Arginyl-glycyl-aspartic acid sequences and fibrinogen binding to platelets

Human fibrinogen has an Arg-Gly-Asp-Ser (RGDS) sequence at residues 572- 575 of its A alpha-chain. Although RGDS-containing peptides inhibit fibrinogen binding to stimulated platelets, these peptides also inhibit platelet binding of human fibrinogen fragment X and rat fibrinogen, which lack RGDS sequences corresponding to A alpha 572-575. Thus competition between free RGD-containing peptides and internal RGDS sequence at A alpha 572-575 is not the basis for their inhibition of fibrinogen binding to platelets. Addition of a Thr to the carboxy- terminus and an Asn to the amino-terminus of the RGDS sequence, the amino acids corresponding to A alpha 576 and 571 respectively, reduced the inhibitory potency of RGDS-containing peptides by fourfold to tenfold. Arg-Gly-Asp-Phe (RGDF) corresponds to A alpha 95-98, and the RGDF peptide was an effective inhibitor of fibrinogen binding, fourfold to fivefold more potent than RGDS. Thus, local primary structure may play an important role in regulating the capacity of RGD sequences in proteins to interact with specific adhesion receptors.     

Residual juvenile chronic myelogenous leukemia cells detected in peripheral blood during clinical remission

At diagnosis peripheral blood (PB) or bone marrow from patients with juvenile chronic myelogenous leukemia (JCML) have shown two reproducible abnormalities when studied in cell culture: impaired growth of normal hematopoietic progenitors and excessive proliferation of malignant monocyte-macrophage elements. We used these findings to assess quality of treatment response by serially studying PB specimens from four JCML patients (patients 5, 7, 8, and 9) in complete chemotherapy-induced remission. PB readily yielded high numbers of monocyte-macrophage colonies in CFU-C and CFU-GEMM assays when cultured in early remission, and the colonies were cytogenetically proven to have arisen from a malignant clone in patient 9. When studied later in remission, the abnormal cell proliferation persisted in three of the four patients, but in patient 8 PB colony growth resembled controls. Similarly, when PB from patient 8 was studied in liquid culture without using added growth factor, cell proliferation declined identical to controls, whereas PB from the other three patients showed exuberant growth of monocyte-macrophage elements. Patient 8 successfully completed therapy and has been in a long-term, disease-free remission. The other three had recurrent, ultimately fatal disease. The cell cultures have allowed detection of residual abnormal cells that circulate in PB of JCML patients in remission. Although patient numbers were small because of the rarity of JCML, the data suggested that persistence of leukemia cells in these patients had a bearing on clinical outcome.     

CD79a: a novel marker for B-cell neoplasms in routinely processed tissue samples

The CD79 molecule, comprising two polypeptide chains, mb-1 (CD79a) and B29 (CD79b), is physically associated in the B-cell membrane with immunoglobulin. It transmits a signal after antigen binding and may, therefore, be considered the B cell equivalent of CD3. It appears before the pre-B-cell stage, and the mb-1 (CD79a) chain can still be present at the plasma cell stage. In this report, we describe a new anti-CD79a monoclonal antibody, JCB117, which reacts with human B cells in paraffin embedded tissue sections, including decalcified bone marrow trephines. When tested on a total of 454 paraffin embedded tissue biopsies, gathered from a number of different institutions, it reacted with the great majority (97%) of B-cell neoplasms, covering the full range of B-cell maturation, including 10 of 20 cases of myeloma/plasmacytoma. It is of interest that the antibody labels precursor B-cell acute lymphoblastic leukemia samples, making it the most reliable B-cell marker detectable in paraffin-embedded specimens in this disorder. All neoplasms of T cell or nonlymphoid origin were negative, indicating that antibody JCB117 may be of value to diagnostic histopathologists for the identification of B-cell neoplasms of all maturation stages.     

