T-cell activation and subset patterns are altered in B-CLL and correlate with the stage of the disease

Two-color FACS analysis was used to study activated and "functional" T and natural killer (NK) cell subsets of circulating lymphocytes in 23 patients with B-type chronic lymphocytic leukemia (B-CLL) and in 30 healthy subjects. As compared with controls, B-CLL patients had increased absolute numbers of phenotypically activated, HLA-DR+ CD4+ and CD8+ cells and T suppressor/effector (CD11b+CD8+) cells. When patients in Rai stages II through IV (n = 11) were compared with cases in Rai stages O through I (n = 12), the former group of patients had higher numbers of activated CD4+ and CD8+ T cells and decreased levels of suppressor/effector T cells. The absolute numbers of T suppressor/inducer (CD45R+CD4+) cells were elevated in patients with stage O through I disease but within normal range in stage II through IV leukemia. We further showed that the absolute numbers of NK-like (CD16+) cells and their activated counterparts (DR+CD16+) are elevated in B-CLL patients as compared with healthy subjects. The comparison of relative T and NK subsets in the blood of patients and controls showed that the proportions of CD4+, CD8+, and CD16+ cells expressing the activation marker HLA-DR were increased in B-CLL. Furthermore, the percentage of T-suppressor/inducer (CD45R+) cells within the CD4+ population was decreased in the patients. The proportion of T- suppressor/effector (CD11b+) cells within the CD8+ subset was reduced in subjects with stage II-IV disease only. When stimulated in vitro with the T-cell-dependent inducer TPA, B-CLL cells from patients in Rai stages II through IV secreted larger amounts of IgM as compared with cells from stage O through I patients. A positive correlation was observed between the degree of phenotypic activation of CD4+ T-helper cells and their functional capacity to augment IgM secretion by autologous B-CLL cells. Our findings indicate a tumor cell-directed regulatory role of T cells (and possibly NK cells as well) in B-CLL. Furthermore, monitoring of phenotypically activated and functional T- cell subsets may be helpful in the prediction of disease progression and timing of therapy in B-CLL.     

Hereditary persistence of fetal hemoglobin or (delta beta)o- thalassemia: three types observed in South-Chinese families

Hematological and hemoglobin composition data, and results from extensive gene mapping, using a battery of restriction enzymes and probes, have been used to distinguish different types of hereditary persistence of fetal hemoglobin (HPFH) (or delta beta-thal) among three Chinese families from the southern part of China. The first (Family Z) is an A gamma-(delta beta)+-HPFH without a detectable deletion and may be the same as, or similar to, that described by Farquhar et al (Am J Hum Genet 35:611, 1983). The second (Family C) resembles a G gamma(A gamma delta beta)o-thalassemia and is characterized by a large deletion of DNA originating 3' to the G gamma globin gene and extending beyond sequences recognized by the pRK28 probe. Data from various digests indicate possible differences in the 3' end of the deletion when compared with data for some other types of G gamma(A gamma delta beta)o- thalassemia, described by Trent et al (Br J Haematol 57:279, 1984). The third (Family Zh) concerns a G gamma A gamma(delta beta)+-HPFH, which is characterized in heterozygotes by a fetal hemoglobin level of 20% to 25% with a G gamma value averaging 60% and by the absence of any DNA deletion detectable by extensive gene mapping analyses. The C----G mutation at position 202 5' to the G gamma globin gene [characteristic for the high G gamma-(delta beta)+-HPFH (Proc Natl Acad Sci USA 81:4894, 1984; Blood 64:1292, 1984)] was absent, but the Xmn I site at position 158 5' to the G gamma globin gene [characteristic for a modest increase in G gamma values and thus and increased G gamma to A gamma ratio (Blood)] was present. No indication has yet been obtained explaining the elevation in both G gamma and A gamma chains; haplotyping showed that the chromosome carrying this G gamma A gamma(delta beta)+ determinant is unusual among the Chinese population.     

Two independent genetic factors in the beta-globin gene cluster are associated with high G gamma-levels in the HbF of SS patients

The gamma-chains of fetal hemoglobin (HbF) of newborn babies are composed of about 70% G gamma and 30% A gamma. In most babies, the G gamma value declines postnatally to 40%, but in about 20% of black SS patients from Georgia, 5 years and older, the G gamma level remains high at 60%. Moreover, some 3% to 4% of black newborns have high G gamma values of 85%. PstI digestion of DNA of one such high G gamma baby and of one normal newborn showed the former to be heterozygous for the -G gamma-G gamma- and -G gamma-A gamma-chromosomes. Only about one fourth of high G gamma SS patients were such heterozygotes, while three fourths were -G gamma-A gamma-/-G gamma-A gamma-homozygotes. Analysis of DNA of 38 SS patients without the -G gamma-G gamma-chromosome showed a correlation of G gamma values with genotype at one polymorphic restriction site: at the HincII site in the psi beta gene, all -G gamma- A gamma-/-G gamma-A gamma-homozygotes with high G gamma were +/- or +/+, while low G gamma individuals were all -/-. Family studies, involving analyses at four polymorphic sites (HindIII sites in the G gamma and A gamma genes and HincII sites in the psi beta gene and 3' to it), suggested the association of an unidentified high G gamma genetic determinant with haplotype + - + +. This indicates that a genetic factor causing high G gamma levels in SS patients is closely linked to the -G gamma-A gamma-psi beta region of the beta-globin gene cluster.     

