Chronic isolated macrothrombocytopenia with autosomal dominant transmission: a morphological and qualitative platelet disorder

Abstract: We studied 47 subjects belonging to 13 unrelated families with a history of mild haemorrhagic diathesis and chronic thrombocytopenia. 36 patients presented some degree of thrombocytopenia: 7/36 (19%) had slight thrombocytopenia (100–150×10⁹/L); 26/36 (72%) had mild thrombocytopenia (50–100×10⁹/L) and 3/36 (8%) had severe thrombocytopenia (<50×10⁹/L). No correlation was observed between platelet count and the degree of haemorrhagic diathesis, which was mild in the majority of patients. Transmission was autosomal dominant. Platelet anisocytosis, increased percentage of large platelets and absence of leukocyte inclusions were observed in 26/30 (87%) of the examined blood smears. The ultrastructural appearance of platelets was normal. Megakaryocytes appeared normal in number in 10/10 patients, but showed asynchronous nuclear-cytoplasm maturation and mainly nonlobulated nuclei. Platelet aggregation was studied in 26 patients and either increased or decreased curves were variably observed in response to different aggregating agents. Platelet-associated IgG (PAIgG) was increased in 18/31 (58%) patients, while serum autoantibodies against platelet glycoproteins Ib/IX or IIb/IIIa were demonstrable in only 1 case. An increased expression of platelet surface glycoproteins Ib and IIb/IIIa, as studied by murine monoclonal antibodies binding in 17 cases, was observed. Platelet survival performed by 111In-oxine-labelled autologous platelets was normal in the 3 studied patients. Congenital macrothrombocytopenia confirms to be a distinct clinical disorder for which the name of “chronic isolated hereditary macrothrombocytopenia” is proposed.     

Immunoglobin synthesis by fresh biopsy cells and established cell lines from Burkitt''s lymphoma

(1) Freshly dispersed cells from four Burkitt's lymphoma biopsies, and three `lymphoblast' cell lines (OB2, OB3 and OB6) derived from the tumour were tested for capacity to synthesize immunoglobulins in vitro.

Using labelled amino acid incorporation, electrophoretic, radio-immunoelectrophoretic and ultracentrifugation techniques, it was possible to demonstrate the release of labelled proteins with antigenic characteristics of immunoglobulins by all four biopsy samples, and the two cell lines tested by these techniques. Newly synthesized labelled proteins were demonstrable in the culture medium within 1 hour of incubation.

In a separate series of experiments, absorption and immunodiffusion techniques were used in the characterization of proteins synthesized by two cell lines (OB2 and OB3) growing as healthy continuous cultures.

(2) Freshly isolated cells from Burkitt's lymphoma are capable of immunoglobulin synthesis in vitro. Cells from all four fresh biopsy materials produced IgG. One cell line (OB2) produced IgG and type-κ light chains, while OB6 cell line produced IgA. Immunoglobulin synthesis was not detected in OB3 cell line by immunodiffusion technique.

(3) Cells from each biopsy specimen or cell line produced not more than one type of immunoglobulin, although there was a wide variation in the sedimentation coefficient of the protein molecules synthesized by one (OB6) and presumably all other cell lines.

     

Glycosylphosphatidylinositol-linked proteins are required for maintenance of a normal peripheral lymphoid compartment but not for lymphocyte development

Surface proteins tethered to the membrane through a glycosylphosphatidylinositol (GPI) anchor are deficient in the blood cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) as result of a somatic mutation, in a hematopoietic stem cell, of the X-linked phosphatidylinositolglycan complementation group A (PIG-A) gene. In PNH patients, compared to the large numbers of GPI-deficient myeloid cells, the proportion of GPI-deficient lymphocytes tends to be low, and therefore the impact of GPI deficiency on immune function has been unclear. We have obtained complementation by Pig-a(-) embryonic stem (ES) cells of Rag(-/-) blastocysts, and we show that Pig-a(-) ES cells are able to reconstitute the T cell and B cell compartments of Rag(-/-) mice. Although these mice were immunologically competent, by comparison with appropriate controls we detected several abnormalities: (1) increased levels of IgG; (2) high frequency/titers of anti-nuclear antibodies; (3) markedly reduced delayed hypersensitivity; and (4) impaired activation-induced lymphocyte death in vitro. In some cases, aging Pig-a(-)/Rag(-/-) chimeric mice developed lymphadenopathy and polyclonal T cell and B cell expansion. Thus, GPI-linked proteins are not required for lymphocyte development but they are required for normal lymphocyte function and for maintaining normal peripheral lymphoid homeostasis.     

