Microenvironmental toxicity of azidothymidine: partial sparing with hemin

Azidothymidine (AZT) is a useful drug in management of AIDS. Nevertheless, its hematologic toxicity such as anemia and neutropenia present further complications to an already compromised hematopoietic state in patients. We studied the effects of AZT on human and murine bone marrow (BM) colony growth as determined by assays of CFU-E, BFU-E, CFU-GM, and fibroblastoid stromal (CFU-Fb) colonies. Cultures were grown in methylcellulose with growth factors and scored after three- to 14-day incubation. In general, murine marrow cultures were more sensitive to AZT as compared with human marrow. Furthermore, interindividual variation in toxicity to AZT was observed between marrow samples; 1 mumol/L AZT inhibited murine CFU-E, BFU-E, and CFU-GM by 98% to 100%, whereas human marrow was inhibited by 52%, 87%, and 65%, respectively. Lower concentrations of AZT (0.1 mumol/L) inhibited murine erythroid colony growth by 85% to 90%, whereas human growth was inhibited by only 39% to 52%. Myeloid colony inhibition was similar for human and murine systems. CFU-Fb growth was markedly suppressed (75%) by 1 mumol/L AZT. Hemin, at a concentration of 10 mumol/L, overcame some of the inhibitory effects of 1 to 0.1 mumol/L AZT without hindering antiviral activity. Inhibition of human CFU-E growth was completely overcome with hemin, whereas CFU-GM growth was recovered to 66% to 74% of control. A similar but less pronounced effect was observed for BFU-E. Furthermore, hemin does not decrease AZT's effects of HIV antigen content in vitro. We conclude that anemia and neutropenia, occurring as a result of AZT, may not be as pronounced in the presence of hemin. Furthermore, CFU-Fb was significantly reduced in the presence of low concentrations of AZT. This may indicate a major target site for BM toxicity since the stromal microenvironment may be responsible for maintaining short- and long-term hematopoiesis.     

Life-threatening transient neonatal Behcet's disease [published erratum appears in Br J Rheumatol 1997 Sep;36(9):1032]

This case report describes transient neonatal Behcet's disease, with life-threatening complications in the neonate. Male Baby R developed blood-streaked diarrhoea 5 days after birth, followed by recurrent severe scarring orogenital ulceration and vasculitic skin lesions. In this sixth week of life, he developed stridor leading to a respiratory arrest and necessitating assisted ventilation. No infective cause was isolated. Baby R responded well to i.v. and subsequent oral steroid therapy. At 8 weeks old he had fully recovered and remains well. Baby R's mother was not previously known to have Behcet's disease. During the pregnancy, she began to suffer orogenital ulceration, associated with skin lesions typical of Behcet's disease. Mild orogenital ulceration has become recurrent.      

Translocation (14;18)-positive cells are present in the circulation of the majority of patients with localized (stage I and II) follicular non- Hodgkin's lymphoma

Stage I and II follicular non-Hodgkin's lymphoma (NHL) is clinically defined as a localized disease. To study the possibility that this disease is in fact disseminated, we used the sensitive polymerase chain reaction (PCR) method using translocation (14;18) as marker. Samples from 21 patients who were clinically diagnosed with stage I or II follicular NHL were analyzed for the presence of t(14;18)-positive cells using PCR. We analyzed (1) the diagnostic lymph node biopsy and (2) the peripheral blood or bone marrow samples from these patients. Translocation (14;18) cells were detected in the diagnostic lymph node biopsies of 12 patients. In 9 of these patients, t(14;18)-positive cells were detected in peripheral blood and/or bone marrow samples at diagnosis and/or after therapy. Thus, in 75% of the follicular NHL patients carrying the t(14;18) as a marker for lymphoma cells, t(14;18)- positive cells were detected in peripheral blood and bone marrow at diagnosis and after therapy. Our results show that t(14;18)-positive cells can be detected in the circulation of patients with stage I and II follicular NHL, indicating that, although diagnosed as localized, the disease is disseminated.     

The genotype and phenotype of T cell and non-T, non-B acute lymphoblastic leukemia

We used Southern blot analysis to investigate the T cell receptor and immunoglobulin (Ig) genes in tumor DNA derived from 13 patients with acute lymphoblastic leukemia (ALL). Rearrangement of both alleles of the T cell receptor beta chain gene was demonstrated in the three individuals with T cell ALL, while the Ig genes remained in germline configuration. In contrast, in nine of ten patients with non-T, non-B ALL (seven common ALL antigen [CALLA]-positive), Ig genes were rearranged while the T cell receptor genes were intact: the noteworthy exception was a CALLA-positive individual in whom one allele of the T cell receptor was rearranged and in whom a minor rearranged Ig band was detected as well. This exception indicates that CALLA-positive non-T, non-B ALL includes proliferations that have undergone an initial genetic step toward T cell differentiation. With probes now available for both the Ig and T cell receptor genes, analysis of genomic tumor DNA is useful in all variants of ALL.     

Characterization of immature T cell subpopulations in neonatal blood

A series of monoclonal antibodies directed against T cell differentiation antigens was used to identify circulating T cells in normal human neonates. Twenty-five cord blood samples, taken after cesarean or vaginal delivery, and 16 venous blood samples from normal adult controls were examined using monoclonal antibodies in an indirect immunofluorescence technique. The percentage of circulating OKT3 positive (pan-T cell) cells was significantly lower in the neonatal blood (52.8%) compared with the adult controls (74.9%) (P less than .001). Of the cord mononuclear cells, 58% showed reactivity with OKT10 (common thymocyte antigen) compared with only 7% in adult controls (P less than .001). The helper:suppressor T cell ratio was lower in cord blood (1:2) as compared with 1:3 for adult blood (P less than .005) as observed in these studies. These figures reflect the presence of a significant population (25%) of immature T cells in cord blood that expresses simultaneously both helper (OKT4) and suppressor (OKT8) phenotypes. Twenty-four percent of the T cells in cord blood also expressed OKT6 antigen (cortical thymocyte), a feature not found in adult blood. Double-labeling studies characterized a previously undescribed blood T cell phenotype, which was simultaneously OKT6 and OKT3 (pan-T cell) positive; of the cells reactive with OKT3, 43% also were positive with OKT6. This study reveals the presence of immature populations of T cells in normal human neonatal blood exhibiting phenotypes characteristic of normal developing thymocytes and a previously undescribed cell phenotype.     