The role of neutrophil membrane glycoprotein GP-150 in neutrophil adherence to endothelium in vitro

We have previously described two patients with a congenital defect in neutrophil function characterized by an inability to form pus. The patients' neutrophils lack a membrane glycoprotein of mol wt 150,000 daltons (GP-150) on analysis by SDS-PAGE. This glycoprotein is part of a membrane antigen complex recognized by the murine monoclonal antibody (MoAb) 60.3. Addition of MoAb 60.3 to normal neutrophils produces defects in chemotaxis and phagocytosis in vitro similar to those observed in the patients. Since neutrophil adherence to vascular endothelium is prerequisite to neutrophil emigration in vivo, we examined the interaction of the patients' neutrophils and normal neutrophils treated with MoAb 60.3 with cultured endothelium. Adherence was determined as the percentage of 51Cr-labeled purified peripheral blood neutrophils which remained adherent to plastic wells or endothelial monolayers after a 45-minute incubation at 37 degrees C. The percentage of neutrophils from patient 1 remaining adherent to uncoated, fibronectin-coated, or laminin-coated plastic was similar to that observed in normal neutrophils (55% to 84% adherence with normal neutrophils v 73% to 78% adherence with the patient's neutrophils and 63% to 82% adherence with MoAb 60.3-treated normal neutrophils). The adherence of the neutrophils from patient 1 and MoAb 60.3-treated normal neutrophils to human or bovine endothelium in serum-free medium was also not significantly different from that observed in normal neutrophils (less than 10% adherence with normal, MoAb 60.3-treated, and patient neutrophils). In medium containing 10% autologous or heterologous human plasma, however, the adherence of neutrophils from patient 1 or MoAb 60.3-treated normal neutrophils to endothelial monolayers was significantly reduced (35% +/- 7% of normal neutrophils in seven experiments). Although phorbol myristate acetate (PMA) (10 ng/mL) and calcium ionophore A23187 (10(-5) mol/L) markedly increased the adherence of normal neutrophils to endothelial monolayers in serum- free medium (40% to 85% adherence), neither agent increased the adherence of the neutrophils from patient 1 or normal neutrophils treated with MoAb 60.3 (less than 5% adherence). The adherence of PMA- activated neutrophils from patient 2 to endothelial monolayers was also markedly decreased when compared with that of normal neutrophils. Postsecretory cell-free supernatants from PMA-activated normal neutrophils failed to augment adherence of neutrophils from patient 1 (less than 5% adherence).(ABSTRACT TRUNCATED AT 400 WORDS)     

Interleukin-3 and interleukin-7 are alternative growth factors for the same B-cell precursors in the mouse

Clones and lines of precursor (pre) B cells can be established by limiting dilutions of unseparated cell suspensions of fetal liver or bone marrow on stromal cells in the presence of interleukin (IL)-7. When IL-3 is used instead of IL-7, cultures are regularly overgrown by different precursor cells of the myeloid lineage, as well as by adherent cells that inhibit pre-B-cell expansion. However, in the presence of either IL-7 or IL-3, clones of pre-B cells can be established on stroma cells at frequencies near one in one when the cultures are initiated with cell sorter purified CD45RO (B220)+/c-kit+ fetal liver or bone marrow derived pre-B cells. Clones grown on stromal cells in the presence of IL-7 can be regrown in IL-3, and vice versa. Pre-B cells that proliferate on stromal cells in the presence of IL-7 or IL-3 have the same phenotype, ie, are B220+ c-kit+, CD43+, and surrogate light chain+. Removal of the growth factors (IL-7, respectively IL-3) from the cultures results in differentiation to surface immunoglobulin (slg) positive, c-kit-, CD43-, surrogate light chain- B cells, a fraction of which is lipopolysaccharide (LPS) responsive as shown by IgM secretion. These results show that IL-7 and IL-3 stimulate largely overlapping populations of precursor B cells from bone marrow to proliferate for long periods of time in the presence of stromal cells. Thus, IL-7 and IL-3 are alternative growth factors for the same pre-BI cell.     

