Characterization of an acute T lymphoblastic leukemia expressing the γ/δ T-cell receptor

Summary Leukemic cells of a 20 year old patient, suffering from acute lymphoblastic leukemia, were characterized by surface marker and functional analysis. A significant cell population within this type of leukemia expresses concomitantly the CD4 and CD8 antigen on the same cell and might represent a new differentiation stage of T-cells with the / receptor. The leukemic cells show a distinct pattern of growth response to mitogens and lymphokines, which might correlate to their differentiation stage. Moreover, a natural killer-like activity can be induced in these cells by IL-2.Abbreviations FITC fluorescein isothiocyanate - PE phycoerythrin - IL-2 interleukin 2; - / TCR gamma/delta T cell receptor - NK natural killer - PBL peripheral blood lymphocytes - T-ALL acute T lymphoblastic leukemia - ConA concanavalin A - PMA phorbol myristate acetate - BM bone marrow - IL-2R IL-2 receptor - TdT terminal deoxynucleotidyl transferase Supported by the Deutsche Forschungsgemeinschaft (DFG Wi-728/3-1)     

Serum cytokines in patients with rheumatoid arthritis

Serum cytokines such as interleukin 1 (IL-1), interferon (IFN-), and tumor necrosis factor (TNF) were measured in 40 patients with rheumatoid arthritis (RA). In the 40 patients studied, serum IL-1 was detected in 5 patients, IFN- in 10 patients, and TNF in 20 patients. The IL-1-positive group showed increased values of activity indices compared to the IL-1-negative group. Values of serum IFN- correlated well with the number of peripheral blood lymphocytes and CD3⁺ cells and with the percentage of CD3⁺ CD26⁺ cells. Values of serum TNF correlated positively with the number of peripheral blood monocytes and the percentage of CD3⁺ HLA-DR⁺ and CD3⁺ CD25⁺ cells. These results indicated that serum IL-1 in RA patients reflects the activity of RA, while the serum IFN- and TNF in RA patients may be related to circulating activated lymphocytes and monocytes, respectively.     

Cytokine Production by Intestinal Intraepithelial Lymphocyte Subsets in Celiac Disease

One of the earliest signs of mucosal immune activation in celiac disease (CD) is an increase in the intraepithelial lymphocyte (IEL) count in the small intestinal epithelium. Though most of those IELs express T cell receptor (TcR)- chains, CD is characterized by an increase in TcR- ⁺ IELs and by the loss of CD3^– IELs. There is currently little evidence that these changes in IEL subset distribution are of relevance in the pathogenesis of CD. We aimed to determine the pattern of cytokine production by IEL subsets isolated from duodenal biopsy specimens from control subjects and CD patients at different stages of the disease. We quantified the capacity of IEL subsets to produce IFN-, TNF-, IL-2, IL-4, and IL-10 by intracellular staining by flow cytometry. All IEL subsets studied displayed a type I cytokine profile in both CD and control subjects, with TcR-⁺ IELs being the main IFN- producers. Untreated CD exhibited a trend toward a superior accumulation of IFN- per cell but a reduced proportion of INF-⁺ cells in vitro in association with a significantly increased apoptotic rate of IELs. IL-4 was almost undetectable in all cases and IL-10 showed a tendency to increase in treated and silent celiac patients. IEL subsets have a similar Th1 profile in controls and CD patients, and the superior in vitro apoptosis of IELs from CD patients may reflect their superior in vivo activation. The induction of IL-10-dependent regulatory Tr1 responses may be of potential clinical significance in this disease and merits further investigation.     

Effect of gold therapy on CD5+ B-cells and TCR γδ + T-cells in patients with rheumatoid arthritis

Summary Circulating CD5⁺ B-cell levels in 15 patients with rheumatoid arthritis (RA) not receiving remittive therapy was significantly increased when compared to 17 normal controls (mean±SE: RA, 19.7±2.85%; controls, 11.6±1.67%; P<0.02). In contrast, 24 patients with RA receiving gold sodium thiomalate therapy (GST) had similar CD5⁺ B-cell levels (11.88±1.65) when compared to controls and significantly reduced levels when compared to the RA group not receiving remittive agents (P<0.01). Furthermore, TCR ⁺ T-cell levels were also assessed in these patients groups. These values were not significantly different between any of the groups (controls, 4.46±1.36%; GST, 6.88±1.73%; RA, 2.73±0.55%), although 42% of the GST treated group had ⁺ T-cell levels higher than the entire untreated RA group. No correlation was observed between the levels of TCR ⁺ T-cells and CD5⁺ B-cells in any of these groups. These results suggested that therapy does influence the level of CD5⁺ B-cells and ⁺ T-cells in these patients.     