Refined mapping of genomic rearrangements involving the short arm of chromosome 9 in acute lymphoblastic leukemias and other hematologic malignancies

Deletions of chromosomal band 9p21 have been detected in various tumor types as well as in more than 20% of acute lymphoblastic leukemia (ALL). These deletions frequently include the entire interferon (IFN) gene cluster as well as the methylthioadenosine phosphorylase (MTAP) gene. Recently, the CDKN2 gene (p16INK4A, MTS I, CDK41) was proposed as a candidate tumor-suppressor gene on 9p21 because it is frequently deleted in cell lines derived from multiple tumor types. To determine if CDKN2 or another closely related gene on 9p is the target of 9p deletions in ALL and other hematologic malignancies, we analyzed 20 primary patient samples (13 ALL, 2 acute myeloid leukemias [AML], and 5 non-Hodgkin's lymphomas [NHL]) with 9p rearrangements using Southern blot analysis, fluorescence in situ hybridization (FISH), and single- strand conformation polymorphism (SSCP) for alterations of CDKN2. Homozygous deletions of the CDKN2/CDKN2B (p15) region were detected in 10 cases (50%; 6 ALL, 2 AML, and 2 NHL). In 1 additional case, the intensity of the Southern blot band was significantly reduced, suggesting a CDKN2 deletion in a subpopulation of the malignant cells. No CDKN2 or CDKN2B rearrangements were seen. The IFN gene cluster was homozygously deleted in 2 of 15 (13%) analyzed cases, whereas the MTAP gene was deleted in 6 of 15 cases (40%). In addition, hemizygous deletions of the CDKN2 region were identified in 6 ALL cases using interphase FISH. No point mutation of the coding region of CDKN2 was detected by SSCP in these cases. We conclude that CDKN2 is the most frequently homozygously deleted marker on 9p. The absence of point mutations in the coding region of CDKN2 in cases with hemizygous 9p deletions and the frequent codeletion of MTAP, CDKN2B, and other yet unidentified neighboring genes suggest that the simultaneous deletion of these genes may be necessary for the selective growth advantage of malignant cells.     

In vitro and in vivo effects of deferoxamine in neonatal acute leukemia

A six week old infant with acute leukemia failed to attain remission with chemotherapy. Because we previously demonstrated that the iron chelator deferoxamine (DFO) has antiproliferative properties and modulatory effects on cell differentiation, a protocol was designed for in vitro study and for clinical use in the patient. At diagnosis, blast cells were morphologically undifferentiated, had nondiagnostic cytochemistry, showed an abnormal karyotype (t[4;11]), expressed markers of B cell lineage, and demonstrated C mu gene rearrangement. Tissue culture of marrow or blood cells yielded colonies of leukemic blasts. Increasing concentrations of DFO produced a dose-dependent suppression of patient's blast colony growth in vitro, and blasts within colonies showed a marked change in surface antigen expression from lymphoid to myelomonocytic markers, became monocytic in appearance, and developed intense staining for nonspecific esterase. When DFO was given intravenously to the patient as a single agent for 48 hours, blasts no longer expressed lymphoid antigens and became strongly positive for myelomonocytic markers, identical to the in vitro findings. Intravenous DFO halted rising peripheral blood blast cell numbers and allowed a several-fold increase in normal hematopoietic progenitor colony growth. When combined with low-dose cytosine arabinoside in the treatment protocol, DFO caused striking leukemic cytoreduction. Our findings indicate that DFO has antileukemic properties by virtue of its effects on proliferation and differentiation, and they prompt further experimental and clinical studies with this agent.     

