Homozygous G γλβ thalassaemia

Summary This report describes the clinical and haematological findings in three siblings homozygous for ^Gγ δβ thalassaemia in an Indian family. There was a mild to moderate anaemia and markedly abnormal red cell morphology. Haemoglobin analysis showed 100% Hb F, solely of the ^Gγ type, with a pancellular but uneven distribution. Considerable chain imbalance was detectable in globin synthesis studies. In contrast to the five previously reported cases, these children were essentially asymptomatic and have never required transfusions.     

Peripheral T cell receptors αβ and γδ in patients with Hodgkin's lymphoma

Antigen recognition by T cells is determined by an antigen specific T cell receptor (TCR). Two heterodimeric TCR structures associated with CD3 have been defined: TCR αβ and TCR γδ. TCR αβ and its function are well described but the role of TCR γδ in normal and lymphoproliferative disorders is not well established. In newly diagnosed or relapsed/refractory Hodgkin's disease (HD), a disease associated with defective T cell functions and increased sIL-2R, We determined levels of seven TCR αβ variable regions [βV5(a), βV5(b), βV6(a), βV12(a), αβV(a), αV2(a)] and TCR γδ by using monoclonal antibodies (MCA). TCR γδ levels did not show any difference, but several variable regions of the TCR αβ differed when groups are compared with each other and the control group.     

δβ -Thalassaemia in Two Yugoslavian Families

Members of two Yugoslavian families were found to have δβ-thalassaemia. Interaction of β-thalassaemia with δβ-thalassaemia occurred in two young children producing a clinical condition which is somewhat less severe than that of homozygous β-thalassaemia. Results from biosynthetic analyses indicate that the degree of globin chain imbalance in double heterozygotes for β- and δβ-thalassaemia is similar to that in homozygous β-thalassaemia. Fetal haemoglobin of all heterozygotes contained ^Gγ and ^Aγ chains in an average ratio of about 2:3 whereas that in the two double heterozygotes had ^Gγ and ^Aγ chains in a ratio of 3:2.     

Association of PPAR‐γ2 and β3‐AR Polymorphisms With Postmenopausal Hypertension

The aim of this study was to test the association of peroxisome proliferator‐activated receptor (PPAR‐γ2) (Pro12Ala, C1431T) and β3‐AR (Trp64Arg) polymorphisms with metabolic, nutritional, and blood pressure parameters in 271 postmenopausal women (151 hypertensive and 120 normotensive controls). The TaqMan genotyping assay and restriction fragment length polymorphism methods were used to determine the distributions of selected alleles and genotype frequencies. Nutritional status was determined by a bioimpedance method and dietary habits were assessed via 7‐day dietary recall. The distribution of selected genotypes and allele frequencies did not differ between hypertensive women and normal controls after analysis by chi‐square test. The postmenopausal hypertensive women were older and had higher body fat mass, serum glucose, and triglyceride levels. The cluster analysis showed that the hypertensive group with Pro12Pro genotype had highest pulse pressure and mean arterial pressure values when compared with Pro12Ala patients. In the logistic regression analysis, blood glucose (Pro12Ala polymorphism) and energy intake (C1431Tand T1431T polymorphisms) determined hypertension.     

Regulation of pulmonary inflammation and fibrosis through expression of integrins αVβ3 and αVβ5 on pulmonary T lymphocytes

Objective

Pulmonary diseases associated with fibrosis, including scleroderma lung disease, are characterized by the accumulation of T cells in the lungs. These cells are thought to facilitate lung fibrosis, but the exact mechanisms of their profibrotic action are not clear. Several αV‐containing integrins, including αVβ3 and αVβ5, have been shown to directly activate transforming growth factor β (TGFβ) and promote collagen accumulation. The aim of this study was to investigate whether pulmonary T cells express profibrotic integrins and regulate collagen accumulation.

Methods

Expression of integrins was assessed by immunohistochemical analysis of lung tissue, by flow cytometry using bronchoalveolar lavage fluid from patients with systemic sclerosis (SSc), and in a CCL18 overexpression animal model of pulmonary T cell infiltration. Experiments in cell cultures were performed to determine whether integrin‐expressing T cells are profibrotic in cocultures with pulmonary fibroblasts and, if so, through what possible mechanism.