Local and Systemic Liberation of Proinflammatory Cytokines in Ulcerative Colitis

Determination of plasma and tissue cytokinelevels in inflammatory bowel disease have frequentlyresulted in conflicting data. In the present study wedetermined in patients with ulcerative colitis (UC), the levels of the proinflammatory cytokinesinterleukin (IL)-1, IL-6, interferon(IFN)-, and tumor-necrosis factor (TNF)-liberated by peripheral blood mononuclear cells (PBMC)and lamina propria mononuclear cells (LPMC) after 48-hrculture with pokeweed mitogen (PWM). IL-1, IL-6,IFN- and TNF- in the supernatant weredetected by ELISA. Results show low basal levels ofIL-1 secretion by PBMC and LPMC, and a considerableincrease after mitogen stimulation. Basal IL-6production by PBMC was higher in UC patients than incontrols [2029 pg/ml, CI₉ (–165 to4223) vs 572 pg/ml (–383 to 1527) respectively, P = 0.05] and also afterPWM activation [14,995 pg/ml (7759 -22230) vs 6598 pg/ml(3240-9956), respectively, P = 0.05]. In LPMC, nodifferences in IL-6 secretion were observed. TNF- in activated PBMC of patients with UC was notsignificantly increased in relation to control (P =0.09). No constitutive secretion of IFN- wasobserved in mononuclear cells. IFN- levelssecreted by activated LPMC were lower in patients withUC than in controls [1571 pg/ml (–108 to 3251) vs7953 pg/ml (3851-12,055), respectively, P = 0.03]. Theseresults suggest that IL-6, IL-1, and TNF- participate as mediators in the inflammatoryphenomena observed in UC. Further studies are necessaryto evaluate the role of IFN- in thiscondition.     

γ/δ Receptor-expressing T-cell clones from a cutaneous T-cell lymphoma suppress hematopoiesis

Summary Recently we described a cutaneous T-cell lymphoma expressing the / T-cell receptor [5]. The patient suffering from this lymphoma showed low numbers of myeloid and T cells in peripheral blood, while B and NK cells were relatively increased. In vitro culture of the patient's bone marrow (BM) cells revealed a significant suppression of myeloid/monocyte colony formation (GM-CFU) compared with normal controls. This was not due to infiltration of the BM with lymphoma cells. We speculated that a soluble factor either secreted or induced by the lymphoma cells might be responsible for the marked suppression of hematopoiesis in this patient. From a skin biopsy with infiltrating / T-lymphoma cells we established T-cell clones bearing the / T-cell receptor and resembling the phenotype of the lymphoma cells. The supernatant (SN) of these / T-cell clones reduced the number of colonies in a CFU-GM assay (using normal control BM) in comparison to SN of / T-cell clones established from the same biopsy. This suppression was seen mainly on day 7 of culture and was not neutralized by the addition of placenta-CM. The main mediator of this suppression seems to be IFN-,since it was detectable in high amounts in the SN of these / T-cell tumor clones as well as in the serum of the patient. In addition, anti-IFN- antibodies can reverse the T-cell SN-mediated suppression of CFU-GM. We conclude that high serum levels of interferon-, which is secreted in high amounts from / T-cells grown from a biopsy of a cutaneous lymphoma, can suppress hematopoiesis.Abbreviations TCR T-cell receptor - IFN- interferon- - SN supernatant - placenta CM placenta conditioned medium - BM bone marrow - CFU-GM myeloid/monocyte colony formation - NK cells natural killer cells - Ab antibody M. Wilhelm was supported by theDeutsche Forschungsgemeinschaft (DFG Wi 728-2)     

