Myeloid glycosylation defects lead to a spontaneous common variable immunodeficiency-like condition with associated hemolytic anemia and antilymphocyte autoimmunity.

Common variable immunodeficiency (CVID), the most frequent symptomatic primary immune deficiency in humans, is a heterogeneous group of immunologic disorders estimated to affect 1:10,000-1:50,000. Although a clear disease etiology remains elusive, a common characteristic of CVID is deficient IgG Ab production in response to infection or vaccination. Patients often also exhibit autoimmune cytopenias with symptoms of abnormal T cell function, including reductions in naive T cells, which correlate with clinical severity. In this study, we discovered that targeted alterations in the glycome of the myeloid lineage lead to spontaneous immunodeficiency characteristic of both humoral and T cell dysfunction regularly found in human CVID. Mice carrying a myeloid-specific knockout of the Mgat2 gene encoding UDP-GlcNAc:α-6-d-mannoside ß-1,2-N-acetylglucosaminyltransferase II enzyme exhibit deficiencies in IgG responses to both protein and polysaccharide conjugate vaccines. Interestingly, the immunodeficiency is associated with decreased T cell activity because of a persistent autoimmune-mediated depletion of naive T cells, which is induced by changes in erythrocyte surface glycosylation. The N-glycosylation dependent autoepitopes that emerge on erythrocytes lead to autoimmune hemolytic anemia, and the causative auto-IgM cross-reacts with naive T cells despite the lack of glycan change on T cells. These findings demonstrate that alterations in erythrocyte glycosylation trigger the development of autoantibodies directed at both erythrocytes and naive T cells, revealing a possible mechanistic link between the induction of autoimmune hemolytic anemia, the reduction in naive T cells, and poor Ab responses to vaccine in severe CVID patients.

TNT003, an inhibitor of the serine protease C1s, prevents complement activation induced by cold agglutinins.

Activation of the classical pathway (CP) of complement is often associated with autoimmune disorders in which disease pathology is linked to the presence of an autoantibody. One such disorder is cold agglutinin disease (CAD), an autoimmune hemolytic anemia in which autoantibodies (cold agglutinins) bind to red blood cells (RBCs) at low temperatures. Anemia occurs as a result of autoantibody-mediated CP activation on the surface of the erythrocyte, leading to the deposition of complement opsonins that drive extravascular hemolysis in the liver. Here we test the effects of TNT003, a mouse monoclonal antibody targeting the CP-specific serine protease C1s, on CP activity induced by cold agglutinins on human RBCs. We collected 40 individual CAD patient samples and showed that TNT003 prevented cold agglutinin-mediated deposition of complement opsonins that promote phagocytosis of RBCs. Furthermore, we show that by preventing CP activation, TNT003 also prevents cold agglutinin-driven generation of anaphylatoxins. Finally, we provide evidence that CP activity in CAD patients terminates prior to activation of the terminal cascade, supporting the hypothesis that the primary route of RBC destruction in these patients occurs via extravascular hemolysis. Our results support the development of a CP inhibitor for the treatment of CAD.

Indoleamine 2,3 dioxygenase contributes to transferable tolerance in rat red blood cell inducible model of experimental autoimmune haemolytic anaemia.

Autoimmune haemolytic anaemia (AIHA) is caused by autoantibodies against red blood cell (RBC) surface antigens that render RBC susceptible to Fc-mediated phagocytosis and complement-mediated lysis. Experimental AIHA can be induced by injection of rat RBC to naive mice, but a lymphocyte-mediated regulatory mechanism eventually suppresses the production of autoantibodies specific for mouse RBC. Critically, this tolerogenic response can be transferred to naive mice by splenocytes from the rat RBC-immunized mouse. Here we investigate whether indoleamine 2,3 dioxygenase (IDO) or the initiators of IDO cascade, including the cytotoxic T lymphocyte antigen (CTLA)-4 receptor and its soluble isoform, contribute to this tolerogenic mechanism. Splenocytes from experimental AIHA mice were transferred adoptively to naive mice under the cover of anti-CTLA-4, anti-soluble CTLA-4 antibodies or IDO inhibitor 1-methyl tryptophan (1-MT). Recipient mice were immunized with rat RBC and levels of antibody against self-RBC and rat-RBC were monitored. Our results indicate that transfer of tolerance to naive recipients is dependent upon IDO-mediated immunosuppression, as mice receiving previously tolerized splenocytes under the cover of 1-MT were refractory to tolerance and developed haemolytic disease upon further challenge with rat RBC. Initiators of IDO activity, CTLA-4 or soluble CTLA-4 did not mediate this tolerogenic process but, on their blockade, boosted antigen-specific effector immune responses.

