Maternal and Fetal Circulating Levels of Thymosin α1 During Parturition

ABSTRACT: The thymus directs T-lymphocyte development and contributes to the maintenance of immune homeostasis, in part, through its production of peptides known as thymosins. In pregnancy, maternal serum levels of thymosin α₁ have been reported to be low at midgestation and to increase by term, suggesting that maternal levels represent fetal levels. To evaluate this further, we obtained maternal venous and newborn mixed cord blood from 90 pregnancies between 20 and 42 weeks of gestation at delivery. An ELISA was used for thymosin α assay, and analysis was by paired t test and regression. Maternal and newborn levels were independent of gestational age, but an apparent association (r = 0.51) between the two was inconclusive. Maternal levels (1,207 ± 947 pg/ml) tended to be higher than those of healthy adults (1,043 ± 576 pg/ml). Mixed umbilical cord serum levels (1,466 ± 940 pg/ml) were higher than maternal levels (P ≤ 0.005). Although maternal thymosin α levels may reflect fetal levels, immunological perturbations related to parturition appear to influence both.     

GROα in the Fetomaternal and Amniotic Fluid Compartments During Pregnancy and Parturition

PROBLEM: The use of monoclonal antibodies for CD45RA and CD45RO antigens is important in defining maturational and functional stages on lymphocytes. METHOD: To characterize distribution of two isoforms of CD45 antigen CD45RA and CD45RO on CD3⁺, CD4⁺, CD8⁺ and CD56⁺ lymphocyte subsets from first trimester human decidua, two-color flow cytometry were used. RESULTS: In decidua, there were much higher levels of CD45RO^a+ and much lower levels of CD45RA⁺ cells among CD3⁺, CD4⁺, and CD8⁺ cells as compared with peripheral blood samples of the same pregnant women. Only ?40% of CD56⁺ cells in decidua expressed CD45RA. Unlike peripheral blood, ?30% of decidual natural killer (NK) cells weakly stained with anti-CD45RO antibodies. Double-negative CD45RA^? CD45RO^? NK cells were also present in decidua. CONCLUSIONS: The significantly raised percentage of intradecidual T cells expressing CD45RO suggest decidual accumulation of antigen-committed memory cells. The patterns of CD45 isoforms expression on decidual CD56⁺ cells are consistent with hypothesis that uterine CD56⁺ lymphocytes are terminally differentiated cells of NK lineage. PROBLEM: GROα/MGSA is a new member of the chemokine superfamily CXC(α) and is produced by a variety of cells including macrophages, fibroblasts, epithelial, and endothelial cells, and keratinocytes. This chemokine has chemoattractant activity and may participate in neutrophil recruitment and activation during the course of intrauterine infection. This study was conducted to investigate the effect of labor and microbial invasion of the amniotic cavity (MIAC) on amniotic fluid, fetal, and maternal plasma GROα concentrations. METHOD: A cross-sectional study was designed using parameters that included gestational age, results of amniotic fluid (AF) cultures, and labor status at the time of amniocentesis. Fluid was retrieved by transabdominal amniocentesis. MIAC was defined as a positive amniotic fluid culture for bacteria. Umbilical cord blood was retrieved at the time of delivery. Amniotic fluid, maternal and fetal plasma GROα concentrations were measured with a sensitive and specific ELISA (Quantikine, R&D Systems, Minneapolis, MN). RESULTS: 1) GROα was detectable in amniotic fluid, umbilical cord, and maternal plasma samples; 2) GROα concentrations in amniotic fluid increased with advancing gestational age; 3) Both term and preterm gestations with MIAC were associated with higher amniotic fluid GROα concentrations than those with sterile amniotic fluid, independent of the labor status (term, MIAC, labor: median 2.7 ng/ml, range 1.4–12.7 vs. term, no MIAC, labor: median 2.1 ng/ml, range 0.7-3.4, vs term, no MIAC, no labor: median 1.9 ng/ml, range 1.8-4.2; P <0.005; preterm: MIAC median 5 ng/ml, range 0.6–47.9 vs. no MIAC: median 2.3 ng/ml, range 0.5–10; P <0.008); 4) A strong correlation was found between umbilical cord plasma GROα concentrations and neonatal neutrophil count, and between GROα concentrations and white blood cell count in the amniotic fluid (r = 0.67, P < 0.0005 and r ? 0.38, P < 0.001, respectively). CONCLUSION: GROα is a physiologic constituent of amniotic fluid and cord blood. Amniotic fluid GROα concentrations increase with gestational age. Intrauterine infection both preterm and at term is associated with an increase in GROα concentrations of amniotic fluid, suggesting that GROα may play an important role in recruitment of neutrophils into the amniotic cavity.     

