Elena E. Solomou

Rabbit ATG but not horse ATG promotes expansion of functional CD4+CD25highFOXP3+ regulatory T cells in vitro.

Regulatory T cells (Treg) play important roles in suppressing immune responses and maintaining tolerance. Rabbit antithymocyte globulin (rATG) and horse ATG (hATG) are widely used in the treatment of immune-mediated syndromes, but their effects on Treg are unknown. We show here that in vitro culture of normal human peripheral blood mononuclear cells (PBMCs) with a low-dose rATG resulted in marked expansion of functional Treg by converting CD4+CD25- T cells to CD4+CD25+ T cells. hATG did not expand but rather decreased Treg. Immuno-blot showed increased expression of FOXP3 and NFAT1 in CD4+CD25- and CD4+CD25+ T cells exposed to rATG. PBMCs treated with rATG displayed increased interleukin-10 in culture supernatants than those treated with hATG. Furthermore, rATG and hATG showed differences in their potential to stimulate CD4+ T cells as examined using different activation markers. Microarray revealed that rATG induced markedly different gene-expression patterns in PBMCs, compared with hATG-treated or untreated PBMCs. Our findings indicate that rATG expanded Treg, probably through transcriptional regulation by enhanced NFAT1 expression, in turn conferring CD4+CD25- T cell FOXP3 expression and regulatory activity. The therapeutic effects of rATG may occur not only because of lymphocyte depletion but also enhanced Treg cell number and function.

Systemic lupus erythematosus serum IgG increases CREM binding to the IL-2 promoter and suppresses IL-2 production through CaMKIV

Systemic lupus erythematosus (SLE) T cells express high levels of cAMP response element modulator (CREM) that binds to the IL-2 promoter and represses the transcription of the IL-2 gene. This study was designed to identify pathways that lead to increased binding of CREM to the IL-2 promoter in SLE T cells. Ca(2+)/calmodulin-dependent kinase IV (CaMKIV) was found to be increased in the nucleus of SLE T cells and to be involved in the overexpression of CREM and its binding to the IL-2 promoter. Treatment of normal T cells with SLE serum resulted in increased expression of CREM protein, increased binding of CREM to the IL-2 promoter, and decreased IL-2 promoter activity and IL-2 production. This process was abolished when a dominant inactive form of CaMKIV was expressed in normal T cells. The effect of SLE serum resided within the IgG fraction and was specifically attributed to anti-TCR/CD3 autoantibodies. This study identifies CaMKIV as being responsible for the increased expression of CREM and the decreased production of IL-2 in SLE T cells and demonstrates that anti-TCR/CD3 antibodies present in SLE sera can account for the increased expression of CREM and the suppression of IL-2 production.

Molecular Basis of Deficient IL-2 Production in T Cells from Patients with Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease characterized by diverse cellular and biochemical aberrations, including decreased production of IL-2. Here we show that nuclear extracts from unstimulated SLE T cells, unlike extracts from normal T cells, express increased amounts of phosphorylated cAMP-responsive element modulator (p-CREM) that binds the −180 site of the IL-2 promoter. Nuclear extracts from stimulated normal T cells display increased binding of phosphorylated cAMP-responsive element binding protein (p-CREB) to the −180 site of the IL-2 promoter, whereas nuclear extracts from stimulated SLE T cells display primarily p-CREM and decreased p-CREB binding. In SLE T cells, p-CREM bound to the transcriptional coactivators, CREB binding protein and p300. Increased expression of p-CREM correlated with decreased production of IL-2. The transcription of a reporter gene driven by the −180 site was enhanced in normal T cells, but was suppressed in SLE T cells. These experiments demonstrate that transcriptional repression is responsible for the decreased production of IL-2 by SLE T cells.

