Tolerogenic dendritic cells induce antigen-specific hyporesponsiveness in insulin- and glutamic acid decarboxylase 65-autoreactive T lymphocytes from type 1 diabetic patients

Tolerogenic dendritic cells (tDC) constitute a promising therapy for autoimmune diseases, since they can anergize T lymphocytes recognizing self-antigens. Patients with type 1 diabetes mellitus (T1D) have autoreactive T cells against pancreatic islet antigens (insulin, glutamic acid decarboxylase 65 -GAD65-). We aimed to determine the ability of tDC derived from T1D patients to inactivate their insulin- and GAD65-reactive T cells. CD14 + monocytes and CD4 + CD45RA- effector/memory lymphocytes were isolated from 25 patients. Monocyte-derived DC were generated in the absence (control, cDC) or presence of IL-10 and TGF-β1 (tDC), and loaded with insulin or GAD65. DC were cultured with T lymphocytes (primary culture), and cell proliferation and cytokine secretion were determined. These lymphocytes were rechallenged with insulin-, GAD65- or candidin-pulsed cDC (secondary culture) to assess whether tDC rendered T cells hyporesponsive to further stimulation. In the primary cultures, tDC induced significant lower lymphocyte proliferation and IL-2 and IFN-γ secretion than cDC; in contrast, tDC induced higher IL-10 production. Lymphocytes from 60% of patients proliferated specifically against insulin or GAD65 (group 1), whereas 40% did not (group 2). Most patients from group 1 had controlled glycemia. The secondary cultures showed tolerance induction to insulin or GAD65 in 14 and 10 patients, respectively. A high percentage of these patients (70–80%) belonged to group 1. Importantly, tDC induced antigen-specific T-cell hyporesponsiveness, since the responses against unrelated antigens were unaffected. These results suggest that tDC therapy against multiple antigens might be useful in a subset of T1D patients.     

In Memoriam

This paper summarizes the results on the epidemiology and molecular basis of thalassemias and other hemoglobinopathies in the Republic of Macedonia. Over the past 40 years, population surveys of more than 22,000 participants (school children and workers) from all over the country, have shown that the average incidence of β-thalassemia (thal) trait is 2.6%, ranging from less than 1% in the northeast to 10% in the south. The frequency of δβ-thal is 0.2%, while the frequency of the Swiss type of hereditary persistence of fetal hemoglobin (HPFH) is 0.3%. Screening of 9,619 newborns has shown that the frequency of α-thal trait is 1.5%, of which α-thal-2 is 1.45% and α-thal-1 is 0.05%. The molecular basis of the different forms of β-thal and other hemoglobinopathies has been completely defined. Among the Macedonians, over 450 β-thal chromosomes have been studied. Fifteen different β-thal mutations have been detected, four of which [IVS-I-110 (G→A), IVS-I-6 (T→C), IVS-I-1 (G→A), codon 39 (C→T)] account for 85% of all β-thal chromosomes. Among the Albanians, 48 β-thal chromosomes have been studied. Eight different mutations have been detected, four of which [codon 39, ?30 (T→A), IVS-I-110, IVS-I-1] account for 85% of all β-thal chromosomes. Four new mutations [?101 (C→A), ?87 (C→G), ?30, polyadenylation signal (poly A) (AATAAA→AATGAA)] have been characterized. Molecular analyses of DNA from over 20 unrelated cases with δβ-thal have shown that this condition is caused by a 13 kb deletion (Sicilian type); in two families a deletion of 18 to 23 kb (Macedonian type of δβ-thal) was discovered. Molecular analyses of α-thal in the Republic of Macedonia have shown the following types of molecular defects: 20.5 kb deletion, 17.5 kb deletion, 3.7 kb deletion, poly A mutation (AATAAA→AATGAA), and Hb Icaria [α142, Term→Lys, TAA→AAA (α2)]. The incidence of abnormal hemoglobins (Hbs) in the Republic of Macedonia is 0.4%. Three different α chain variants among 10 families, seven different β chain variants among 33 families, two γ chain variants in two newborns, one variant with an extended α chain, and Hb Lepore among 105 families, have been observed. Structural analysis of numerous cases with Hb Lepore showed that the variant was of the Washington-Boston type.     

IDENTIFICATION OF TWO NEW β-THALASSEMIA SPLICE MUTATIONS: IVS-I-1 (G → C) AND IVS-I (−2) (A → C)

Hb Constant Spring [Hb CS; α142, Term→Gln (TAA>CAA in α2)] is a nondeletional α-thalassemia (α-thal) defect difficult to detect on conventional electrophoresis because of its small amount in heterozygotes. We have found that individuals with an Hb CS trait could efficiently be detected using the Sebia capillarys 2 system. In the present study, we have confirmed this method in a cohort of 23,842 individuals from Guangdong Province (South China). Hb CS was detected in 71 (0.3%) of the cases. The levels of Hb CS in heterozygotes ranged from 0.1–1.0% with an average of 0.6%. We propose the reported 0.3% as a realistic figure for the prevalence of Hb CS in South China.     