Exome sequencing and unrelated findings in the context of complex disease research: ethical and clinical implications.

Exome sequencing has identified the causes of several Mendelian diseases, although it has rarely been used in a clinical setting to diagnose the genetic cause of an idiopathic disorder in a single patient. We performed exome sequencing on a pedigree with several members affected with attention deficit/hyperactivity disorder (ADHD), in an effort to identify candidate variants predisposing to this complex disease. While we did identify some rare variants that might predispose to ADHD, we have not yet proven the causality for any of them. However, over the course of the study, one subject was discovered to have idiopathic hemolytic anemia (IHA), which was suspected to be genetic in origin. Analysis of this subject's exome readily identified two rare non-synonymous mutations in PKLR gene as the most likely cause of the IHA, although these two mutations had not been documented before in a single individual. We further confirmed the deficiency by functional biochemical testing, consistent with a diagnosis of red blood cell pyruvate kinase deficiency. Our study implies that exome and genome sequencing will certainly reveal additional rare variation causative for even well-studied classical Mendelian diseases, while also revealing variants that might play a role in complex diseases. Furthermore, our study has clinical and ethical implications for exome and genome sequencing in a research setting; how to handle unrelated findings of clinical significance, in the context of originally planned complex disease research, remains a largely uncharted area for clinicians and researchers.

Hemolysis and elevated transaminases imitating severe preeclampsia.

BACKGROUND: Illnesses coincident with pregnancy may present similarly to preeclampsia or may be mistaken for severe preeclampsia or hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome. Distinguishing these imitators from preeclampsia is important to allow for appropriate treatment and to avoid unnecessary delivery. CASE: A primigravida at 32 2/7 weeks of gestation transferred to our institution with flu-like symptoms, anemia, jaundice, and elevated liver function tests. The team caring for her was concerned about HELLP or acute fatty liver. After extensive workup, she was found to have a cold agglutinin. Her laboratory test results improved, and she was discharged undelivered with a presumptive diagnosis of cold agglutinin autoimmune hemolytic anemia. CONCLUSION: When patients present with atypical features of HELLP syndrome, clinicians should take time to consider other "imitators of preeclampsia" before rapidly progressing to delivery to avoid inappropriate treatment of the disorder.

Differential expression of carbonic anhydrase isoenzymes in various types of anemia.

BACKGROUND: The aim of the present study was to determine the concentrations of cytosolic carbonic anhydrase (CA) isoenzymes in erythrocytes of patients with aplastic, autoimmune hemolytic, iron deficiency or beta-thalassemia anemia. METHODS: Western blotting and CA esterase activity analysis were used to analyze cytosolic erythrocyte CA isoenzymes in 118 subjects with various types of anemia and 35 healthy controls. RESULTS: Total CA activity and CAII concentration of anemia patients were significantly higher than that of the control subjects while CAI concentration was significantly lower in patients of autoimmune hemolytic anemia (P<0.01). Compared with controls, CAIII concentration was lower in iron deficiency anemia (P<0.01), but higher in beta-thalassemia anemia (P<0.001). CONCLUSIONS: Carbonic anhydrase isoenzymes may contribute differently to various types of anemia. CAI may be an indicator to differentiate autoimmune hemolytic anemia from other types of anemia. CAII provides the CA activity necessary for maintaining ion transport in erythrocytes while CAIII may play an agent against oxidative damage in iron deficiency and beta-thalassemia anemia.

Ciprofloxacin-induced acute interstitial nephritis and autoimmune hemolytic anemia.

