Beta-globin gene cluster polymorphisms are strongly associated with severity of HbE/beta(0)-thalassemia.

We evaluated the contribution of 67 single nucleotide polymorphisms (SNPs) within the beta-globin gene cluster to disease severity in groups of 207 mild- and 305 severe unrelated patients from Thailand with Hemoglobin E (HbE)/beta(0)-thalassemia and normal alpha-globin genes. Our analysis showed that these SNPs comprise two distinct linkage disequilibrium blocks, one containing the beta-globin gene and the other extending from the locus control region (LCR) to the delta gene, which are separated by a recombination hotspot in the narrow region of the beta-globin gene promoter. Forty-five SNPs within the interval including the LCR region and the delta gene showed strong association with disease severity. The strongest association was observed with the XmnI polymorphism located 158-bp upstream to the G gamma gene (p = 4.6E-12). Carriers of the T allele of XmnI were more likely to have a milder disease course and higher level of fetal hemoglobin (HbF) in both the mild (p = 0.005) and severe (p = 8.7E-06) patient groups. Haplotype analysis revealed that the T allele of XmnI was nearly always in cis with the HbE allele. The high frequency of this haplotype may be favored by positive selection against malarial infection. Further studies are needed to validate this hypothesis and determine whether XmnI or another closely linked variant modulates severity and HbF levels in patients with beta(0)-thalassemia/HbE disease.

ADP-ribosylation of rho by C3 ribosyltransferase inhibits IL-2 production and sustained calcium influx in activated T cells.

Activation of the T lymphocyte induces dramatic cytoskeletal changes, and there is increasing evidence that disruption of the cytoskeleton inhibits early and late events of T cell signal transduction. However, relatively little is known about the signaling molecules involved in activation-induced cytoskeletal rearrangement. The rho family of small GTP-binding proteins, which include rho, rac, and cdc42, regulates the cytoskeleton and coordinates various cellular functions via their many effector targets. In prior studies, the Clostridium botulinum toxin C3 exoenzyme has been used to ADP-ribosylate and inactivate rho. In this study, we demonstrate that treatment of T cells with C3 exoenzyme inhibits IL-2 transcription following ligation of the TCR. Inhibition of IL-2 expression correlated with loss of sustained increase in [Ca+2]i and mitogen activated protein kinase (MAPK/Erk) activity, but not with activation of the tyrosine kinase, lck. These findings are the first to show that ADP-ribosylation of rho by C3 ribosyltransferase (exoenzyme) inhibits IL-2 production due, in part, to the requirement for sustained calcium influx and MAPK activation after Ag receptor ligation.

TJ6: the pregnancy-associated cytokine.

These studies have demonstrated that TJ6m protein can be measured in women prior to a spontaneous abortion based on expression of TJ6 on CD56-positive NK cells. This suggested to us a possible regulator function for TJ6 during pregnancy. We have shown that in T cells only crosslinking of the T-cell receptor can upregulate TJ6 expression and the other activators, such as mitogens, do not. A clear discrepancy in the pattern of expression of TJ6 on CD56 and CD19-positive cells was noted between successful and unsuccessful pregnancies. A successful pregnancy is denoted by TJ6 expression on B cells, whereas in a potential spontaneous abortion there is TJ6 expression on both NK and B cells.

Mast cell mediators regulate vascular permeability changes in Arthus reaction.

Plasma exudation characterizes the early phase of acute inflammation. The possible role of mast cells and their mediators in this event in immune complex-induced injury was studied. Dye exudation was assessed from 5 min to 2 hr after initiating reverse passive Arthus reaction in mast cell-deficient mice, WBB6F1-W/Wv (W/Wv), and their normal congenic controls, WBB6F1-+/+ (+/+). The response to antibody (10, 30 and 100 micrograms/site, i.d.) was dose- and time-dependent in both groups of mice. At the lower doses of antibody, 10 and 30 micrograms/site, exudation was significantly less (30% and 40%, respectively) in W/Wv as compared to +/+ mice between 15 to 45 min. With 100 micrograms of antibody/site, significant differences between W/Wv and +/+ mice were noted only at 15 and 30 min. The deficit in permeability changes in W/Wv mice was reversed by local mast cell reconstitution. In +/+ mice, pyrilamine and methysergide pretreatment reduced vascular permeability to the same extent by 70, 60 and 35% when stimulated for 30 min with 10, 30 and 100 micrograms of antibody/site, respectively. An equivalent inhibition was observed with the 5-lipoxygenase inhibitor A-63162. None of the inhibitors decreased plasma permeation in W/Wv mice. These results indicate that the mast cell mediators histamine and serotonin regulate vascular permeability early during an immune complex-mediated inflammation. The data also suggest the involvement of leukotrienes and the importance of mast cells in their synthesis. The profile of inhibition in +/+ mice agrees well with the difference in exudation observed between normal and mast cell-deficient mice.

Expression of TJ6 during pregnancy.

PROBLEM: TJ6 will be one of the molecules involved in fetal-specific immune suppression during pregnancy. In the mouse and human decidua, the regulation of uterine natural killer (uNK) cells is important during pregnancy. METHOD OF STUDY: To further understand the possible functions of TJ6 during pregnancy, syngeneic, allogeneic, and mutant mice were examined for TJ6 expression. RESULTS: Immunoblotting showed that TJ6 protein was expressed on most of the placenta-associated mononuclear cells, and the size was 70-72 kDa at all stages of pregnancy. The expression of TJ6 mRNA was studied by a ribonuclease protection assay in syngeneic and allogeneic matings, and in immune-deficient mice of genotypes scid/scid and scid/scid.bg/bg. CONCLUSIONS: Genetic disparity, lack of T and B lymphocytes, and loss of NK lytic function had no significant effect on the expression of TJ6 mRNA.

Expression of a membrane form of the pregnancy-associated protein TJ6 on lymphocytes.

TJ6 is a novel protein which has immunosuppressive activity and may have a functional role in fetal allograft survival during pregnancy. Initial studies indicated that when mice were treated with an anti-TJ6 binding mAb early in pregnancy, the pregnancies were completely ablated and that TJ6 expression is enhanced dramatically during pregnancy. In addition we have cloned the cDNA for TJ6 which encodes a possible transmembrane domain that may include six to seven transmembrane regions. Therefore, we examined TJ6 expression on PBL of pregnant and non-pregnant women and found that TJ6 is expressed primarily on CD19+ B cells from pregnant but not nonpregnant women. TJ6 was not expressed on CD3+ lymphocytes from either group but was expressed on CD56+ cells from a small population of pregnant women which preliminary data indicate may correlate with the occurrence of spontaneous abortion in these women. Here we also show that TJ6 transcripts are highly expressed in the developing fetoplacental unit as well as in the developing thymus. We also begin to characterize the expression of TJ6 isoforms in an acute lymphocytic leukemia cell line (SB), murine thymus, and the developing murine fetoplacental unit, as well as the expression of a membrane form of TJ6 present on human lymphocytes during pregnancy. All of these cells and tissues expressed TJ6 proteins which were smaller than predicted based on either the cDNA sequence or the in vitro translation even though they expressed mRNA similar in size. The TJ6 isoforms varied in size from the 45-kDa isoform in SB cells to the 52-kDa isoform of the fetoplacental unit to a 70-kDa isoform in murine thymus. Flow cytometric analysis also demonstrated that similar to the CD19+ B cells from pregnant women, TJ6 is expressed on the surface of SB cells.