The regulatory role of interleukin 2-responsive T lymphocytes on human marrow granulopoiesis

The effect of recombinant interleukin 2 (IL2) on marrow CFU-C colony formation was evaluated to define the role for T lymphocytes in human marrow granulopoiesis. The colony-stimulating factor (CSA) used in our experiments was found to contain IL2. IL2 depletion from CSA resulted in a reduction in CFU-C colony proliferation. Addition of exogenous IL2 caused an increase in CFU-C colony numbers in a dose-dependent manner. This increase could be prevented by anti-Tac, a monoclonal antibody (MoAb) to the IL2 receptor. Moreover, anti-Tac in the absence of exogenous IL2 resulted in an overall decrease in colony numbers. Depletion of either adherent cells or T lymphocytes abolished the effect of IL2 and anti-Tac on colony growth. In the presence of IL2, re- addition of T lymphocytes to the T-depleted marrow or adherent cells to adherent cell-depleted marrow resulted in a significant increase in CFU- C colony numbers, whereas no significant effect was found when IL2- depleted CSA was used. Although T lymphocytes were not themselves essential for CFU-C colony growth, our studies indicate that IL2 and IL2-responsive T cells can regulate in vitro granulopoiesis.     

Deficiency of plasma plasminogen activator inhibitor 1 results in hyperfibrinolytic bleeding

A 63-year-old man was evaluated for a lifelong history of bleeding commencing with frequent epistaxis as a child; all previous routine coagulation parameters were within the normal range. The patient's hemorrhagic disorder is characterized predominantly by delayed bleeding at surgical sites. In the resting state, there was no clinical or laboratory evidence of excessive fibrin(ogen)olysis. Bleeding was not caused by disseminated intravascular coagulation, factor XIII deficiency, alpha 2-antiplasmin deficiency, or dysfibrinogenemia. It was found that the patient was deficient in plasma PAI-1 antigen and activity but with approximately half normal antigen and normal activity of platelet PAI-1. The low concentration of plasma PAI-1 was insufficient to neutralize circulating t-PA, resulting in high t-PA activity with normal antigen and causing the hyperfibrinolytic activity observed. Studies on seven family members of the proband indicated autosomal inheritance of plasma PAI-1 deficiency. Studies on this patient emphasize a clear correlation between decreased plasma PAI-1 activity and hyperfibrinolytic bleeding and also emphasize the unique role of plasma PAI-1 in the balance between the coagulation and fibrinolytic mechanisms.     

Monocyte-macrophage-derived acidic isoferritins: normal feedback regulators of granulocyte-macrophage progenitor cells in vitro

The recent identification of a leukemia-associated inhibitory activity (LIA) against granulocyte-macrophage progenitor cells (CFU-GM) as acidic isoferritins has now led to detection of this activity in normal bone marrow and blood cells. Detection of this activity depends on stimulation of CFU-GM by granulocyte-macrophage colony stimulatory factors (GM-CSF), and some conditioned media (CM) sources of GM-CSF (human placental and monocyte, mouse macrophage and WEHI-3) contained low levels of acidic isoferritin that lowered colony formation. Inactivation or removal of this activity increased the stimulatory capacity of the CM. CM depleted of acidic isoferritins or CM originally devoid of this activity (human GCT, 5637, Mo, lymphocytes: mouse L cells or pokeweed-mitogen-stimulated spleen cells) increased the sensitivity of the assay to detect acidic isoferritin inhibitory activity. This activity was selectively contained and released from normal monocytes and macrophages. Restriction of this activity to mononuclear phagocytes was substantiated, as only continuous cell lines of monocytes and macrophages or lines capable of induction to this lineage contained and released acidic isoferritin inhibitory activity. The cells of origin and target cells of action suggest that acidic isoferritin-inhibitory activity can be considered as a negative feedback regulator, at least in vitro.     

A child with bronchiectasis,chronic mucocutaneous candidiasis,and hypothyroidism secondary to STAT1 gain‐of‐function mutation: A case report and review of the literature

STAT 1 GOF mutations are a rare cause of childhood primary immunodeficiency. Recurrent mucocutaneous candidiasis, chest infections, and autoimmune disease are all classic phenotype presentations. Rapid identification and diagnosis of this debilitating disease using whole exon sequencing may improve outcomes and minimize long‐term sequelae.