Ciprofloxacin has been associated with several side effects including interstitial nephritis and hemolytic anemia. The combination of both side effects is extremely rare. In this report, we describe a case of ciprofloxacin-induced interstitial nephritis and autoimmune hemolytic anemia. Hemolytic anemia improved after stopping the drug and initiation of steroid therapy. Unfortunately, acute interstitial nephritis was irreversible and the patient developed end-stage renal disease.

Pyrimidine nucleotides impair phosphoribosylpyrophosphate (PRPP) synthetase subunit aggregation by sequestering magnesium. A mechanism for the decreased PRPP synthetase activity in hereditary erythrocyte pyrimidine 5'-nucleotidase deficiency.

The activity of phosphoribosylpyrophosphate (PRPP) synthetase (ATP: D-ribose-5-phosphate pyrophosphotransferase, EC 2.7.6.1) is decreased in the erythrocyte in hereditary pyrimidine 5'-nucleotidase (P5N) deficiency. Given the increased pyrimidine nucleotide content of the P5N-deficient erythrocyte, we evaluated the effects of prototypic pyrimidine nucleotides on the activity of PRPP synthetase. In normal hemolysate a 1.0 mM combination of cytidine tri-, di- and monophosphate (CTP/CDP/CMP) inhibited PRPP synthetase activity and changed the ribose 5-phosphate (R5P) saturation curve from a hyperbola to a biphasic shape. Untreated crude hemolysate from P5N-deficient erythrocytes showed a biphasic R5P kinetic curve. Since the activity of PRPP synthetase is dependent on its state of subunit aggregation, we examined PRPP synthetase subunit aggregation using gel permeation chromatography. P5N-deficient erythrocytes had a decreased absolute amount of aggregated PRPP synthetase and almost a total loss of disaggregated PRPP synthetase. Using normal hemolysate, 1 mM CTP/CDP/CMP interfered with the ability of 1.0 mM ATP and 2.0 mM MgCl2 to promote PRPP synthetase subunit aggregation. Increasing the MgCl2 to 6.0 mM overcame the inhibitory effect of CTP/CDP/CMP. Thus, the decreased PRPP synthetase activity of the P5N-deficient erythrocyte is due, at least in part, to the ability of the accumulated pyrimidine nucleotides to sequester magnesium and to interfere with the subunit aggregation of PRPP synthetase.

[The course of acquired C 1 esterase inhibitor deficiency in lymphoproliferative syndrome]. / Evolution d'un déficit acquis en inhibiteur de C 1 estérase au cours d'un syndrome lympho-prolifératif.

Acquired deficiency in C1 esterase inhibitor (C1 INH) was first described by Caldwell in 1972. Since that date, about 30 cases have been reported, in most cases during proliferative lymphocyte B syndromes. The acquired C1 INH deficiency can provoke episodes of angioneurotic edema as in hereditary AE. The complement profile differs, notably by the usual sudden fall in C1. Documented data suggest consumption of complement and therefore of C1 INH. The present case is the first reported of an acquired C1 INH deficiency during a cold hemagglutinin disease. The activation of complement by the classical pathway appears provoked by tumoral cells rather than a humoral factor, as suggested by the efficacy of anti-tumoral therapy in contrast to plasmapheresis in the present case. A possible mechanism for the C1 INH deficiency is discussed.

Unique profile for erythrocyte membrane acetylcholinesterase in hereditary spherocytosis.