Influence of insulin on cyclic 3′,5′-AMP phosphodiesterase activity in liver,skeletal muscle,adipose tissue,and kidney

Summary Influence of insulin on liver glycogen metabolism and on lipolysis appears to be mediated by a decreased intracellular 3,5-AMP concentration. Reduced formation of 3,5-AMP had been shown in adipose tissue incubated with insulin. The influence of insulin on 3,5-AMP degradation has been investigated. — 3,5-AMP phosphodiesterase (PDE) activity was reduced in liver, adipose tissue and, insignificantly, in skeletal muscle of insulin deficient, i.e. alloxan diabetic or starved rats. I.V. injection of a low dose of insulin (0.5 U/kg) or stimulation of endogenous insulin secretion by injection of glucose led to a rapid increase of PDE activity in these tissues. 15 min after insulin injection liver PDE activity was increased. The maximal effect occurred after 30–45 min. Renal PDE activity was not decreased in alloxan diabetes, insulin injection has been found ineffective. —In vitro, there was an activating effect of crystalline insulin on PDE purified from beef heart. Insulin concentration required for duplication of enzyme activity was of the order of 2 · 10^–5 M. Treatment with actinomycin D nearly prevented stimulation of liver PDE by insulin. This may indicate that the action of insulin on PDE activity is essentially based on an increased enzyme synthesis. — Owing to the influence of insulin secretion on liver and adipose tissue 3,5-AMP concentration, glycogen metabolism and lipolysis can be quickly adapted to food intake.

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Der Einfluß von Insulin auf die 3,5-AMP-Phosphodiesterase-Aktivität in Leber, Skeletmuskulatur, Fettgewebe und Niere

Zusammenfassung An der Steigerung der Glykogensynthese der Leber und der Verminderung der Lipolyse durch Insulin ist eine Abnahme der 3,5-AMP-Konzentration wesentlich beteiligt. Die 3,5-AMP-Bildung ist in Fettgewebe, das mit Insulin inkubiert wird, vermindert. Insulin beeinflußt jedoch auch den 3,5-AMP-Abbau. -Die 3,5-AMP-Phosphodiesterase (PDE)-Aktivität des Fettgewebes, der Leber und, in geringerem Grade, der Skeletmuskulatur ist im Insulinmangel vermindert, d.h. bei alloxandiabetischen oder hungernden Ratten. I.v. Injektion von 0,5 E/kg Insulin oder eine erhöhte Abgabe von Insulin aus dem Pankreas nach Glucoseinjektion führen in diesen Geweben zu einem raschen Anstieg der PDE-Aktivität. Dieser ist in der Leber schon 15 min nach Insulingabe nachweisbar und erreicht nach 30–45 min sein Maximum. In der Niere ist kein Einfluß von Insulin auf die PDE-Aktivität nachweisbar. — Aus Rinderherz isolierte PDE wirdin vitro durch Insulin aktiviert, jedoch werden2 · 10^–5 M zur Verdopplung der Aktivität benötigt. Actinomycin D verhindert die Steigerung der Leber-PDE-Aktivität nach Insulininjektion. So kann die Wirkung des Hormons im wesentlichen auf eine gesteigerte PDE-Synthese zurückgeführt werden. — Durch diesen Einfluß der Insulininkretion auf die 3,5-AMP-Konzentration in Leber und Fettgewebe können Glykogenstoffwechsel und Lipolyse rasch an die Nahrungsaufnahme angepaßt werden.

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Influence de l'insuline sur l'activité de la 3,5-AMP-phosphodiestérase dans le foie, le muscle strié, le tissu adipeux et le rein