Low Periconceptional Maternal Serum Thymosin α1 Levels Are Associated With Blighted Pregnancies

OBJECTIVE: The thymus-derived peptides, thymosin α₁ and thymosin (β₄, are believed to contribute to the maintenance of immune homeostasis. They are also associated with the hypothalamic-pituitary-adrenal-gonadal axis and may play a role in reproduction. STUDY DESIGN: Patients were recruited from a university hospital setting. Eligible candidates were 24 to 38 years old who were being seen in an ovulation induction and in vitro fertilization program. Serial maternal serum thymosin α₁ and β₄ levels were assayed preconceptual and then twice in the first trimester by ELISA in 28 women with known ovulation dates who successfully conceived as demonstrated by positive serum β human chorionic gonadotropin (hCG). Thymosin α₁ and β₄ serum levels for viable pregnancies (group I; N = 19) were compared to pregnancies that aborted (group II; N = 9) using repeated measures of multivariate analysis of variance (MANOVA). Periconceptional (preovulatory and early pregnancy) thymosin α₁ and β₄ values between groups I and II were compared using repeated measure one-way ANOVA. RESULTS: Thymosin α₁ levels from pregnancies that remained viable were significantly higher than those from pregnancies that spontaneously aborted. Preovulation thymosin α₁ levels also tended to be lower in pregnancies that subsequently aborted. Thymosin β₄ levels were similar between the two groups. CONCLUSIONS: Decreased maternal serum thymosin α₁ levels may be associated with periconceptional endocrine and/or immune disturbances preceding miscarriage.     

Molecular characterization and clinical presentation of HKαα and anti‐HKαα alleles in southern Chinese subjects

The HKαα allele is a rearrangement occurring in the α‐globin gene cluster containing both the ‐α^3.7 and ααα^anti4.2 unequal crossover junctions. The anti‐HKαα allele is the reciprocal product containing both the ‐α^4.2 and ααα^anti3.7 unequal crossover junctions, which had been predicted but had not been detected previously. The phenotypic feature and population frequency of these two unusual alleles were not described. We report the identification of nine individuals carrying the HKαα allele and two individuals carrying the anti‐HKαα allele in southern China and describe their phenotype and haplotype data. The molecular structures of HKαα allele and anti‐HKαα allele were confirmed by two‐round nested polymerase chain reaction assay. The mechanism of origin of both alleles is related to probably simultaneous double crossover. Heterozygotes of HKαα or anti‐HKαα allele show a normal hematological phenotype. Finally, we report the carrier rates of these both alleles in the Guangxi Zhuang Autonomous Region of southern China, namely, ∼0.07% for the HKαα allele and ∼0.02% for the anti‐HKαα allele.     

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.     

Expression of HNF-1α and HNF-1β in various histological differentiations of hepatocellular carcinoma

Hepatic nuclear factor 1 (HNF-1) regulates genes in a hepatocyte-specific manner. It has been previously reported that the ratio of HNF-1α and HNF-1β mRNA is related to histological differentiation hepatocellular carcinoma (HCC). In this study, the expression levels of the HNF-1α and HNF-1β proteins were analysed relatively and quantitatively in various histologically differentiated HCC and surrounding non-cancerous tissues, and HNF-1α binding activity for the AT element of the B domain of the human α-fetoprotein enhancer was examined. Western blot analysis demonstrated that HNF-1α protein was expressed at a higher level in well-differentiated HCC tissues than in the surrounding non-HCC tissues; on the other hand, the HNF-1α protein was expressed at lower levels in moderately and poorly differentiated HCCs than in the surrounding non-HCC tissues. The levels of HNF-1β expression in well-differentiated and poorly differentiated HCCs were similar to and higher than those found in the respective surrounding non-cancerous portions. In binding assays, HNF-1 binding activity was high in well-differentiated HCC and lower in moderately and poorly differentiated HCCs. Most well-differentiated HCC cases showed immunohistochemical expression of HNF-1α. These findings show that poor histological differentiation of HCC correlates with decreases in the level and activity of HNF-1α proteins. © 1998 John Wiley & Sons, Ltd.     

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.     

α-thalassaemia due to a single codon deletion in the α-1-globin gene. Computational structural analysis of the new α-chain variant

A new unstable α-globin chain associated with α-thalassemia phenotype has been found in a Spanish patient. Molecular analysis of the α-globin gene complex using PCR and non-radioactive single-strand conformation analysis, allowed to identify a new mutation in the second exon of the α-globin gene. Direct sequencing of the abnormal fragment revealed a 3 bp deletion, which led to the loss of a single codon corresponding to a Lys (K) residue at position 60 or 61 DK60 or DK61. Theoretical structural analysis, performed by computational methods, indicated that the loss of an amino acid residue at this position disturbed the contact region between the B and E-helices, affecting the overall stability of the molecule. Therefore, the DK60 or DK61 results in a structurally abnormal α-globin chain, not previously described, named Hb Clinic, which leads to the α-thalassemia phenotype in the heterozygote patient. No abnormal hemoglobin was detected by standard electrophoretic procedures, suggesting that this α-globin chain variant is so unstable that it may be catabolized immediately after its synthesis. This mutation was confirmed by PCR using an allele specific primer. Hum Mutat 11:412, 1998. © 1998 Wiley-Liss, Inc.     