Ultrastructural alterations induced in quail ciliary neurons by postganglionic nerve crush and by Ricinus toxin administration, separately and in combination

The response to postganglionic nerve crush and Ricinus toxin administration by the ciliary neurons of the quail ciliary ganglion was investigated at the ultrastructural level. The toxin was either applied at the crush site on the postganglionic nerves or injected into the anterior eye chamber without any other operative intervention. Crush of postganglionic nerves without toxin administration and saline injection into the anterior eye chamber served as controls for the two toxin administration procedures. Postganglionic nerve crush caused a distinct chromatolytic reaction, accompanied by massive detachment of the preganglionic axon terminals from the ciliary neurons and loss of most of the synapses, both chemical and electrical. This process does not induce cell death and is reversible. Saline injection in the anterior eye chamber caused a moderate retrograde reaction in some of the ciliary neurons, presumably as a consequence of paracentesis. The changes consisted mainly of an increase of perikaryal neurofilaments with, at most, a minor detachment of the preganglionic boutons from a small portion of the cell body at the nuclear pole. Ricinus toxin administration induced neuronal degeneration following a pattern common to both delivery modes. The degenerative process consisted of disruption and detachment of polyribosomes from the rough endoplasmic reticulum, an increase of smooth cisterns and tubules, a dramatic increase of neurofilament bundles, compartmentalization of the cytoplasmic organelles and, finally, karyorrhexis and cell lysis. The final stages of Ricinus toxin degeneration involve a progressive accumulation of extracellular flocculo-filamentous material and cell lysis. After administration of Ricinus toxin to the crush site, ricin-affected neurons showed withdrawal of the preganglionic boutons from a portion of the ciliary neuron, especially at the nuclear pole. After Ricinus toxin injection into the anterior eye chamber, however, the bouton shell surrounding the affected ciliary neurons remained intact in the early stages of degeneration. Detachment of the preganglionic terminals and disruption of the cell junctions, therefore, is the consequence of nerve crush and not of the toxin itself.

This study demonstrates that quail ciliary neurons are a suitable model for experimental neuropathology and neurotoxicology.     

The "n-1" model for myelodysplastic syndromes.

Current models of leukaemogenesis tend to visualize this process as consisting of several discrete steps. Since myelodysplastic syndromes (MDS) are, almost by definition, pre-leukaemic disorders, we expect that bone marrow from patients with MDS must contain cells which are one step short of full leukaemic transformation: we designate these cells, for convenience, as "n-1". It should be possible therefore to obtain leukaemic transformation in vitro by introducing into n-1 cells a gene with an appropriate leukaemogenic mutation. We have found, by using as a model system the retrovirus VSN-2 (which carries the neomycin-phosphotransferase gene, neo, which confers resistance to the antibiotic G418), that human bone marrow cells can be successfully transfected in microcultures. Indeed, G418-resistant CFU-CM have been recovered from these cultures for a period of several weeks.     

Specific defect in N-acetylglucosamine incorporation in the biosynthesis of the glycosylphosphatidylinositol anchor in cloned cell lines from patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal disorder arising in a multipotent hemopoietic stem cell. PNH manifests clinically with intravascular hemolysis resulting from an increased sensitivity of the red cells belonging to the PNH clone to complement-mediated lysis. Numerous studies have shown that surface proteins anchored to the membrane via a glycosylphosphatidylinositol (GPI) anchor (including proteins protecting the cell from complement) are deficient on the cells of the PNH clone, leading to the notion that GPI-anchor biosynthesis may be abnormal in these cells. To investigate the biochemical defect underlying PNH we have used lymphoblastoid cell lines (LCLs) with the PNH phenotype obtained by Epstein-Barr virus immortalization of lymphocytes from nine patients with PNH. By labeling cells with myo-[3H]inositol we have found that PNH LCLs produce phosphatidylinositol normally. By contrast, PNH LCLs fail to incorporate [3H]mannose into GPI anchor precursors. When cell-free extracts of PNH LCLs and normal LCLs obtained from the same patients (and expected therefore to be isogeneic except for the PNH mutation) were incubated with uridine diphospho-N-acetyl[3H]glucosamine (UDP-[3H]GlcNAc), we observed complete failure or marked reduction in the production of N-acetylglucosaminyl(alpha-1,6)phosphatidylinositol and glucosaminyl(alpha-1,6)phosphatidylinositol by the PNH LCLs in all cases. These findings pinpoint the block in PNH at an early stage in the biosynthesis of the GPI anchor, suggesting that the defective enzyme is UDP-GlcNAc:phosphatidylinositol-alpha-1,6-N- acetylglucosaminyltransferase. The existence of PNH type III cells and type II cells is probably explained by the transferase deficiency being total or partial, respectively.     