Results

Lymphocytes and integrin‐positive cells were present in the lungs, and pulmonary T cells expressed integrins αVβ3 and αVβ5 in patients with SSc and in the animal model. Systemic administration of neutralizing anti–integrin αV antibody or a genetic deficiency of integrin β3 in the CCL18 overexpression model significantly attenuated CCL18‐driven pulmonary lymphocytic infiltration and collagen accumulation. Jurkat T cells overexpressing integrin αVβ3 or integrin αVβ5 in cocultures with primary pulmonary fibroblasts stimulated collagen accumulation and Smad2 nuclear translocation. Neutralizing anti‐TGFβ antibody attenuated the profibrotic effect of integrin‐expressing T cells.

Conclusion

Pulmonary infiltrating T lymphocytes may express integrins αVβ3 and αVβ5 that are necessary for lymphocytic infiltration and T cell–associated TGFβ activation and collagen accumulation.

     

New indices from the H*2 analyser improve differentiation between heterozygous β or δβ thalassaemia and iron-deficiency anaemia

Summary The two main causes of microcytic and hypochromic anaemia are iron deficiency (IDA) and thalassaemia (THAL) traits. In the Mediterranean area there is a high prevalence of β and δ-β THAL minor. The differentiation between these causes of microcytosis can be significantly improved with two new indices, percentage of microcytes (%Mi) and percentage of hypochromic red blood cells (%Hy), and the direct determination of MCHC, provided by the technological advances of the H*2 analyser. Our discriminant analysis, based on the minimization of Wilk's lambda (A) criterion, was used to select the best predictive variables to differentiate between IDA and THAL and has resulted in the highest diagnostic efficiency published to date. The discriminant function obtained is a simple linear combination of the following variables: D = 1.145 RBC-0.174 MCV+0.091 MCHC+0.787 √(%Hy/%Mi)-22.119. The overall correct classification was 97.6% on the training sample (79 THAL and 90 IDA) and 96.7% on a validation sample of microcytic patients (72 THAL and 80 IDA). The sensitivity and diagnostic specificity were 97.5% and 97.8%, respectively, for the training sample, and 95.8% and 97.5% for the control group.     

Hemoglobinopathies in the dogon country: Presence of βs, βc,and δa'genes

The population of the Dogon, located in Mali, is divided in an endogamic Noble class and two endogamic servant castes (Tanners and Blacksmiths). We find that the polymorphic frequencies of β^c, β^s, and, unexpectedly, a mutation of the δ-chain (δ^A'), are geographically (valley vs. plateau) as well as social status dependent. © 1994 Wiley-Liss, Inc.     

Involvement of αvβ5 integrin–mediated activation of latent transforming growth factor β1 in autocrine transforming growth factor β signaling in systemic sclerosis fibroblasts

Objective

To confirm the involvement of αvβ5 in the self‐activation system in systemic sclerosis (SSc) fibroblasts.

Methods

Levels of αvβ5 expression were analyzed by immunoprecipitation. The promoter activity of the human α2(I) collagen gene was determined by transient transfection assay. Phosphorylation levels and DNA binding ability of Smad3 were investigated by immunoprecipitation and DNA affinity precipitation, respectively. The localization of active transforming growth factor β (TGFβ) was determined by coculture assay using TMLC cells (mink lung epithelial reporter cells that stably express a portion of the plasminogen activator inhibitor 1 promoter). The morphologic features of cells were determined by immunofluorescence analysis.

Results

Levels of αvβ5 expression were significantly elevated in SSc fibroblasts compared with normal fibroblasts. Treatment with anti‐αvβ5 antibody or β5 antisense oligonucleotide significantly reduced human α2(I) collagen gene promoter activity in SSc fibroblasts. In SSc fibroblasts pretreated with TGFβ1 antisense oligonucleotide, the exogenous latent TGFβ1 stimulation significantly increased human α2(I) collagen gene promoter activity; this effect was significantly reduced in the presence of anti‐αvβ5 antibody. Phosphorylation levels and DNA binding ability of Smad3 in SSc fibroblasts were significantly reduced by treatment with β5 antisense oligonucleotide. The luciferase activity of TMLC cells cocultured with SSc fibroblasts was significantly elevated compared with that of TMLC cells cocultured with normal fibroblasts and was significantly reduced in the presence of anti‐αvβ5 antibody. Anti‐αvβ5 antibody reversed the myofibroblastic features of SSc fibroblasts.