Résumé L'influence de l'insuline sur le métabolisme du glycogène hépatique et sur la lipolyse semble s'exercer par l'intermédiaire d'une diminution de la concentration de 3,5-AMP intracellulaire. Onamontré une diminution de la formation de 35-AMP dans le tissu adipeux incubé avec de l'insuline. L'influence de l'insuline sur la dégradation du 3,5-AMP est étudiée. — L'activité de la 3,5-AMP-phos-phodiestérase (PDE) est diminuée dans le foie, le tissu adipeux et, de façon non-significative, dans le muscle strié des rats qui manquent d'insuline, c-à-d les rats rendus diabétiques par l'alloxane ou les rats privés de nourriture. L'injection intraveineuse d'une faible dose d'insuline (0.5 U/kg) ou la stimulation de la sécrétion d'insuline endogène par une injection de glucose provoquent une augmentation rapide de l'activité de la phosphodiestérase dans ces tissus. 15 min après l'injection d'insuline, l'activité de la phosphodiesterase du foie est augmentée. L'effet maximum est atteint après 30–45 min. L'activité de la phosphodiestérase rénale n'est pas diminuée dans le diabète alloxanique, l'injection d'insuline s'est avérée inefficace.In vitro, l'insuline cristalline a un effet activant sur la phosphodiestérase purifiée du coeur de boeuf. La concentration d'insuline requise pour doubler l'activité de l'enzyme est de l'ordre de 2 · 10^–5 M. Le traitement avec actinomycin D empêche la stimulation par l'insuline de la PDE dans le foie. Ceci peut indiquer que l'action de l'insuline sur l'activité de la phosphodiestérase est essentiellement basée sur une synthèse accrue de l'enzyme. A cause de l'influence de la sécrétion d'insuline sur la concentration en 3,5-AMP du foie et du tissu adipeux, le métabolisme du glycogène et la lipolyse peuvent s'adapter rapidement à la prise de nourriture.

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Non-Standard Abbreviations G 6 P Glucose-6-phosphate - UDPG UDP-glucose - FFA non-esterifled, free fatty acids - 3,5-AMP cyclic adenosine-3,5-monophosphate - PDE 3,5-AMP phosphodiesterase This study was supported by the Deutsche Forschungsgemeinschaft.Deceased October 31, 1967.     

Majority of Gliadin-Specific T-Cell Clones from Celiac Small Intestinal Mucosa Produce Interferon-γ and Interleukin-4

An abnormal mucosal cell-mediated immune response plays a fundamental role in the pathogenesis of celiac disease. To characterize locally infiltrating T cells, gliadin-specific T-cell clones were isolated from two treated celiac patients. Mucosal biopsies were cultured in vitro for 24 hr with a peptic-tryptic digest (PT) of gliadin. T-cell clones (TCC) were then isolated by limiting dilution. The production of interferon- (IFN-) and interleukin-4 (IL-4) was evaluated by ELISA in culture supernatants obtained after a short incubation with anti-CD3 and PMA, or with antigen. Twenty-two TCC were specific for gliadin and/or PT. All were CD3⁺, CD4⁺, CD8⁺, TCR ⁺. In one such clone the PT-specific response was inhibited by an anti-DQ, but not by an anti-DR antibody. Of the five gliadin-specific TCC examined, four produced IL-4 and high levels of IFN-; the remaining one initially produced only IL-4, but subsequently also IFN-. All clones obtained from the celiac mucosa, including the gliadin-specific ones, produced high levels of IFN-, in most cases with IL-4. This cytokine profile could explain most of the immunological features of the celiac mucosa.     

Triplication (/ααα<Superscript>Anti3.7</Superscript>) or deletion (-α<Superscript>3.7</Superscript>/) association in Argentinian β-thalassemic carriers

Prevalence of alpha gene triplication or deletion in -thalassemia carriers was studied in 109 unrelated individuals in Rosario, Argentina. In different populations -^3.7 allele presents a higher prevalence than ^anti3.7; thus, -thalassemia associated with -thalassemia is more frequently observed. Nevertheless, this event was detected in only one case (0.9%), while the association with alpha triplication was present in two subjects (1.8%).     

Bone marrow and peripheral blood globin chain biosynthesis in iron deficiency

Summary Globin chain synthesis was studied in 13 iron-deficient patients. The mean whole-cell globin / ratio in the peripheral blood of 11 patients was 1.05±0.06 which is similar to the value 0.99±0.08 obtained for 10 controls. The ratios odtained for stroma-free globin were not significantly different from those of whole cell preparations. In contrast, the / ratio of bone marrow was 0.73±0.14 in 10 iron deficient patients, which is significantly lower than that of controls. Two other patients had decreased / ratios in the peripheral blood, probably because of the presence of an -thalassemia gene. These results demonstrate a reduced rate of synthesis of chains relative to that of chains in the bone marrow of iron-deficient patients that is not demonstrable in the peripheral blood.This work was partly supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil     