Evidence that Dkk‐1 is dysfunctional in ankylosing spondylitis

OBJECTIVE Dkk-1 is an inhibitory molecule that regulates the Wnt pathway, which controls osteoblastogenesis. This study was undertaken to explore the potential role of Dkk-1 in ankylosing spondylitis (AS), a prototypical bone-forming disease. METHODS Serum Dkk-1 levels were measured in 45 patients with AS, 45 patients with rheumatoid arthritis (RA), 15 patients with psoriatic arthritis (PsA), and 50 healthy subjects by sandwich enzyme-linked immunosorbent assay (ELISA). A functional ELISA was used to assess the binding of Dkk-1 to its receptor (low-density lipoprotein receptor-related protein 6). Furthermore, we studied the effect of sera from patients with AS and healthy subjects on the activity of the Wnt pathway in the Jurkat T cell model, with and without a neutralizing anti-Dkk-1 monoclonal antibody, by Western immunoblotting. RESULTS Serum Dkk-1 levels were significantly increased in patients with AS (mean +/- SEM 2,730 +/- 135.1 pg/ml) as compared with normal subjects (P = 0.040), patients with RA (P = 0.020), and patients with PsA (P = 0.049). Patients with AS receiving anti-tumor necrosis factor alpha (anti-TNFalpha) treatment had significantly higher serum Dkk-1 levels than patients with AS not receiving such treatment (P = 0.007). Patients with AS studied serially prior to and following anti-TNFalpha administration exhibited a significant increase in serum Dkk-1 levels (P = 0.020), in contrast to patients with RA, who exhibited a dramatic decrease (P < 0.001). Jurkat cells treated with serum from AS patients exhibited increased Wnt signaling compared with cells treated with control serum. In that system, Dkk-1 blockade significantly enhanced Wnt signaling in control serum-treated, but not AS serum-treated, Jurkat T cells. CONCLUSION Our findings indicate that in patients with AS, circulating bone formation-promoting factors functionally prevail. This can be at least partially attributed to decreased Dkk-1-mediated inhibition.

Granulocyte colony-stimulating factor preferentially stimulates proliferation of monosomy 7 cells bearing the isoform IV receptor.

Granulocyte colony-stimulating factor (GCSF) administration has been linked to the development of monosomy 7 in severe congenital neutropenia and aplastic anemia. We assessed the effect of pharmacologic doses of GCSF on monosomy 7 cells to determine whether this chromosomal abnormality developed de novo or arose as a result of favored expansion of a preexisting clone. Fluorescence in situ hybridization (FISH) of chromosome 7 was used to identify small populations of aneuploid cells. When bone marrow mononuclear cells from patients with monosomy 7 were cultured with 400 ng/ml GCSF, all samples showed significant increases in the proportion of monosomy 7 cells. In contrast, bone marrow from karyotypically normal aplastic anemia, myelodysplastic syndrome, or healthy individuals did not show an increase in monosomy 7 cells in culture. In bone marrow CD34 cells of patients with myelodysplastic syndrome and monosomy 7, GCSF receptor (GCSFR) protein was increased. Although no mutation was found in genomic GCSFR DNA, CD34 cells showed increased expression of the GCSFR class IV mRNA isoform, which is defective in signaling cellular differentiation. GCSFR signal transduction via the Jak/Stat system was abnormal in monosomy 7 CD34 cells, with increased phosphorylated signal transducer and activation of transcription protein, STAT1-P, and increased STAT5-P relative to STAT3-P. Our results suggest that pharmacologic doses of GCSF increase the proportion of preexisting monosomy 7 cells. The abnormal response of monosomy 7 cells to GCSF would be explained by the expansion of undifferentiated monosomy 7 clones expressing the class IV GCSFR, which is defective in signaling cell maturation.

Phosphorylation and O-Linked Glycosylation of Elf-1 Leads to Its Translocation to the Nucleus and Binding to the Promoter of the TCR ζ-Chain

Elf-1, a member of the E 26-specific transcription factor family with a predicted molecular mass of 68 kDa, is involved in the transcriptional regulation of several hematopoietic cell genes. We demonstrate that Elf-1 exists primarily as a 98-kDa form in the nucleus and as an 80-kDa form in the cytoplasm. Phosphorylation and O-linked glycosylation contribute to the increased posttranslational molecular mass of Elf-1. The 98-kDa Elf-1 is released from the cytoplasm tethering retinoblastoma protein and moves to the nucleus, where it binds to the promoter of the TCR ζ-chain gene. Finally, the cytoplasmic 98-kDa form enters the proteasome pathway and undergoes degradation. In conclusion, different forms of Elf-1 are the products of posttranslational modifications that determine its subcellular localization, activity, and metabolic degradation.