Conclusion

Up‐regulated expression of αvβ5 contributes to the establishment of autocrine TGFβ signaling in SSc fibroblasts through activation of endogenous latent TGFβ1.

     

Genetic interactions in thalassemia intermedia: Analysis of β-Mutations, α-Genotype, γ-Promoters,and β-LCR hypersensitive sites 2 and 4 in Italian patients

In order to verity the genetic factors influencing the clinical expression of β-thalassemla we have studied 292 Kalian patients, 165 with thalassemia intermedia and 127 with thalassemia major. The β-globin gene mutations were defined in all cases. The number of α-globin genes and the integrity of specific control regions of the β-globin cluster—γ promoters and β-Locus Control Region (β-LCR)—were studied in selected cases. Homozygosity for mild mutations (group I) accounts for 24% of the intermedia patients and it is not represented among major patients. Forty-four percent of intermedia patients had combinations of mild/severe (group II) mutations and 32% had homozygosity or double heterozygosity for severe mutations (group III). Seventy-six percent of patients with thalassemia major were classified in group III end 24% in group II. Deletion type —α^3.7 thalassemia, assessed in a part of the cases, was found in 5% of thalassemia major and 19.5% of Intermedia patients in groups II and III. Structural analysis of γ promoters and β-LCR HS2 and HS4 regions, carried out in order to look for alterations associated with Hb F increase, did not reveal new mutations. Only rare polymorphic changes were observed at the HS2 and HS4 level. The ? 158 γ C T change was found with an increased incidence in intermedia patients in groups II and III. A subset of 10 β-thalassemia heterozygotes with mild intermedia phenotype resulted from coinheritance of a triplicated α-locus. We have been unable to find a molecular basis for the benign clinical course in approximately 20% of patients with thalassemia intermedia. Other genetic or acquired factors must be hypothesized which ameliorate the clinical condition.     

Increased usage of vβ2 and vβ6 in rheumatoid synovial fluid t cells

Objective. To determine if the T cell antigen receptor V_β usage of unstimulated rheumatoid arthritis (RA) synovial fluid (SF) T cells is biased compared with those in peripheral blood (PB). Methods. Freshly isolated, matched synovial fluid and peripheral blood T cells were analyzed for V_β gene expression using quantitative polymerase chain reaction (PCR) methods. Ten synovial fluid samples from the knees of 7 patients with RA were studied. The PCR assay used 26 V_β primers with a constant region C_β primer, and 2 C_α primers that co-amplified a product that served as an internal standard. Cycle number and complementary DNA content were controlled to ensure the linear accumulation of PCR products. Labeled products were separated on 10% polyacrylamide gels and counted with a Betascope blot analyzer. Results. There were consistent differences between the V_β gene usage of SF and PB T cells directly isolated from patients with RA, regardless of HLA–DR haplotype. In all synovial specimens, V_β2 was increased relative to the peripheral blood, while V_β13.1 and V_β13.2 were decreased. V_β6 and V_β21 were increased in 9 of the 10 synovial samples. Analyses of bilateral SF specimens from 2 subjects and serial specimens from the same knee of 1 subject revealed virtually identical patterns in each patient. The SF V_β bias was not solely due to differences in the proportion of CD4+ and CD8+ cells, because the CD4:CD8 ratios in SF and PB were similar. However, V_β gene usage of separated CD4+ and CD8+ synovial T cells showed that V_β2 and V_β6 were more highly expressed on CD4 cells. Conclusion. Freshly isolated synovial T cells from inflamed (not end-stage) knees of patients with RA have a remarkably consistent biased V_β gene usage compared with PB T cells. V_β2 and V_β6 are uniformly increased, and this increase is primarily in CD4+ T cells. The same V_β bias in the SF T cells of several RA patients suggests that shared antigens may be stimulating the T cell response.     