Effects of Voltage-Sensitive Calcium Channel Blockers on Extracellular Dopamine Levels in Rat Striatum

Various subtypes of voltage-sensitive calcium channels (VSCCs) support the release of dopamine (DA) in the central nervous system. Using in vivo microdialysis, we investigate the influence of these subtypes of calcium channels on dopaminergic terminals in the rat striatum. L-type (nifedipine-sensitive), N-type (-conotoxin GVIA-sensitive), or N- and P/Q-type (-conotoxin MVIIC-sensitive) Ca²⁺ channels were blocked using selective antagonists injected locally, and K⁺-evoked DA release was measured in freely moving animals. K⁺(100 mM) induced a massive increase of basal DA extracellular levels (930%) and was without significant effect on extracellular levels of DA metabolites DOPAC and HVA, and on the serotonin metabolite 5HIAA. -Conotoxin GVIA (1 M) and -conotoxin MVIIC (1 M) significantly reduced the K⁺-evoked DA release by 55 and 62%, respectively. The simultaneous application of the two conotoxins at the same concentration reduced K⁺-evoked DA release by 66%. Nifedipine (10 M) had no significant effect on K⁺-evoked DA release, while neomycin, a nonspecific VSCC blocker, produced a highly significant decrease when applied at 250 and 500 M (56 and 75%, respectively). The compounds, however, had no effect on basal DA release and on the levels of extracellular DOPAC, HVA, and 5HIAA. These results suggest that under high and persistent conditions of membrane depolarization (15 min, 100 mM K⁺), striatal DA release is mainly mediated by N-type VSCCs.     

Regulation of the release of tumour necrosis factor (TNF)α and soluble TNF Receptor by γ irradiation and interferon γ in Ewing's sarcoma/peripheral primitive neuroectodermal tumour cells

This study analyses the production of tumour necrosis factor (TNF) and soluble TNF receptor (sTNF-R) before and after exposure to irradiation and interferon (IFN) in 12 cell lines derived from Ewing's sarcoma (ES)/peripheral primitive neuroectodermal tumours (pPNET). Supernatants from ES/pPNET cell cultures were tested in a TNF-specific amplified enzyme-linked immunosorbent assay (ELISA), a bioassay, and sTNF-R_p55 and sTNF-R_p75 ELISA. The tumour cell lines released minimal amounts of TNF, prominent amounts of sTNF-R_p55 (7/12 cell lines) and no sTNF-R_p75. Exposure to irradiation (5 Gy) either induced (3/12) cell lines) or up-regulated (3/12 cell lines) TNF release without changing sTNF-R_p55 and sTNF-R_p75 levels. Priming of cultures with recombinant human IFN (rhIFN) markedly enhanced TNF secretion in the radiation-responsive cell lines and had no influence on sTNF-R_p55 and sTNF-R_p75 levels. rhIFN affected the magnitude rather than the sensitivity of the radiation response. The TNF secreted was bioactive, as shown by its cytotoxic effect of WEHI-164 cells, and neutralization of its activity by anti-TNF monoclonal antibody. Herbimycin A (a tyrosine-specific protein kinase inhibitor) but not calphostin C (a protein kinase C inhibitor), H89 (a protein kinase A inhibitor), AACOCF3 (a specific inhibitor of phospholipase A₂) and MK-886 (a specific inhibitor of 5-lipoxygenase) abrogated -irradiation-stimulated TNF release. The antioxidantsN-acetylcysteine, nordihydroguaiaretic acid and mepacrine dose-dependently inhibited -irradiation-mediated TNF production. Collectively our findings indicate that IFN priming potentiates the secretion of bioactive TNF by ES/pPNET cells in response to irradiation without affecting sTNF-R release. The data suggest a requirement for protein tyrosine kinase activity and a role for reactive oxygen species in the -irradiation-mediated intracellular signalling pathway leading to TNF production.     