Localization of factor VIII-procoagulant antigen: an immunohistological survey of the human body using monoclonal antibodies

Various organs, including liver, spleen, heart, lung, kidney, intestines, lymph nodes, pancreas, bone marrow, and thymus, were investigated for the presence of factor VIII-procoagulant antigen (VIIICAg) and factor VIII-related antigen (VIIIRAg), using a panel of monoclonal antibodies directed to factor VIII-von Willebrand factor in combination with a sensitive immunoperoxidase staining technique. In addition to hepatic sinusoidal endothelial cells, the presence of VIIICAg was demonstrated in mononuclear cells sporadically present in lymph nodes, in the alveolar septa of lung, and in the red pulp of spleen. The identity of these mononuclear cells could not be unequivocally determined. Based on morphological criteria, however, it is tentatively concluded that these cells are nonlymphoid and belong to the mononuclear phagocyte system. The presence of VIII-RAg was confined to vascular endothelial cells, hepatic sinusoidal endothelial cells, cells lining the venous sinuses of the red pulp of the spleen, cells lining renal glomeruli and lung capillaries, platelets, and megakaryocytes.     

Characterization of 25 monoclonal antibodies to factor VIII-von Willebrand factor: relationship between ristocetin-induced platelet aggregation and platelet adherence to subendothelium

We have studied the role of factor VIII-von Willebrand factor (FVIII- vWF) in both platelet adherence to subendothelium and ristocetin- induced platelet aggregation using monoclonal antibodies to human FVIII- vWF. Twenty-five monoclonal antibodies were obtained, two of which were directed to the factor VIII moiety of FVIII-vWF; one of these two completely inhibited the procoagulant activity (FVIII:C). The remaining 23 monoclonal antibodies were directed to the von Willebrand factor moiety of FVIII-vWF. The ability of the latter monoclonal antibodies to inhibit platelet adherence to arterial subendothelium was investigated with a perfusion model. According to the number of platelets adhering to the subendothelium, three groups of monoclonal antibodies could be discerned: (A) antibodies not affecting platelet adherence; (B) antibodies that inhibited platelet adherence to the level as observed when von Willebrand's disease plasma was tested; and (C) antibodies that completely inhibited both platelet adherence to subendothelium and ristocetin-induced platelet aggregation. The two antibodies present in group C competed for the same or closely related epitope(s) present on FVIII-vWF. These results demonstrate that a domain is present on the FVIII-vWF molecule that is associated both with ristocetin-induced aggregation and with the ability of FVIII-vWF to support platelet adherence to the subendothelium. Based on these observations, it is concluded that ristocetin-induced binding of FVIII-vWF to platelets reflects, at least in part, a physiologic mechanism regulating the function of FVIII-vWF in primary hemostasis.     

The rare alpha-thalassemia-1 of blacks is a zeta alpha-thalassemia-1 associated with deletion of all alpha- and zeta-globin genes

Restriction endonuclease mapping with alpha and zeta-globin gene probes showed differences between the alpha-thalassemia-1 (alpha-thal-1) condition in two patients with HbH disease. One patient had the rare black type of alpha-thal-1 together with alpha-thal-2 and HbS heterozygosities. The second patient was a Laotian child with HbE, Hb Constant Spring (alpha-thal-2), and alpha-thal-1 heterozygosities. The diagnoses were based on clinical, hematologic, and biochemical data. Whereas DNA fragments hybridizing to a zeta-probe were obtained from the Laotian type of alpha-thal-1, neither alpha nor zeta-gene fragments could be identified deriving from the black type of alpha-thal-1. Therefore, the black type of alpha-thal-1 is associated with a deletion of the entire zeta 2-psi zeta-psi alpha-alpha 2-alpha 1 gene complex and can be considered a zeta alpha-thal-1. It is likely that homozygosity for such a condition will lead to embryonic wastage, explaining the absence of hydrops fetalis in blacks.     

Serological confirmation of human T-lymphotropic virus type I infection in healthy blood and plasma donors

We wished to develop criteria for serological confirmation of human T- lymphotropic virus type I (HTLV-I) infection in healthy donors. Selected serum or plasma samples reactive by HTLV-I enzyme immunosorbent assay or gel-agglutination assays with at least one viral- specific band on Western immunoblot (WIB) were tested in six laboratories by four WIBs and four radioimmunoprecipitation assays (RIPAs) for antibodies to HTLV-I proteins encoded by gag (p19 and p24), env (gp46 and/or gp61), and tax (p40x) genes. One hundred forty-two donor sera were obtained from 38 Japanese, 69 American, and 35 Caribbean blood or plasma donors. Among these samples, WIB assays appeared more sensitive to p24 antibodies, whereas RIPAs were significantly more sensitive to gp61 antibodies. All sera (137) with gp61 antibodies had p24 antibodies. Of the 137 sera positive for p24 and gp61 antibodies, p19 antibodies were detected in 129 sera, and p40x antibodies were detected in 108. In sera with p19 antibodies and antibodies to env- or tax-encoded proteins, p24 antibodies were always present. Antibodies to p40x were not found in the absence of gp61 antibodies. Virological evidence of infection was found in seven American donors by lymphocyte coculture (one HTLV-I, one HTLV-II) or by polymerase chain reaction (three HTLV-I, two HTLV-II). Sera from all seven donors showed p24 and gp46 and/or gp61 antibodies. We suggest that seroreactivity to both p24 and gp46 and/or gp61 by WIB or RIPA or both are suitable criteria to confirm but not to distinguish HTLV-I and HTLV-II infections.