They also express various MHC class I-specific inhibitory receptors (IR), in particular CD94/NKG2-A heterodimers, which participate in the fine tuning of their TCR-mediated activation threshold. Most mature Vγ9/Vδ2 T cells express surface CD94 receptors, unlike cord blood or thymus-derived Vγ9/Vδ2 clones, thus suggesting a role for the microenvironment in IR expression. In the present study we show that most CD94⁻ Vγ9Vδ2 PBL ex vivo express an intracellular pool of CD94/NKG2-A receptors that is translocated to the cell surface upon activation by phosphoantigens or IL-2. In stark contrast, intracellular CD94/NKG2-A complexes are undetectable in CD94⁻ thymus or PBL-derived mature Vδ2 T cell clones, and no surface induction is observed following phosphoantigen activation of T cell clones. Altogether these results provide new insights into the regulation of CD94/NKG2-A expression on T lymphocytes and suggest the existence of distinct mechanisms controlling in vivo and in vitro induction of IR on these cells.     

Changes in thymic export of γδ and αβ T cells during fetal and postnatal development

The thymus plays an essential role in the generation and selection of T cells and exports approximately 0.5–1% of thymocytes per day in young animals and considerably fewer in older animals. To date there have been no studies directly examining fetal thymic export in any species. Using the technique of intrathymic injection of fluorescein isothiocyanate, followed by an assay for green fluorescent cells in the periphery and for the expression of cell surface antigens on these cells, we have compared directly the export of T cells from the fetal and postnatal ovine thymus. While the thymus exports both αβ and γδ T cells, our results demonstrate that the proportion of thymic γδ T cells that are exported per day is much higher than that of thymic αβ T cells. Moreover, the export rate of γδ T cells increased from approximately 1 in every 60 γδ thymocytes per day emigrating from the fetal thymus to 1 in every 20 from the postnatal thymus. In addition, we identify a population of CD5⁺CD4^?CD8^?γδ^? T cells emigrating from the fetal thymus but greatly reduced among thymic emigrants after birth. These findings have several implications regarding the mechanisms and control of selection of both γδ and αβ T cells.     

The role of γδ T cells in priming macrophages to produce tumor necrosis factor-α

The secretion of tumor necrosis factor (TNF)-α from macrophages is regulated by both priming and triggering signals. We found that macrophages from mice lacking γδ T cells [T cell receptor (TCR) δ^?/- mice], which lack the gene encoding the δ chain, produced only small amounts of TNF-α in response to lipopolysaccharide (LPS) and showed a reduced level of expression of CD14. Pre-incubation of macrophages from TCR δ-/- mice with γδ T cells from their TCR δ^+/- littermates restored their capacity to produce TNF-α in response to LPS. The priming activity of γδ T cells was in part inhibited by neutralizing anti-interferon (IFN)-γ monoclonal antibodies. Collectively, these results suggest that γδ T cells play a role in priming macrophages to a steady state of activation via IFN-γ secretion, which allows them to produce TNF-α when exposed to LPS.     

β2-adrenergic control of plasma volume in hemorrhage

Hemorrhage is associated with absorption of extravascular fluid from skeletal muscle to blood in order to compensate for the loss of intravascular volume. Our previous studies have shown that this fluid gain is mainly linked to β-adrenergic microvascular adjustments leading to decrease in capillary hydrostatic pressure and to precapillary ‘sphincter’ mediated increase in the capillary surface area available for fluid exchange. In the present study the importance of β-adrenergic control of plasma volume in bleeding was confirmed by measurement of changes in plasma volume after graded hemorrhage in animals with intact and blocked vascular β₂-adrenoceptors (i. v. administration of the ‘selective’β₂-blocking agent ICI 118, 551). With intact β₂-adrenoceptors plasma volume was gradually restored after bleeding so that about 50% of the shed plasma volume (about 35% of the shed blood volume) had been compensated for at two hours after exsanguination of 20% as well as 40% of the blood volume. The corresponding figures in animals with blocked β₂-adrenoceptors were only 14% of the shed plasma volume and 8% of the shed blood volume at both degrees of hemorrhage.     

Evidence that productive rearrangements of TCR γ genes influence the commitment of progenitor cells to differentiate into αβ or γδ T cells

Two models have been considered to account for the differentiation of γδ and αβ T cells from a common hematopoietic progenitor cell. In one model, progenitor cells commit to a lineage before T cell receptor (TCR) rearrangement occurs. In the other model, progenitor cells first undergo rearrangement of TCRγ, δ, or both genes, and cells that succeed in generating a functional receptor commit to the γδ lineage, while those that do not proceed to attempt complete β and subsequently α gene rearrangements. A prediction of the latter model is that TCRγ rearrangements present in αβ T cells will be nonproductive. We tested this hypothesis by examining Vγ2-Jγ1Cγ1 rearrangements, which are commonly found in αβ T cells. The results indicate that Vγ2-Jγ1Cγ1 rearrangements in purified αβ T cell populations are almost all nonproductive. The low frequency of productive rearrangements of Vγ2 in αβ T cells is apparently not due to a property of the rearrangement machinery, because a transgenic rearrangement substrate, in which the Vγ2 gene harbored a frame-shift mutation that prevents expression at the protein level, was often rearranged in a productive configuration in αβ T cells. The results suggest that progenitor cells which undergo productive rearrangement of their endogenous Vγ2 gene are selectively excluded from the αβ T cell lineage.