Erythrocytosis in a Child due to Hb Andrew-Minneapolis [β144(HC1)Lys→Asn (AAG>AAT or AAC)] Associated with a Spanish (δβ)0-Thalassemia

We report a rare association of δβ-thalassemia (δβ-thal) and a hemoglobin (Hb) variant with high oxygen affinity in a Spanish newborn. The proband had no Hb A and showed microcytosis and hypochromia; the peripheral blood smear was compatible with a thalassemia trait. Molecular studies revealed that the proband had a Spanish (δβ)⁰-thal (inherited from his father) and also carried a de novo variant (Hb Andrew-Minneapolis) because from the point of hematology, his mother was quite normal.

The hemoglobinopathies with high affinity for oxygen constitute an infrequent cause of secondary congenital erythrocytosis. The degree of erythrocytosis and the resulting clinical manifestations are highly variable, depending on the degree of altered oxygen affinity and the presence of thalassemic genes. Thus, when these variants are associated with β⁰- or δβ-thal, as in our case, the proportion of abnormal Hb is ～100.0%, which may cause polycythemia, hyperviscosity, and iron deficiency. This type of association is very rare and few have been described, especially in children, as they would normally be detected in adults as the increased packed cell volume (PCV) also increases blood viscosity and causes the typical symptoms (cephalalgia, drowsiness, dizziness).

The association of a high oxygen affinity Hb and a δβ-thal presents a greater degree of erythrocytosis than when this same variant is associated with a β⁰-thal, mainly because the Hb F percentage is usually greater in the δβ-thal, and Hb F normally shows a greater affinity for oxygen and a reduced P₅₀, although one must always take into account the degree of oxygen affinity of the Hb variant. Familial erythrocytosis and an abnormal electrophoresis finding are indicative of a high affinity Hb. However, the absence of these findings does not reject the possibility of hemoglobinopathies, and in these cases, functional and molecular studies would be justified and should be mandatory for the differential diagnosis of erythrocytosis.     

A New High Affinity Variant Hb Aurillac (β141Leu→Val)

Hemoglobin (Hb) variant β141(H19)Leu→Val (HBB:c.424C>G), one of the two mutations defining Hb Kochi [the other one being β144(HC1)Lys-Tyr-His→0 (HBB:c.433A>T)], was found as an isolated mutation. In contrast to what was suggested for Hb Kochi, the new variant was not clinically silent. It displayed increased oxygen affinity and was associated with mild erythrocytosis.     

Clinical Genotyping by Next Generation Sequencing Reveals a Novel,De Novo β-Globin Gene Mutation Causing Hemolytic Anemia in a Chinese Individual

Abstract

Abnormal hemoglobins (Hbs) are one of the most common hemoglobinopathies worldwide. Some Hb gene mutations may produce unstable, abnormal Hbs causing macrocytic hemolysis. We identified a novel, de novo deletion/frameshift mutation at nucleotide position 408 in exon 3 of the β-globin gene (HBB: c.408delT) compound with an Hb F-associated regulatory single nucleotide polymorphism (rSNP) (rs368698783) through next generation sequencing (NGS). This β-globin gene variant was identified in a 5-year-old Chinese girl with splenomegaly, jaundice and macrocytic, hemolytic anemia. This variant causes a new stop codon to be formed in the 3' untranslated region (3'UTR) of the HBB gene at amino acid position 158, consequently leading to a β-sheet disruption of the last α helix of this abnormal β-globin chain. We named this variant Hb Urumqi after the proband’s current city of residence.     

A New Highly Unstable α Chain Variant Causing α+‐Thalassemia: Hb Zurich Albisrieden [α59(E8)Gly→Arg (α2)]

A new α‐globin mutation causing persistent mild hypochromic microcytosis and erythrocytosis is described. Hb Zurich Albisrieden [α59(E8)Gly→Arg (α2)] is not detected at the protein level and leads to α⁺‐thalassemia (thal).     

The ‘Hot Spot’ of Hb E [β26(B8)Glu→Lys] in Southeast Asia: β‐Globin Anomalies in the Lao Theung Population of Southern Laos

Hb E [β26(B8)Glu→Lys], is the most common abnormal hemoglobin (Hb) in Southeast Asian populations. The hitherto highest frequencies of the Hb E gene (HBB^*E) in large population samples, ～?0.3, were observed in the southern part of northeastern Thailand. The finding of even higher frequencies in a small, isolated Austroasiatic group in Northeast Thailand prompted us to examine samples of three Austroasiatic populations in southern Laos (official designation: Lao Theung), an area inhabited by numerous ethnic groups belonging to the Mon–Khmer branch. Blood samples were collected from a total of 603 adult subjects. The HBB^*E frequencies were 0.426 in the So of Khammuan Province, 0.433 in the Alak/Ngeh of Sekong Province and 0.253 in the Oy of Attapeu Province. The HBB^*E frequencies in the So and Alak/Ngeh are the highest observed in Southeast Asia in representative population samples. None of the common Southeast Asian β‐thalassemia (thal) mutations were found. The results are discussed with respect to natural selection by malaria, selection time, effects of β‐thal and the ethnic history of the population of Southeast Asia.