The clinical spectrum of postpartum thyroid disease

The clinical and biochemical features of postpartum thyroid disease were analysed in 152 antithyroid peroxidase antibody-positive (anti TPO+ve) women and compared with 239 anti-TPO-ve age-matched control postpartum women. All were assessed monthly for up to 12 months postpartum. Seventy three anti-TPO+ve women developed post-partum thyroiditis (PPT): 19.2% hyperthyroid alone, 49.3% hypothyroid alone, and 31.5% characterized by hyper- followed by hypothyroidism. None of the antibody-negative women developed any thyroid dysfunction. A significant increase in many of eleven symptoms of hypothyroidism and some of eight symptoms of hyperthyroidism compared to control women was observed in all anti-TPO+ve women, independent of thyroid status. This was particularly seen in women who later developed PPT when they were euthyroid, but was also observed in euthyroid anti-TPO+ve women who showed no decline of thyroid function during the postpartum period. Although PPT is usually transient, this condition, and the euthyroid antibody-positive state, may be associated with significant symptomatology, including an increased incidence of minor to moderate depression. Early recognition of this syndrome by antenatal screening of thyroid antibodies may contribute to improved management of women during the postpartum period.      

Monocyte‐derived dendritic cells can induce autoreactive CD4+ T cells showing myeloid lineage directed reactivity in healthy individuals

T cells against self‐antigens can be detected in peripheral blood of healthy individuals, although intrathymic negative selection removes most high‐avidity T cells specific for self‐antigens from the peripheral repertoire. Moreover, spontaneous T‐cell proliferation following stimulation with autologous monocyte‐derived dendritic cells (autoDCs) has been observed in vitro. In this study, we characterized the nature and immunological basis of the autoDC reactivity in the T‐cell repertoire of healthy donors. We show that a minority of naive and memory CD4⁺ T cells within the healthy human T‐cell repertoire mediates HLA‐restricted reactivity against autoDCs, which behave like a normal antigen‐specific immune response. This reactivity appeared to be primarily directed against myeloid lineage cells. Although cytokine production by the reactive T cells was observed, this did not coincide with overt cytotoxic activity against autoDCs. AutoDC reactivity was also observed in the CD8⁺ T‐cell compartment, but this appeared to be mainly cytokine‐induced rather than antigen‐driven. In conclusion, we show that the presence of autoreactive T cells harboring the potential to react against autologous and HLA‐matched allogeneic myeloid cells is a common phenomenon in healthy individuals. These autoDC‐reactive T cells may help the induction of primary T‐cell responses at the DC priming site.     

Terminal deoxynucleotidyl transferase expression can precede T cell receptor beta chain and gamma chain rearrangement in T cell acute lymphoblastic leukemia

The nuclear enzyme terminal deoxynucleotidyl transferase (TdT) is thought to contribute to the diversity of certain immunoglobulin and T cell receptor gene rearrangements through the addition of random nucleotides at their variable (V)-joining (J) region junctions. An acute lymphoblastic leukemia (ALL) with an immature T cell phenotype (CD7+, CD5+, CD1+/-, CD2+/-, CD3-, CD4-, CD8-) was found to be TdT+ with germline immunoglobulin heavy chain, T cell receptor beta chain, and T cell gamma chain genes. The data indicate that TdT expression can precede T gamma and T beta rearrangement during T lymphoid ontogeny consistent with its proposed association with the T cell receptor rearrangement process. Southern analysis of certain cases of T-ALL may not result in the detection of a monoclonal population of cells.     

Effects of recombinant human granulocyte-macrophage colony-stimulating factor on intracellular pH in mature granulocytes

We studied the effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSFrh) on the internal pH of granulocytes using the fluorescent probe BCECF. GM-CSFrh did not directly alter the resting pH of granulocytes isolated from the peripheral blood; however, when the cells were preincubated for 90 minutes with the growth factor and then activated with the chemotactic peptide N-formyl met leu phe (fMLP), they exhibited both an acceleration in the initial rate of acidification and a marked delay in realkalinization. The kinetic changes both in initial acidification and in subsequent realkalinization induced by GM-CSFrh priming were not prevented by protein synthesis inhibitors and were observed in granulocytes harvested from patients with both sex-linked and autosomal recessive chronic granulomatous disease (CGD). By directly quantitating H+ ion secretion, by monitoring the effects of sodium repletion on intracellular pH, and through use of the sodium channel inhibitors amiloride and dimethyl amiloride and the Na+/K+-ATPase inhibitor ouabain, we showed that the altered kinetics of intracellular acidification and alkalinization following fMLP stimulation of GM-CSFrh- primed granulocytes could not be accounted for by changes in transmembrane proton exportation regulated by the Na+/H+ antiport channel. Although the initial acidification following fMLP was abrogated by 2-deoxy-D-glucose in both GM-CSFrh-pretreated and GM-CSFrh- untreated granulocytes, retardation of the subsequent phase of alkalinization was observed in GM-CSFrh-primed cells even after inhibition of both glycolytic and mitochondrial metabolism. Our data indicate that the increased cytosolic acidification following fMLP stimulation in granulocytes "primed" with GM-CSFrh does not result from disordered proton excretion but instead from increased release of intracellular free acid which is only partially coupled to glucose catabolism or to the generation of superoxide anion (O2-).     

