bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasminogen activator inhibitor: a regulator of ancrod-induced fibrin deposition in rabbits
Authors: Krishnamurti C , Barr CF , Hassett MA , Young GD , Alving BM
Published: October 2019

Plasma levels of a fast-acting plasminogen activator inhibitor (PAI), which neutralizes both tissue plasminogen activator (t-PA) and urokinase, are markedly increased in endotoxin-treated rabbits. The ability of this inhibitor to prevent the fibrinolysis that occurs after a thrombogenic stimulus was investigated in a rabbit model. Normal and endotoxin-treated male New Zealand rabbits were infused with ancrod, an enzyme that causes noncrosslinked fibrin formation in vivo. Ancrod stimulated t-PA activity by 90% in normal rabbits and caused hypofibrinogenemia but did not increase PAI levels or induce fibrin deposition in target organs. Rabbits injected with endotoxin (10 micrograms/kg) showed an increase in PAI from less than 1 to 32 U/mL 4 hours later. When ancrod was infused at this time, 90% of the rabbits developed renal fibrin thrombi. Fibrin deposition was recorded in 40% of the rabbits that received a lower dose of endotoxin (1.0 microgram/kg) and had a PAI level of 14 U/ml at the time of ancrod infusion 4 hours later. Fibrin deposition did not occur in the endotoxin-treated rabbits that received normal saline. These data suggest that high levels of PAI inhibit fibrinolysis in vivo, thereby promoting fibrin clot deposition following a thrombogenic stimulus.
Monoclonal anti-light chain idiotype as a tumor-specific probe for human neoplastic B lymphocytes
Authors: Wrightham M , Tutt AL , Glennie MJ , Hamblin TJ , Stevenson GT , Stevenson FK
Published: October 2019

Tumor cells from patients with B cell neoplasms often secrete small amounts of free monoclonal light chains that can be found in the urine. Such tumor-derived light chains of the lambda type from a patient with typical chronic lymphocytic leukemia have been used to raise mouse monoclonal antibodies (MoAbs). A hybridoma-secreting antibody that recognized the idiotypic lambda chain but not normal lambda chains by a preliminary screen but which also reacted with idiotypic IgM from the patient's tumor cells was selected. This MoAb in fact recognized 1 in 20 X 10(3) molecules of pooled normal lambda chains, thus establishing its specificity for a private idiotypic determinant. It failed to give a detectable reaction with normal IgM, normal serum, or a panel of IgM paraproteins. The antibody bound to the patient's neoplastic B cells but not to normal tonsillar cells. The site of binding of the antibody to idiotypic IgM is clearly separate from that of another MoAb specific for idiotypic determinants on heavy plus light chains, since the two showed additive binding curves. The determinant also appeared to be less available in dimeric lambda chains than in monomeric lambda chains or in idiotypic IgM. Antibodies to idiotypic determinants on light chains show some technical advantages and should be useful for monitoring and possibly treating B cell tumors, either alone or together with the more conventional anti-idiotypic antibodies that usually recognize the heavy and light chain combination.
Blast cell colony assay for umbilical cord blood and adult bone marrow progenitors
Authors: Leary AG , Ogawa M
Published: October 2019

We previously described candidate human blast cell colonies in culture of umbilical cord blood cells. However, their replating efficiencies were low, and we were unable to grow colonies from adult marrow cells. We report here a consistent method of growth and identification of human blast cell colonies that are supported by low serum culture and by delayed addition of medium conditioned by a T lymphoblast cell line, C5MJ. Nonadherent mononuclear cord blood and bone marrow cells were prepared by use of Ficoll-Paque and overnight adherence to plastic. Bone marrow cells were further enriched for progenitors by panning with monoclonal anti-My-10 antibody. Cells were plated in methylcellulose culture containing 2% fetal calf serum and supplemented with bovine serum albumin, lecithin, cholesterol, and transferrin. On day 14 of culture, concentrated C5MJ-conditioned medium was carefully added to each dish. Blast cell colonies consisting of 18 to 100 cells were detected on days 21 to 28. Forty percent to 75% of the blast cell colonies in individual samples yielded secondary colonies upon replating (positive colonies). The replating efficiency of the positive colonies ranged from 3% to 100%. The largest secondary colony contained 7,800 cells. In addition to single-lineage colonies, multilineage colonies revealing two to five lineage combinations were seen. These results suggest that human primitive progenitors are dormant in cell cycle and that they survive in the absence of colony-stimulating factors. Human blast cell colonies may provide a unique population of progenitors for studies of the early process of human hemopoiesis.
T cell gamma gene rearrangements in hematologic neoplasms
Authors: Asou N , Matsuoka M , Hattori T , Kawano F , Maeda S , Shimada K , Takatsuki K
Published: October 2019

