Phenotypic and clonal analysis of T lymphocytes in childhood immune thrombocytopenic purpura

In an attempt to identify and characterize T-lymphocyte immunoregulatory abnormalities in immune thrombocytopenic purpura (ITP), we have performed phenotypic and clonal analysis on peripheral T lymphocytes from 23 children with ITP. Quantitation of lymphocyte subpopulations showed that children with acute ITP had higher numbers of CD45RA+ and lower numbers of CD45RO+ T cells than children with chronic ITP or controls, but these differences may be age related. Analysis of T-cell receptor variable beta gene usage identified 2 boys with chronic ITP and elevated numbers of V beta 8+ T cells. Eight T- cell clones were established (6 CD4+, 4B4+ helper-inducer lines and 2 CD8+ lines) that showed in vitro proliferation against allogeneic platelets. The addition of autologous antigen-presenting cells enhanced the proliferation of six clones, but not for two clones that coexpressed natural killer (NK) markers. Four of seven positive clones also had measurable interleukin (IL)-2 secretion following platelet stimulation, providing further evidence for T-cell reactivity. Our results provide the first evidence that patients with ITP may have platelet-reactive T lymphocytes identifiable at the clonal level, supporting the hypothesis that autoreactive peripheral T lymphocytes may mediate or participate in the pathogenesis of this disorder.     

Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding to human platelets

A large number of glycoprotein (GP) IIb/IIIa receptors are present on the surface of platelets. Studies to define precisely the number of GPIIb/IIIa receptors using specific monoclonal antibodies (MoAbs) or fibrinogen binding have, however, yielded varying estimates of receptor number. To refine the quantitative estimation of GPIIb/IIIa receptors on resting platelets, we have used the MoAb 7E3, which has high affinity for GPIIb/IIIa. Quantitative binding studies were performed using radiolabeled conjugates of 7E3 IgG, as well as fragments and derivatives of 7E3. For platelets obtained from any single individual, the numbers of 7E3 F(ab')2 and IgG molecules bound per platelet were equivalent (approximately 40,000), whereas the number of Fab molecules bound per platelet was consistently approximately twofold higher (approximately 80,000). To investigate the basis of the quantitative disparity in binding of intact 7E3 and 7E3 F(ab')2 versus 7E3 Fab, we studied the binding of a newly constructed, bispecific (Fab')2 molecule containing only a single 7E3 combining site. Because this construct bound to the same extent as the Fab species, the larger size of the intact 7E3 and 7E3 F(ab')2 molecules could not explain the reduced number of molecules that bound per platelet compared to the Fab fragment. Rather, it appears that the valency of the antibody is the critical factor determining the number of antibody molecules bound per platelet. Thus, we conclude that the binding of 7E3 Fab corresponds most closely with surface GPIIb/IIIa number and that the number of GPIIb/IIIa receptors is approximately 80,000 per platelet.     

Lymphocyte dysfunction in chronic graft-versus-host disease

Three recipients of HLA-identical bone marrow transplants developed chronic graft-versus-host disease (cGVHD) and hypergammaglobulinemia. All three had evidence of abnormal B-lymphocyte function, including a polyclonal increase in immunoglobulins (Ig), antinuclear antibodies, rheumatoid factor, lymphocytotoxins, and increased immune complexes. T- lymphocyte function was also abnormal, including decreased mitogen reactivity and delayed cutaneous hypersensitivity. The cellular basis of these immune abnormalities was studied in an in vitro system in which we analyzed spontaneous pokeweed mitogen (PWM) driven Ig synthesis. Multiple defects in both T- and B-lymphocyte function were detected. In contrast to normal B cells, circulating B cells from all three patients with cGVHD spontaneously synthesized in vitro greater than 200 ng of IgG and in two of the three greater than 175 ng of IgM. This increase in spontaneous Ig synthesis was not due to a deficiency of regulatory cells, since T cells from the three patients suppressed spontaneous Ig synthesis in a normal fashion. In contrast to this increased spontaneous Ig synthesis, the response of the patients' B cells to PWM-driven Ig synthesis was normal. Using the PWM system we demonstrated several defects in these patients' T cells, including increased suppressor activity and decreased helper cell activity. These data indicate that some patients with cGVHD have multiple defects in both T- and B-cell function that may contribute to their profound immune deficiency.     

Six point mutations that cause factor XI deficiency

We have identified six novel types of mutation that cause factor XI deficiency, an inherited bleeding disorder. Two are point mutations that interfere with the normal splicing of exons in the mRNA and four are point mutations that result in amino acid substitutions. One of these amino acid substitutions (Asp 16-->His) is near the amino terminal end of the protein. The other three amino acid substitutions (Leu 302-->Pro, Thr 304-->Ile, and Glu 323-->Lys) are in the fourth apple domain, a region that mediates dimerization of identical subunits of factor XI. All four amino acid substitutions cause a reduction in the amount of factor XI secreted from cells grown in vitro.     