Lymphosarcoma cell leukemia: the contribution of cell surface study to diagnosis

Cell surface immunoglobulin, complement receptor, and spontaneous rosette formation with sheep erythrocytes were investigated in 43 patients with malignant lymphoma, including 13 with lymphosarcoma cell leukemia, and in 59 patients with chronic lymphocytic leukemia. The quantity of immunoglobulin on the lymphocyte surface was estimated from the intensity of fluorescent staining with fluorescein-conjugated anti- immunoglobulin antisera. At least two, and probably three, B cell species could be recognized by cell surface study. Cells from chronic lymphocytic leukemia and diffuse well-differentiated lymphocytic lymphoma had sparse amounts of surface immunoglobulin, while the cells of diffuse poorly differentiated lymphocytic lymphoma had large quantities of this material. Nodular lymphoma probably represented a third B-cell subtype with intermediate amounts of surface immunoglobulin. The lymphocytes of chronic lymphosarcoma cell leukemia exhibited the intense surface staining, which was characteristic of the underlying poorly differentiated lymphocytic lymphoma (diffuse or nodular), and could be readily distinguished from the faint-staining chronic lymphocytic leukemia cells.     

Antigen receptor nonresponsiveness in chronic lymphocytic leukemia B cells

B chronic lymphocytic leukemia (B-CLL) are clonal populations of mIgM+ or mIgM+/mIgD+ CD5+ B cells that appear to be arrested in the follicular mantle-zone B-cell stage. Functional analyses have shown two groups of B-CLL that can be distinguished based on their capacity to proliferate in response to B-cell antigen receptor complex (BCR) cross- linking. To investigate the molecular basis for this phenomenon, we have analyzed both architecture and functional properties of BCR complexes on these two groups of B-CLL. Both groups were found to express structurally similar BCR. However, protein tyrosine kinase (PTK) activity associated with and specific for BCR constituents was strongly diminished in nonresponsive B-CLL. Moreover, the PTK-dependent assembly of Shc/Grb2 complexes, which may couple the BCR to p21ras, was absent in these B-CLL. Finally, of all PTKs tested, the expression of PTK syk was found to be considerably lower in nonresponsive B-CLL. Thus, absence of mitogenic responses upon BCR cross-linking in particular B-CLL was found to be strictly correlated with diminished induction of BCR-associated PTK activity and lower levels of PTK syk. Because nonresponsive B-CLL closely resembles tolerant autoreactive B cells both functionally and biochemically, distinction between B-CLL with respect to functional properties in vitro may be determined by differences in antigen encounter in vivo.     

Intracellular ferriprotoporphyrin IX is a lytic agent

Human erythrocytes were treated with menadione to oxidatively denature hemoglobin and release ferriprotoporphyrin IX (ferriheme, FP) intracellularly. The high affinity of FP for chloroquine was used to detect its release. After incubation for 1 hr at 37 degrees C and pH 7.4 with 0.5 mM menadione, erythrocytes bound 14C-chloroquine with an apparent dissociation constant of 10(-6)M. Untreated erythrocytes did not bind chloroquine with high affinity. At a chloroquine concentration in the medium of 2 microM, for example, menadione-treated erythrocytes bound 70 mumole chloroquine/kg and untreated erythrocytes bound 13.4 mumole/kg. The intracellular location of FP released by menadione was verified by finding that Tween 80 did not prevent chloroquine binding. By contrast, Tween 80 inhibited the binding of chloroquine to erythrocytes treated with extracellular FP. The hemolytic response to menadione was characteristic of the hemolytic response to FP. Thus, 5 microM chloroquine caused hemolysis to increase to 60% from baseline values of 5% in experiments using erythrocytes treated either with 0.5 mM menadione or with 5 microM FP; and, in both cases, the potentiating effect of chloroquine was inhibited by 1 microM mefloquine or 10 microM quinine. Higher concentrations of menadione caused hemolysis in the absence of chloroquine. We conclude that FP released by menadione exists intracellularly in a form that is accessible to bind chloroquine and to express its lytic activity.     

A unique 7p/12q chromosomal abnormality associated with recurrent abortion and hypofibrinogenemia

Recurrent first trimester abortions led to evaluation of a 25-year-old woman. Studies revealed she had hypofibrinogenemia (68 mg/dL) without evidence of dysfibrinogenemia or increased fibrinogen turnover. She was also found to have a unique 46,XX, t(7;12) (p 15.2;q24.31) karyotype. Hypofibrinogenemia and identical chromosomal abnormalities were found in other members of her kindred. Southern blots of genomic DNA from the patient, her mother, and her daughter hybridized to human fibrinogen probes showed alpha, beta, and gamma fibrinogen genes to be present and without structural alterations when compared to normal controls. We conclude that the chromosomal abnormality and the hypofibrinogenemia are related but in an unclear manner. Because fibrinogen infusion in the proposita was associated with successful gestation, we also concluded that the chromosomal abnormality itself was not responsible for the repeated abortions but that fibrinogen concentration may be critical in securing implantation.