Estrogen and the brain: beyond ER-α and ER-β

17β-Estradiol is a greatly under-appreciated neural growth and trophic factor for the mammalian brain of all ages. Like other growth factors, such as the neurotrophins, 17β-estradiol influences neurogenesis, neuronal differentiation, and neuronal survival of its targets throughout life. Estrogen elicits developmentally regulated differentiative effects, which are not normally seen in the adult brain. However, re-expression of this developmental response occurs in the adult, following loss of trophic support, whether induced by estrogen deprivation or brain injury. In addition to the classical intranuclear estrogen receptors (ER) ER-α and ER-β, we have recently identified a novel, plasma membrane-associated, putative ER that is neither ER-α nor ER-β, which we have designated ‘ER-X’. ER-X is a developmentally regulated estrogen-binding protein, present in wild-type, ER-α gene-disrupted (αERKO) and ER-α null mice, which is re-expressed following ischemic brain injury. The preferred ligand of ER-X is 17α-estradiol. Although ER-X shares some homology with the C-terminus of ER-α, it is not an alternative splicing variant and may be a new gene. While ER-X appears to mediate 17α- and 17β-estradiol activation of the MAPK cascade, ER-α, in contrast, is inhibitory to its activation. Estradiol activation of MAPK/ERK may be particularly relevant for neuroprotection during aging and Alzheimer's disease.     

α-Thalassemia and β-thalassemia in a turkish family

A Turkish family is described in which three children have a clinical picture similar to that of thalassemia major, with typical red cell morphology and indices, and with about 10% Hb Bart's but without measurable amounts of Hb H. Hematological evaluation of six members of this family that included in vitro hemoglobin synthesis suggests that β- (or δβ-) thalassemia, β-silent thalassemia, and mild and severe α-thalassemia genes are present in different combinations. The data indicate that β/α chain ratios in patients with more than one type of thalassemia should be evaluated in relationship to values obtained for several relatives even though some of the thalassemia determinants may be silent in the parents.     

β Globin messenger RNA content of bone marrow erythroblasts in heterozygous β-thalassemia

RNA from bone marrow erythroblasts and peripheral blood reticulocytes of patients with heterozygous β-thalassemia was analyzed for relative content of α and β globin messenger RNA by molecular hybrization. Erythroblasts from nonthalassemic patients exhibited approximately the same α and β globin mRNA content (β/α mRNA ratio = 0.8–1.0) as circulating reticulocytes (β/α mRNA ratio = 0.74–1.2). The mRNA ratios corresponded well to levels of globin synthesis observed in bone marrow and peripheral blood. Erythroblasts from four patients with heterozygous β-thalassemia also exhibited approximately the same β/α mRNA ratios in bone marrow erythroblasts (0.34–0.59) as in reticulocytes (0.34–0.4): β globin mRNA was clearly deficient in bone marrow erythroblasts. Globin biosynthesis by erythroblasts of β-thalassemia heterozygotes was balanced despite the mRNA deficiency (β/α = 0.9–1.0), suggesting that post-translational phenoma (eg, proteolysis of free globin chains), rather than instability of β mRNA, accounts for the balanced globin chain synthesis frequently observed in bone marrow erythroblasts of patients with β-thalassemia trait.     

T cell receptor vβ repertoire of double-negative α/β t cells in patients with systemic sclerosis

Objective. To analyze the T cell receptor V_β gene on double-negative (DN) α/β T cells, which are increased in number, on peripheral blood lymphocytes (PBL) from patients with systemic sclerosis (SSc). Methods. The DN α/β T cells were sorted by flow cytometry from PBL obtained from 3 patients with SSc. The V_β repertoire was analyzed by polymerase chain reaction. Results. Only 1 or 2 V_β genes (V_β5/7, 5, or 17) were predominantly expressed on DN α/β T cells from these 3 patients. Conclusion. The V_β repertoire on DN α/β T cells in PBL from patients with SSc is rather restricted.