Rearrangements of the T cell gamma (T gamma) gene were studied in primary neoplastic cells from 75 patients with leukemia or lymphoma. T gamma gene rearrangements were observed in 19 of 21 T cell neoplasms; 14 of 21 immature B cell leukemias, including 4 out of 5 patients with rearrangements of both immunoglobulin heavy-chain (JH) and T cell receptor beta chain (T beta) genes; none out of 16 nonlymphoid leukemias. Thus, T gamma gene rearrangement is frequently found in immature B cells and is not always found in T cells showing T beta gene rearrangement, but it is not detected in nonlymphoid cells. Furthermore, T gamma gene rearrangement in cells with the germline configuration of the JH and T beta genes was observed. These results indicate that the detection of T gamma gene rearrangement does not allow a clear assignment to a particular lineage. However, an analysis of T gamma gene rearrangement provides a further potential tool to establish the lymphoid cellular origin and clonality of hematologic neoplasms and identify the normal stages of lymphocyte differentiation.
Total body irradiation and high-dose etoposide: a new preparatory regimen for bone marrow transplantation in patients with advanced hematologic malignancies [published erratum appears in Blood 1987 Jun;69(6):1789]
Authors: Blume KG , Forman SJ , O'Donnell MR , Doroshow JH , Krance RA , Nademanee AP , Snyder DS , Schmidt GM , Fahey JL , Metter GE
Published: October 2019

In a phase I/II study, 47 patients (median age, 24 years) with hematologic malignancies (33 patients with acute leukemia not in first remission and 14 patients with other advanced malignant hematologic disorders) were treated with total body irradiation and high doses of etoposide (VP16–213) followed by bone marrow transplantation. At the time of analysis, 21 patients were alive, and 19 of them were in continued complete remission for 101 days to greater than 40 months (median, 12 months). The actuarial disease-free survival rate of the 33 acute leukemia patients is 43% (2 SEM, 18%) and the actuarial relapse rate is 32% (2 SEM, 20%). Five of the 14 patients with the other hematologic malignancies are alive, and four of them continue to be free of disease for 8 to 27 months. Pharmacokinetic studies established a strong correlation between the administered drug doses and their plasma levels and also demonstrated complete drug clearance prior to marrow grafting. An etoposide dose of 60 mg/kg body weight was found to be the maximum tolerated dose. This new preparatory regimen was well tolerated and was not associated with specific acute or long-term regimen-related toxicities. Our data suggest that total body irradiation with high-dose etoposide presents a viable alternative to other preparatory regimens. The role of this novel combination remains to be defined by future prospective randomized trials.
Development of T3/T cell receptor gene expression in human pre-T neoplasms
Authors: Pittaluga S , Uppenkamp M , Cossman J
Published: October 2019

Acquisition of mature T cell function and the T cell antigen receptor repertoire occur in the thymus. In an effort to delineate the cascade of events leading to T cell maturation, we analyzed a series of clonal human precursor T cell neoplasms representing early, middle, and late stages of intrathymic differentiation. Rearrangements of the T cell receptor beta and gamma genes appear concurrently and are preceded by surface expression of the 3A1 (CD7) molecule. Subsequent transcription of the beta gene is coordinated with surface expression of T1 (CD5) and T11 (CD2), transcription of T3 delta mRNA, and the appearance of intracellular T3 (CD3) protein. As late events, T alpha gene transcripts appear and, finally, T3, the multichain complex linked to the T cell receptor, is presented on the cell surface. Findings reported here provide a model of the developmental orchestration of genes encoding antigen recognition in human T cells.
Early T cell differentiated chronic myeloid leukemia blast crisis with rearrangement of the breakpoint cluster region but not of the T cell receptor beta chain genes
Authors: Gramatzki M , Bartram CR , Muller D , Walter M , Tittelbach H , Kalden JR
Published: October 2019

Early T cell differentiation is described in a case of Philadelphia chromosome-positive chronic myeloid leukemia (CML) in blast crisis, supporting multi-lineage differentiation potential of CML precursor cells. In the absence of myeloid markers, strong positivity for terminal deoxynucleotidyl transferase (TdT) and reactivity with T cell antibody 3A1, but lack of more mature T cell antigens, provided evidence for immature T cell differentiation. Molecular analysis of the breakpoint cluster region (bcr) in chromosome 22 revealed a rearrangement and thus confirmed the CML origin of the early T cell blasts. T cell receptor beta chain sequences were found in germline configuration and therefore suggest a very immature stage of T cell differentiation in the CML blasts.
In vitro action of tumor necrosis factor on myeloid leukemia cells
Authors: Munker R , Koeffler P
Published: October 2019