Monoclonal antibody BA-1 does not bind to hematopoietic precursor cells

Monoclonal antibody BA-1 binds to B lymphocytes, to cells from most cases of non-T acute lymphoblastic leukemia (ALL), and weakly to neutrophils. To determine whether BA-1 also reacts with hematopoietic progenitor cells (HPC), we studied the effect of removal of BA-1+ cells from human bone marrow on the proliferation in vitro of the trilineage precursor cell CFU-GEMM, and on the committed progenitor cells of granulopoiesis (CFU-C) and erythropoiesis (BFU-E/CFU-E). Complement- mediated cytotoxicity using BA-1 at concentrations far beyond those required to lyse BA-1+ bone marrow cells and ALL cells did not result in inhibition of colony formation in any of the assays. A rosette separation method, using ox red blood cells coated with BA-1, resulted in enrichment of HPC in the BA-1-depleted interface, whereas very few HPC were found in the BA-1-enriched pellet. Both methods indicate that BA-1 does not bind to HPC, although binding of the antibody to the lymphohematopoietic stem cell cannot be excluded yet. The high cytotoxic capacity of the IgM antibody BA-1, and the lack of reactivity with HPC, make the antibody particularly suitable for use in autologous bone marrow transplantation for patients with ALL.     

Human acute leukemia cell line with the t(4;11) chromosomal rearrangement exhibits B lineage and monocytic characteristics

A cell line, designated RS4;11, was established from the bone marrow of a patient in relapse with an acute leukemia that was characterized by the t(4;11) chromosomal abnormality. The cell line and the patient's fresh leukemic cells both had the t(4;11)(q21;q23) and an isochromosome for the long arm of No. 7. Morphologically, all cells were lymphoid in appearance. Ultrastructurally and cytochemically, approximately 30% of the cells possessed myeloid features. The cells were strongly positive for terminal deoxynucleotidyl transferase. They were HLA-DR positive and expressed surface antigens characteristic for B lineage cells, including those detected by anti-B4, BA-1, BA-2, and PI153/3. Immunoglobulin gene analysis revealed rearrangements of the heavy chain and kappa chain genes. The cells lacked the common acute lymphoblastic leukemia antigen and antigenic markers characteristic of T lineage cells. The cells reacted with the myeloid antibody 1G10 but not with other myeloid monoclonal antibodies. Treatment with 12-O-tetradecanoyl- phorbol-13-acetate induced a monocyte-like phenotype demonstrated by cytochemical, functional, immunologic, and electron microscopic studies. The expression of markers of both early lymphoid and early myeloid cells represents an unusual phenotype and suggests that RS4;11 represents a cell with dual lineage capabilities. To our knowledge, RS4;11 is the first cell line established from t(4;11)-associated acute leukemia.     

Use of multiple T cell-directed intact ricin immunotoxins for autologous bone marrow transplantation

The monoclonal antibodies (MoAb) T101, G3.7, 35.1, and TA-1 were conjugated to intact ricin using a thioether linkage. These MoAb detect, respectively, the CD5[gp67], CD7[p41], CD2[p50], and [gp95, 170] determinants that are found in the vast majority of cases of T cell acute lymphocytic leukemia (T-ALL). The resulting immunotoxins (ITs) and an equimolar mixture of these ITs were evaluated as potential purgative reagents for autologous transplantation in T-ALL. Leukemic cell lines were used to compare the kinetics of protein synthesis inactivation mediated by each IT. The cells were treated with IT in the presence of lactose in order to block the native binding of ricin. The observed rates of protein synthesis inactivation correlated with target antigen expression detected by fluorescence-activated cell sorter analysis. Of the four ITs, T101-ricin (T101-R) exhibited the fastest rate of inactivation, followed in order by G3.7-ricin, TA-1-ricin, and 35.1-ricin. At concentrations greater than 300 ng/mL, a cocktail containing an equimolar amount of all four ITs (referred to as the four- IT cocktail) exhibited kinetics that were as fast or faster than those of T101-R. The long-term cytotoxic effects of individual ITs and the four-IT cocktail were evaluated using a sensitive clonogenic assay. Each IT was specifically cytotoxic and inhibited 1 to 4 logs of clonogenic leukemic cells at doses (300 to 600 ng/mL) that can be used clinically. The four-IT cocktail was highly cytotoxic; a concentration of 300 ng/mL inhibited greater than 4 logs of leukemic cells while sparing the majority of committed (CFU-GM, CFU-E) and pluripotent (CFU- GEMM) hematopoietic stem cells. The determination of both short-term kinetics of protein synthesis inactivation and longer-term inhibition of clonogenic growth allowed new insight into cell killing by IT. Our results suggest that ITs continue to act on clonogenic target cells for a period of three to five days. Interestingly, the four-IT cocktail was not as potent against clonogenic leukemic cells as T101-R alone, although it exhibited kinetics of protein synthesis inhibition that were as fast as those of T101-R alone. This finding suggests that internalized ITs may differ in the length of time they remain active within the cell. Our results also demonstrate the importance of using several different assays to evaluate IT reagents.     

Polymorphic and monomorphic HLA-DR determinants on human hematopoietic progenitor cells

The expression of monomorphic Ia-like antigens and polymorphic (allotypic) HLA-DR determinants on CFU-GM, BFU-E, CFU-E, and CFU-GEMM was studied in bone marrow and peripheral blood cells from normal healthy individuals. Using various polyclonal and monoclonal anti-Ia- like antibodies, the presence of HLA-DR backbone antigens was shown on all hematopoietic progenitor cells (HPC) studied, both in complement- dependent cytotoxicity assays and in fluorescence-activated cell sorting (FACS). The expression of allotypic determinants was demonstrated on all HPCs, using the HLA-DR typing sera anti-HLA-DR1, 2, 3, 4, 5, and 7. The Class II antigen MT-2 was also shown on all HPCs, using both monoclonal and alloantisera, whereas the MB-1 (DC-1) determinant could not be demonstrated on HPCs. This might open the possibility of removing MB-1-positive malignant cells from the graft in autologous bone marrow transplantation.