Postjunctional α1- and α2-adrenoceptors mediating contraction in isolated human groin arteries and veins

By means of selective agonists and antagonists for α₁- and α₂-receptors, the α-receptor subtypes in human groin arteries and veins were characterized and compared. In the arteries the α₁-receptor blocker prazosin caused a concentration-dependent parallel displacement of the noradrenaline (NA) concentration-response (cr) curve without reduction of maximum (pA₂=9.86); the selective α₂-receptor antagonist rauwolscine in the concentration 10^-8 M caused a right-ward shift of the NA cr-curve without reduction of E_max, but 10^-7 M and 10^-6 M caused little or no further shift. In the veins, the two antagonists had the opposite effects. Rauwolscine caused a concentration-dependent right-ward shift of the NA cr-curve without depression of maximum (pA₂=9.03); prazosin 10^-9 M significantly displaced the NA cr-curve, whereas 10^-8 M and 10^-7 M caused little or no further shift. The responses to the α₂-receptor agonist clonidine in the arteries were too small to allow calculations of pEC50 values; in the veins contractions were elicited in all vessel segments investigated (pEC₅₀=6.24). Phenylephrine, selective for α₁-receptors, was significantly more potent in arteries than in veins. NA was significantly more potent in veins than in arteries. It is concluded that in human groin vessels, there is a functional predominance of arreceptors in the arteries and of a2-receptors in the veins.     

High frequency of CD8αα homodimer-bearing T cells in human fetal intestine

Immunohistochemistry on frozen sections was used to identify CD8αα cells and CD8αβ cells in human intestine. As observed previously, CD8αβ cells predominate (>95%) in tonsil and post-natal intestine. However in human fetal intestine (16–24 weeks gestation), almost half the CD8⁺ cells in the lamina propria are CD8αα, and many CD8αα cells can be identified in the epithelium. In contrast, in the T cell zones of the Peyer's patches, CD8αβ cells are dominant. The CD8αα cells are virtually all αβ T cell receptor positive. By analogy with the murine system, these CD8αα cells in the fetal gut may be directly derived from the marrow, undergoing thymus-independent differentiation in the gut mucosa.     

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.     

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.     

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.     

Distribution of α4β7 and αEβ7 integrins on thymocytes,intestinal epithelial lymphocytes and peripheral lymphocytes

β₇ is expressed on subsets of thymocytes, while T and B lymphocytes show heterogeneous expression of β₇. Here, we examine the phenotype of the thymocyte and lymphocyte subsets which express α₄β₇ and α_Eβ₇ using mAb against α_Eβ₇ and mAb DATK32 which recognizes a combinatorial epitope on α₄β₇. β₇⁺ thymocytes have a mature phenotype: TcR⁺, CD11a^hi CD44^hi HSA^dull. Small subsets of double-negative CD4_?CD8_?, single-positive CD4⁺ and CD8⁺ thymocytes express α₇, while double-positive CD4⁺ CD8⁺ thymocytes are β₇. However, two integrins α_Eβ₇ and α₄β₇ recognized by anti-β₇ are not expressed on an identical subpopulation of thymocytes, as β_Eα₇⁺α₄β₇^?, α_Eβ₇⁺α₄β₇⁺ and α_Eβ₇^?α₄β₇⁺ thymocyte subsets are evident. Similarly, intraepithelial lymphocytes express high levels of α_Eβ₇ but little α₄β₇. In the spleen, Peyer's patches and lymph nodes, α₄β₇ is expressed at higher levels on most B lymphocytes than on the majority of T lymphocytes, while a small subset of T lymphocytes, which includes both CD4⁺ and CD8⁺ lymphocytes, express high levels of β₇ in the form of α₄β₇ and α_Eβ₇, although, as observed with lymphocytes, not all α₄β₇^hi CD4^? lymphocytes expressed α₄β₇. The population of α₄β₇^hi CD4 lymphocytes are enriched in Peyer's patches and form subsets of the memory CD4⁺ lymphocyte population, which can be further subdivided on the basis of α_Eβ₇, L-selectin and α₄ expression. Therefore, memory CD4⁺ lymphocytes are highly heterogeneous in their expression of adhesion receptors, and presumably these subpopulations will exhibit very different trafficking properties.