Effects of glycosaminoglycans and protamine chloridrate on platelet aggregation induced by collagen and thrombin

The effects of heparin (HE), dermatan sulfate (DS), heparan sulfate (HS) and protamine chloridrate (PC) on platelet aggregation were studied. Both PC and the three glycosaminoglycans (GAGs) did not influence collagen-induced platelet aggregation. In contrast, all the tested GAGs blocked thrombin-induced platelet aggregation. HE and HS were equivalent and very effective, while DS was also but to a lesser extent. This could be because HE and HS act via both antithrombin III and heparin-cofactor II, whereas DS exerts its action on the latter only. PC, too, inhibited, in a dose-dependent fashion, thrombin-induced platelet aggregation, probably by competing with the thrombin affinity binding sites on the platelet surface. When the GAGs were tested together with PC, HE was shown to be the most effective: on a weight-for-weight basis, an identical amount of PC was unable to counteract the inhibitory effect of HE, while it partially reversed those of DS and HS. A full reversal of the inhibitory effect of DS and HS was never observed, in spite of adding increasing amounts of PC. It seems likely that plasma components may preserve the bindings of such GAGs with their cofactors.     

Expression of Plasmodium falciparum G6PD-6PGL in laboratory parasites and in patient isolates in G6PD-deficient and normal Nigerian children

As the production of NADPH in the pentose phosphate pathway is the main antioxidant defence mechanism available to the Plasmodium falciparum, we have studied the expression of P. falciparum glucose 6-phosphate dehydrogenase-6-phosphogluconolactonase (PfG6PD-6PGL) in G6PD-deficient and normal erythrocyte host cells. Both erythrocytes infected in vitro with a laboratory isolate and erythrocytes from natural human infections were used. Total RNA was prepared from parasites collected from five G6PD-deficient and nine G6PD-normal children in Ibadan, Nigeria, selected after screening 189 rural schoolchildren and 68 clinical malaria patients, and was subjected to Northern blot analysis. The probe was a cDNA fragment of the G6PD domain of the PfG6PD-6PGL gene, with an internal control probe of P. falciparum 18S ribosomal RNA. Quantification was performed using a phosphoimager. Relative to internal control, the abundance of PfG6PD-6PGL mRNA (mean +/- standard deviation) was lower in parasites from G6PD-deficient children (0.29 +/- 0.27) than in G6PD-normal control subjects (0.74 +/- 0.26) (P = 0.014, Mann-Whitney U-test). Although confirmation in a larger study is required, our results suggest a lower relative abundance of PfG6PD-6PGL, and presumably antioxidant activity, in malaria parasites from G6PD-deficient hosts, thus extending the current knowledge of the mechanism of G6PD-deficiency related host protection.     

Red cell glucose-6-phosphate dehydrogenase status and pyruvate kinase activity in a Nigerian population

Glucose-6-phosphate dehydrogenase A- (G6PD A-) deficiency is a common enzymopathy in Africa that sporadically leads to manifest haemolytic anaemia. It is not exactly known how far the haematological status of individuals with either homozygous or heterozygous G6PD A- deficiency differs from that of individuals with normal G6PD activity. In a field study in Nigeria, we determined G6PD gene variants, the corresponding G6PD and pyruvate kinase (PK) activities, and basic haematological parameters in clinically healthy individuals, who were, in part, asymptomatically infected by malaria parasites. Red blood cell counts and haemoglobin levels were lower in G6PD A- deficient than in G6PD normal subjects. PK activities were higher in G6PD deficients, indicating a younger red cell population in these individuals. These findings suggest that G6PD A- deficiency is accompanied by chronic subclinical haemolysis. As a consequence, the reduced life span of red cells leads to an impaired diagnosis of G6PD heterozygosity when applying routine biochemical methods.     

Two distinct patterns of glycosylphosphatidylinositol (GPI) linked protein deficiency in the red cells of patients with paroxysmal nocturnal haemoglobinuria

We have studied three glycosylphosphatidylinositol (GPI) linked proteins on the erythrocytes of 14 patients with paroxysmal nocturnal haemoglobinuria (PNH). The pattern observed was bimodal in 12 of the patients and trimodal in two. Ten patients had a red cell population with normal CD59 antigen (membrane inhibitor of reactive lysis, MIRL), decay accelerating factor (DAF or CD55) and lymphocyte function-associated antigen (LFA-3 or CD58) and a second abnormal PNH population with absent CD59 antigen, DAF and LFA-3. The other two patients with a bimodal pattern had a red cell population with normal CD59 antigen, DAF and LFA-3 and an abnormal population with reduced, but not absent, CD59 antigen and DAF. The LFA-3 on the abnormal red cells in these two patients appeared to be only slightly reduced. The two patients with a trimodal pattern had a normal population, a population with reduced, not absent, CD59 antigen and DAF, and a population with complete absence of CD59 antigen, DAF and LFA-3. The accuracy of the Ham test in estimating the proportion of red cells with the PNH defect in the two types of PNH was assessed. The case of one patient who appeared to be 'rescued' from severe aplastic anaemia by the development of PNH is described.