Specific CTL Activity of CD8+ TCR Vβ14+ T Cell in Mouse 2, 4, 6-Trinitrobenzene Sulfonic Acid-Induced Colitis

We analyzed the functional role of CD8⁺ T-cell receptor (TCR) V14⁺ T cells, which increased specifically in the lamina propria in 2,4,6-trinitrobenzene sulfonic acid (TNBS) -induced colitis. Cytotoxic activity and cytokine production in CD8⁺ TCR V14⁺ T-cell clones were analyzed by ⁵¹Cr release assay and enzyme-linked immunosorbent assay, respectively. Cell transfer studies using these clones were performed. Established T-cell clones showed specific cytotoxic activity against TNBS-conjugated self spleen cells, and this cytotoxicity was completely inhibited by anti-TCR V14 monoclonal antibody. These clones produced interferon (IFN) - in their culture supernatant, but neither interleukin (IL) - 2 nor IL-4. Histological findings of the colon in mice, which received clone transfer after enema with suboptimal doses of TNBS, showed massive colitis. Our results indicate that CD8⁺ TCR V14⁺ T cells had a cytotoxic T-lymphocyte function induced by Th-1 T-cell response and played a pathogenic role in the development of TNBS-induced colitis.     

B cell adrenoceptors and sulphonylurea-induced insulin release in mouse islets

Summary Interactions of tolbutamide and glibenclamide with B cell adrenoceptors have been reported. This study evaluated the possible role of such interactions in the stimulation of insulin release. Mouse islets were incubated in the presence of 10 mmol/l glucose alone or with tolbutamide (10 mol/l) or glibenclamide (0.02 mol/l). At 0.01–10 mol/l, blockers of ₂-adrenoceptors (yohimbine, idazoxan) or ₁-adrenoceptors (prazosin) had practically no effect on glucose-induced insulin release and did not affect its potentiation by sulphonylureas, except for a slight increase by 10 mol/l prazosin and idazoxan. Nonspecific -blockers (phentolamine, dihydroergotamine) increased control release at 10 mol/l, but only the latter amplified the response to tolbutamide. Blockers of -adrenoceptors were tested at 0.1–100 mol/l: propranolol (₁, ₂), metoprolol (₁) and compound ICI 118-551 (₂). They increased glucose-induced insulin release at 100 mol/l but variably altered the effect of sulphonylureas. Blockers of adrenoceptors have, thus, no effect on insulin release in vitro at therapeutic concentrations. At high concentrations, they non-specifically affect the action of sulphonylureas. We conclude that an interaction with B cell adrenoceptors is not involved in the insulinotropic action of sulphonylureas.     

<Superscript>13</Superscript>C NMR analysis reveals a link between L-glutamine metabolism,D-glucose metabolism and γ-glutamyl cycle activity in a clonal pancreatic beta-cell line

Aims/hypothesis Pancreatic islet cells and clonal beta-cell lines can metabolise L-glutamine at high rates. The pathway of L-glutamine metabolism has traditionally been described as L-glutamineL-glutamate2-oxoglutarateoxidation in TCA cycle following conversion to pyruvate. Controversially, the metabolism of D-glucose to L-glutamate in beta cells is not widely accepted. However, L-glutamate has been proposed to be a stimulation-secretion coupling factor in glucose-induced insulin secretion. We aimed to investigate the metabolism of glutamine and glucose by using ¹³C NMR analysis.Methods BRIN-BD11 cells were incubated in the presence of 16.7 mmol/l [1-¹³C]glucose, 2 mmol/l [2-¹³C]L-glycine or 2 mmol/l [1,2-¹³C]glutamine in the presence or absence of other amino acids or inhibitors. After an incubation period the cellular metabolites were extracted using a PCA extract procedure. After neutralisation, the extracts were prepared for analysis using ¹³C-NMR spectroscopy.Results Using ¹³C NMR analysis we have shown that L-glutamine could be metabolised in BRIN-BD11 cells via reactions constituting part of the -glutamyl cycle producing glutathione. Moderate or high activities of the enzymes required for these pathways of metabolism including glutaminase, -glutamyltransferase and -glutamylcysteine synthetase were observed. We additionally report significant D-glucose metabolism to L-glutamate. Addition of the aminotransferase inhibitor, aminooxyacetate, attenuated L-glutamate production from D-glucose.Conclusion/interpretation We propose that L-glutamine metabolism is important in the beta cell for generation of stimulus-secretion coupling factors, precursors of glutathione synthesis and for supplying carbon for oxidation in the TCA cycle. D-glucose, under appropriate conditions, can be converted to L-glutamate via an aminotransferase catalysed step.Abbreviations NMR Nuclear Magnetic Resonance     