Acetylcholinesterase of human erythrocytes from healthy donors and from patients with hematological disorders was analysed in a search for differential membrane parameters. Two substrates were used to estimate the exposure of acetylcholinesterase active site in the membrane: phenylacetate, a hydrophobic substrate, to determine total enzyme activity, and acetylcholine, an ionic substrate, to measure the externally reactive enzyme. The sensitivity of acetylcholinesterase to added stearic acid was also analysed. Three categories of the disorders studied were discerned: (a) The erythrocyte acetylcholinesterase profile was indistinguishable from normal control in beta-thalassemia minor and groups of patients with autoimmune hemolytic anemia or congenital dyserythropoietic anemia type II. (b) A marked decline in acetylcholinesterase with both substrates and reduced sensitivity to stearic acid were exhibited by the erythrocytes of paroxysmal nocturnal hemoglobinuria, beta-thalassemia major and other autoimmune hemolytic anemia and congenital dyserythropoietic anemia type II patients. Normal erythrocytes, either aged or pretreated to 50 degrees C, also showed similar characteristics. (c) Hereditary spherocytosis was singly differentiated by an elevated acetylcholinesterase activity with acetylthiocholine and by a vastly diminished sensitivity to stearic acid, while activity with phenylacetate was equal to control. This distinct profile may reflect the unique organization of the erythrocyte membrane in hereditary spherocytosis.

Increased erythrocyte superoxide dismutase activities in beta 0-thalassaemia/haemoglobin E and in haemoglobin H diseases.

Erythrocyte superoxide dismutase activities were measured in 45 subjects, 15 each of beta 0-thalassaemia/haemoglobin (Hb) E disease, Hb H disease, and normal. The erythrocyte superoxide dismutase activities were significantly higher in the patients with beta 0-thalassaemia/Hb E and Hb H diseases than in the normal subjects. The increase of erythrocyte superoxide dismutase activities is most likely due to abnormalities specific to thalassaemic red cells rather than an increased number of younger red cells for reticulocytes and nucleated red blood cells did not affect the enzyme activity. Patients with beta 0-thalassaemia/Hb E disease with lower haemoglobin concentration had significantly higher superoxide dismutase activities. In all 45 subjects haemoglobin concentrations and superoxide dismutase activities were inversely correlated (r = -0.60 (p less than 0.001)). This indicates that the amounts of superoxide generated in the red cells may, at least partly, determine severity of red cell damage and thus severity of disease; the increased superoxide dismutase activity in thalassaemia is a response to superoxide generated in greater amounts because of accumulation of excessive globin chains and iron in the red cells. The superoxide dismutase activities in Hb H disease, an alpha-thalassaemic disease, were found to be strikingly increased, higher than in beta 0-thalassaemic disease or other conditions.

Study of a case with severe red-cell pyrimidine 5'-nucleotidase deficiency.

A deficiency of erythrocyte pyrimidine 5'-nucleotidase was found in a 23-year-old male suffering from severe congenital hemolytic disease. Results from exhaustive metabolic exploration are given and compared with reticulocyte-rich blood from subjects with auto-immune hemolytic disease. Evidence is given that the 20% apparent P5N residual activity corresponds to a non-specific acid phosphatase.

[Acetylcholinesterase of erythrocytes in various blood diseases]. / Atsetilkholinesteraza eritrotsitov pri nekotorykh zabolevaniiakh sistemy krovi

Appearance of low molecular components of acetylcholinesterase from erythrocyte membranes was found by gel filtration on Sephadex G-200 and polyacrylamide gel disc electrophoresis. Content of these components was distinctly increased within ghe acute period of Marchiafava-Micheli disease.

Phosphorylase activity in chronic erythremic myelosis.

Phosphorylase activity was detected in the cytoplasm of erythroid precursors of 6 of 7 patients with chronic erythremic myelosis (Di Guglielmo syndrome), in proerythroblasts and megaloblasts from 3 patients with pernicious anemia and in 2 patients with severe folate deficiency in neoplastic lymphocytes from 2 patients with acute lymphoblastic leukemia, and in 1 patient with leukemic lymphosarcoma. In all of these patients, most of the erythroid precursors and/or neoplastic lymphocytes contained increased amounts of glycogen when stained with the PAS reagent. Phosphorylase activity was not detected in erythroid precursors obtained from 6 presumed normal individuals or from 3 of 7 patients with a variety of other types of anemia in which the erythroid precursors were PAS-negative. Similarly, phosphorylase activity was absent in lymphocytes obtained from presumed normal individuals. Although the mechanisms responsible for the pathogenesis of PAS positivity are unclear, it is possible that the increased phosphorylase activity found in cells that are PAS-positive may reflect a disorder in the biosynthetic pathway of glycogen.