Protein Kinase C-θ Participates in the Activation of Cyclic AMP-Responsive Element-Binding Protein and Its Subsequent Binding to the −180 Site of the IL-2 Promoter in Normal Human T Lymphocytes

IL-2 gene expression is regulated by the cooperative binding of discrete transcription factors to the IL-2 promoter/enhancer and is predominantly controlled at the transcriptional level. In this study, we show that in normal T cells, the −180 site (−164/−189) of the IL-2 promoter/enhancer is a p-cAMP-responsive element-binding protein (p-CREB) binding site. Following activation of the T cells through various membrane-initiated and membrane-independent pathways, protein kinase C (PKC)-θ phosphorylates CREB, which subsequently binds to the −180 site and associates with the transcriptional coactivator p300. Rottlerin, a specific PKC-θ inhibitor, diminished p-CREB protein levels when normal T cells were treated with it. Rottlerin also prevented the formation of p-CREB/p300 complexes and the DNA-CREB protein binding. Cotransfection of fresh normal T cells with luciferase reporter construct driven by two tandem −180 sites and a PKC-θ construct caused a significant increase in the transcription of the reporter gene, indicating that this site is functional and regulated by PKC-θ. Cotransfection of T cells with a luciferase construct driven by the −575/+57 region of the IL-2 promoter/enhancer and a PKC-θ construct caused a similar increase in the reporter gene transcription, which was significantly limited when two bases within the −180 site were mutated. These findings show that CREB plays a major role in the transcriptional regulation of IL-2 and that a major pathway for the activation of CREB and its subsequent binding to the IL-2 promoter/enhancer in normal T cells is mediated by PKC-θ.

Malaria: a haematological disease

Abstract Plasmodium falciparum malaria remains a major cause of mortality throughout the tropical world. Haematological abnormalities are considered a hallmark of malaria, bearing an impact on final outcome and representing indices of prognostic and follow-up value. These include severe anaemia, coagulation disturbances, leukocyte numerical or functional changes and spleen involvement. Anaemia involves red blood cell lysis due to parasite invasion, as well as mechanisms of intravascular haemolysis and decreased erythropoiesis. Exchange or blood transfusion is mainly recommended in the management of these patients. Haemorrhagic complications in severe malaria are relatively rare despite prominent thrombocytopenia and dysfunction in the coagulation pathway. Numerical, as well as functional changes in the white blood cell are less dramatic than other blood cell series, but still, remain a significant index of disease progression and ultimate prognosis. Finally, the role of the spleen in severe malaria is multifactorial. Care and vigilance should be taken against splenic rupture which is fatal and can occur despite appropriate antimalarial prophylaxis and treatment.

Decreased TCR ζ-chain expression in T cells from patients with acquired aplastic anaemia

In aplastic anaemia, T cells have a central role in the pathophysiology of bone marrow destruction. This study showed that T cells from patients with aplastic anaemia expressed decreased T‐cell receptor (TCR) ζ‐chain protein and mRNA levels compared to healthy controls. Patients with decreased TCR ζ‐chain showed an abnormal response in intracellular calcium following stimulation through the TCR. We also observed an altered pattern of the transcription factors CREMα and Elf‐1 that are implicated in ζ‐chain transcription. We concluded that TCR ζ‐chain expression was decreased in the majority of patients with aplastic anaemia, regardless of disease activity or treatment status.

Complete remission of recalcitrant viral warts under oral isotretinoin in a patient with low-grade B-cell lymphoma.

Sir, Viral warts are the most frequent clinical manifestations of the ubiquitous human papillomaviruses (HPV) infection. Particularly in immunocompromised patients they may become extensive in both size and area of involvement, representing a frustrating problem, and can progress to squamous cell carcinomas (1, 2). We report herein the complete remission of recalcitrant and extensive warts in a patient with low-grade B-cell small lymphocytic lymphoma under treatment with oral isotretinoin (13-cis retinoic acid).