Thalassemia intermedia: compound heterozygous β∘/β+-thalassemia and co-inherited heterozygous α+-thalassemia

Summary The relative excess of - over -globin chains in the erythroid precursors is the chief pathophysiological factor of homozygous -thalassemia. The clinical picture is usually characterized by a transfusion-dependent dyserythropoietic anemia (thalassemia major). However, some patients present with moderate anemia that does not require regular blood transfusions (thalassemia intermedia). The molecular heterogeneity of -thalassemia mutations and changes of - and -globin gene expression play an important role in modifying the clinical phenotype. We report here on a female Greek patient with homozygous -thalassemia but normal growth and development, excellent exercise tolerance, and no need of blood transfusions. She is thus mildly affected clinically, although there is marked pallor, jaundice, and hepatosplenomegaly. These signs correspond to her marked hypochromic, microcytic anemia with erythroid hyperplasia of the bone marrow. -Globin genotyping shows her to be compound heterozygous for the codon 39 C T -nonsense mutation and for the T C ⁺-mutation at position 6 of the splice consensus at the exon 1/intron 1 junction (CD39 C T/IVS 1–6 T C). -Globin gene mapping demonstrates the presence of a 3.7-kb ⁺-thalassemia deletion on one allele (–^3.7/). Taken together, this study identifies a complex interaction of genetic factors that do not significantly alter the clinical phenotype when present alone but ameliorate the course of homozygous -thalassemia when inherited in combination.Abbreviations Hb hemoglobin - Hct hematocrit - HPFH hereditary persistence of fetal hemoglobin - IVS intervening sequence - MCH mean corpuscular hemoglobin - MCV mean corpuscular volume - PCR polymerase chain reaction     

Intratumoral injection of interferon-gamma gene-modified dendritic cells elicits potent antitumor effects: effective induction of tumor-specific CD8<Superscript>+</Superscript> CTL response

Purpose To examine the antitumor efficacy of intratumoral injection of interferon-gamma gene-modified dendritic cells (DC-IFN-) in a B16 melanoma model and to investigate its related immunological mechanisms.Methods C57BL/6 mice-derived DC were transfected with adenovirus encoding IFN- or -galactosidase (DC-LacZ). Secretion of IFN- and TNF- by DC was detected by ELISA. Nitric oxide (NO) production was measured by Griess reaction. Cytotoxicity of DC against tumor cell lines and activity of cytotoxic T lymphocytes (CTLs) were determined by ⁵¹Cr-release assay. TRP-2aa180–188-specific CD8⁺ CTLs in tumor-bearing mice with different treatment were determined by ELISPOT.Results DC-IFN- could secrete high levels of IFN-, NO and TNF-. DC-IFN- were cytolytic to B16 melanoma cells in vitro, but DC-LacZ and DC were not. Significant inhibition of tumor growth and prolonged survival were achieved in tumor-bearing mice intratumorally injected with DC-IFN- when compared with those in tumor-bearing mice intratumorally injected with DC, DC-LacZ, fibroblasts, IFN- gene-modified fibroblasts or PBS. After treatment with DC-IFN-, enhanced Th1 and decreased Th2 responses were observed, and B16 melanoma antigen TRP-2aa180–188-specific CD8⁺ CTLs were induced significantly in the tumor-bearing mice.Conclusions Intratumorally injected DC-IFN- can uptake tumor antigens in situ and cross-present tumor antigens to specific CD8⁺ T cells, hereby eliciting effective antitumor effects in murine model with preestablished B16 melanoma.     