The effect of recombinant human granulocyte macrophage colony- stimulating factor on neutrophil activation in patients with refractory carcinoma

Patients with refractory carcinoma were treated with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) by intravenous (IV) infusion. During the period of treatment, studies of polymorphonuclear leukocyte superoxide (O2-) release in response to formylmethionylleucylphenylalanine (fMLP) and phorbol myristate acetate (PMA) and studies of chemotaxis in response to fMLP and C5a were performed. We observed that patients receiving rhGM-CSF in vivo exhibited primed O2- release after stimulation both with fMLP and PMA. Chemotaxis, however, was not enhanced by the treatment. These data suggest that host defenses may be enhanced by this treatment and that rhGM-CSF may be a useful therapeutic adjunct in compromised patients.     

Compound heterozygosity for hemoglobin C and Korle-Bu: moderate microcytic hemolytic anemia and acceleration of crystal formation [corrected] [published erratum appears in Blood 1994 May 15;83(10):3105]

We report here that compound heterozygosity for hemoglobin Korle-Bu (HbKB) and HbC (beta 6 Glu-->Lys) is associated with moderate chronic hemolytic anemia with microcytosis. To understand the pathogenesis of this syndrome, we have studied the effect of Hb Korle-Bu (KB = beta 73 Asp-->Asn) on the crystallization of HbC. We have previously established that fetal Hb (HbF) inhibits the crystallization of HbC. In contrast, HbS accelerates crystallization affecting the pathogenesis of SC disease. We now report on in vitro crystallization of mixtures of HbKB, HbC, and various amounts of HbF and the native hemolysate of a child who is a compound heterozygote for HbKB and HbC. At 6 months of age, the propositus' hemolysate contained 55% HbKB, 39% HbC, and 6% HbF. Crystal formed within 2 minutes compared with 30 minutes for the mixture of 40% HbC:60% HbS and with 180 minutes for 40% HbC:60% HbA. The morphology of the crystals formed was cubic, in contrast with the tetragonal crystals observed in CC and SC disease. Early crystals did not exhibit "sharp edges" until 45 minutes. Purified HbKB formed aggregates but not crystals after 24 hours. Isopycnic gradients showed that the KB/C compound heterozygotes have red blood cell (RBC) densities intermediate between the AC and CC phenotype and similar to SC disease. The surface residue beta 73, known to participate in areas of interaction of the deoxy HbS polymer, can now be assigned to areas of contact in HbC containing crystals. The hemolysis observed in the HbKB/C compound heterozygote is likely to be secondary to the acceleration of Hb crystallization. The microcytosis and increased RBC density is clearly the consequence of the presence of HbC, but the basis of the increased RBC pathology compared with AC trait, despite the low proportion of HbC (35% to 40%), remains to be elucidated.     

THE RELATIONSHIP BETWEEN ENDOGENOUS HYPERPROLACTINAEMIA AND PLASMA ALDOSTERONE

It has been suggested that prolactin is a regulator of aldosterone secretion. In order to test this hypothesis, we measured prolactin, thyrotrophin and aldosterone by radioimmunoassay and plasma renin activity by the radioimmunoassay of angiotensin I in eight normal women before and after the intravenous injection of 200 microgram of thyrotrophin releasing hormone (TRH). Prolactin increased from 4.1 +/- 1.1 ng/ml (mean +/- SE) to a peak of 27.4 +/- 3.8 (P less than 0.005) at 15 min following TRH. Plasma renin activity was not different from control levels (1.0 +/- 0.2 ng/ml/h) during the first hour following the administration of TRH, nor did the plasma aldosterone concentration differ significantly from the control levels (39 +/- 7 pg/ml) during this period. However, with upright posture, an increase in aldosterone (from 31 +/- 3 pg/ml at 1 h to 68 +/- 9 at 2 h, P less than 0.005) and in plasma renin activity (from 0.9 +/- 0.2 ng/ml/h at 1 h to 2.0 +/- 0.5 at 2 h, P less than 0.05) was noted, demonstrating a normal capacity to secrete aldosterone in these subjects. Similarly, no change in aldosterone was seen in nine patients with primary hypothyroidism given TRH, despite the fact that the increase in prolactin was greater than normal. Chronic hyperprolactinaemia was not associated with hyperaldosteronism in six patients with pituitary tumour. These data demonstrate that acutely or chronically elevated serum prolactin levels do not result in increased plasma aldosterone levels in humans.