Liver microsomal Δ6 and Δ5 linoleic acid desaturation in female BB rats

Summary Rat liver microsomal 6 and 5 desaturation are defective in experimental diabetes, but this defect is correctable with insulin treatment. Rat liver fatty acid composition and 6 and 5 desaturation were studied in the spontaneously diabetic adult female Bio-Breeding (BB) rat. Control Wistar rats and BB rats (4 weeks of diabetes), that received insulin (1 IU·100 g body weight^–1·day^–1), were killed 20 h after the last insulin injection. 6 and 5 desaturase activities were estimated from the incubation of liver microsomes with (1-¹⁴C) 18:2, n–6 or (2-¹⁴C) 20:3, n–6, respectively, and the fatty acid composition of the liver and microsomal liver lipids were investigated. Under experimental conditions 6 and 5 desaturase activities were unchanged in the BB rats when compared to the control rats. Impairment of the liver fatty acid composition of diabetic BB rats is not consistent with normal desaturase activity and may be explained by factors other than desaturation disturbance.     

Antiproliferative effects of interferonγ in combination withα-difluoromethylornithine on human carcinoma cell cultures

The antiproliferative effects of human recombinant interferon (IFN) in combination with -difluoromethylornithine (DFMO) or as single agents were assessed on human cell cultures derived from carcinomas of the breast (MCF-7), the ovary (EFO-27) or the kidneys (EGI-4). Results were obtained in proliferation assays by direct cell counting. The cell lines differed considerably in their sensitivities to the antiproliferative effect of IFN as compared by the 50% inhibition doses of the growth (ID₅₀). In contrast to the findings with IFN, similar antiproliferative effects resulted from the application of comparable doses of DFMO. While IFN induced cytotoxic effects in EGI-4 cells, DFMO produced only cytostatic actions in the cell lines analyzed. Synergistic growth inhibition resulted from the combined application of IFN and DFMO in EFO-27 cell cultures. This finding was most pronounced after treatment with IFN or DFMO doses below the respective ID₅₀ values. However, antagonistic effects occurred in cells of the line EGI-4 after DFMO had been combined with IFN at concentrations below the cytotoxic dose range. Within the sensitivity of our proliferation assay, no synergistic interactions were found in MCF-7 cell cultures. In the cell lines tested, no relation between the sensitivity for the single agents and the effectivity of the drug combination was identified. Despite promising synergistic effects in the moderately IFN-sensitive ovarian carcinoma cell line EFO-27, the efficacy of the IFN/DFMO combination was restrained by possible antagonistic effects as demonstrated in the highly IFN-sensitive EGI-4 renal carcinoma cell cultures. We conclude that the differential interaction patterns in the cell cultures analyzed preclude general suggestions for clinical studies using IFN and DFMO.Abbreviations IFN interferon - DFMO difluoromethylornithine This work was part of the doctoral thesis of Mariam Klouche     

γ-Glutamyl transpeptidase-cellular expression in populations of normal human mononuclear cells and patients suffering from leukemias

The expression of the ectoenzyme-glutamyl transpeptidase (EC2.3.2.2.,GT) was investigated by flow cytometry on populations of peripheral blood mononuclear cells (PBMC) from healthy subjects and patients suffering from several types of leukemia before and under chemotherapy. In unstimulated PBMC, 28% of these cells were found to beGT positive. The highest expression was measured on monocytes (CD14/GT⁺ cells: 60%). Within the subsets of T lymphocytes (CD3/GT⁺ cells: 18%) we saw no clear differences between CD4⁺ and CD8⁺ cells. B lymphocytes, NK cells, and activated cells showed low expressions (up to 10%). Treatment of PBMC with mitogens, -IFN, IL-2, and GM-CSF did not affect the enzyme expression on normal mononuclear cells (MNC). However, a rapid increase of yGT⁺ cells was found in the presence of glutathione (GSH) and n-acetyl cysteine (nAC), particularly on monocytes, B cells, and NK cells. Comparing 40 healthy subjects and untreated patients suffering from leukemias, a significantly higher expression ofGT⁺ cells in the total MNC populations (B-CLL: 57%, CML: 62%GT⁺ cells) was observed in B-chronic lymphocytic leukemia (B-CLL) and chronic myelogenous leukemia (CML), whereas other leukemias did not show clear differences. Most interestingly, theGT expression was diminished in all populations of CML cells after 5 h of incubation in the presence of 10 units/ml IFN-. These data suggest a possible protective role ofGT in MNC and a regulatory function of this enzyme in the development of CML.