We investigated the in vitro action of recombinant tumor necrosis factor alpha (TNF) on the clonal growth of normal and malignant myeloid cells. Clonogenic cells from six of nine myeloid leukemia cell lines were very sensitive to the effects of TNF with 50% of the colonies inhibited (ED50) by concentrations of TNF that ranged between 6 and 150 U/mL. A decrease in DNA, RNA, and protein synthesis and in cloning efficiency occurred within three hours of exposure of HL-60 promyelocytes to TNF. The TNF in combination with recombinant interferons produced an additive or synergistic inhibition of colony formation of HL-60 and THP-1 myelomonoblasts. Normal human CFU-GM are sensitive to TNF (ED50 between 100 and 50,000 U/mL), but their sensitivity to TNF depends on the source of colony stimulating factor (CSF) with T lymphocyte derived GM-CSF (recombinant or natural) partially protecting the CFU-GM from the suppression exerted by TNF (and interferons). In eight of 15 cases the clonogenic myeloid leukemia cells from patients with acute or chronic myeloid leukemia were more sensitive than normal CFU-GM using GM-CSF as a source of colony stimulating activity. Further studies showed that the action of TNF on myeloid leukemia cells probably can be only partially explained by differentiation. Our finding of a possible selective cytotoxicity to leukemic clonogenic cells by TNF suggests that TNF may have value in the therapy of some patients with myeloid leukemia.
Increased susceptibility of peripheral mononuclear cells of leukemic patients to HTLV-I infection in vitro
Authors: Graziani G , Pasqualetti D , Lopez M , D'Onofrio C , Testi AM , Mandelli F , Gallo RC , Bonmassar E
Published: October 2019

Peripheral mononuclear cells (MNC) collected from 12 healthy donors and 44 leukemic patients at various stages of the disease were tested for natural killer (NK) activity and for their susceptibility to HTLV-I infection in vitro, measured in terms of percentage of p19 positive cells. MNC from leukemic donors at any stage of leukemia (ie, onset or relapse, ON/REL; complete remission or off-therapy, CR/OT donors) were highly susceptible to HTLV-I infection. This was true for acute leukemias of lymphoblastic (ALL) or nonlymphoblastic (ANLL) type. MNC of ON/REL patients were more susceptible to HTLV-I than those of CR/OT donors. In addition, leukemic blasts were more rapidly infected (ie, within five to seven days) than the HTLV-I-susceptible normal cord- blood lymphocytes. However, the presence of circulating blasts was not essential to virus susceptibility, since CR/OT MNC, presumably free of leukemic blasts, were still more susceptible to HTLV-I than normal cells. Basal NK function of MNC from leukemic patients was significantly lower than that detectable in healthy controls. However, no correlation was found between susceptibility to HTLV-I infection and NK activity.
The syndrome of T8 hyperlymphocytosis: variation in phenotype and cytotoxic activities of granular cells and evaluation of their role in associated neutropenia
Authors: Grillot-Courvalin C , Vinci G , Tsapis A , Dokhelar MC , Vainchenker W , Brouet JC
Published: October 2019

We performed a longitudinal study of the phenotype and functions of granular lymphoid cells from seven patients with T8 hyperlymphocytosis and neutropenia. Whereas cells retained a T3+ T8+ (six cases) or T3- T8+ (one case) phenotype at different examinations, the expression of natural killer (NK)-related antigens (HNK1- and Leu11-defined antigens) exhibited striking variations, some of which were also observed after in vitro culture. Similarly, natural or antibody-mediated cytotoxic activities fluctuated in vivo and in vitro. Cells from the patient with T3- T8+ proliferation were able to inhibit directly the growth of early CFU-GM, CFU-E, and BFU-E and to a lesser extent of late CFU-GM, as shown by cultures of autologous blood or marrow progenitors after depletion (and subsequent addition) of granular cells. In the other six patients with T3+ T8+ cells, no such effect was found. However, after a 24-hour incubation of the progenitors with the granular cells, CFU-GM growth was clearly inhibited; this was not observed in all experiments, a finding which may be related to the spontaneous variations of cell killer functions. Finally, no correlation was noted between the clinical course or extent of lymphoid proliferation and cell function or phenotype or with the monoclonal (two cases), polyclonal (three cases) or germ-line (one case) patterns of T cell receptor beta gene configuration.
Mother-to-offspring transmission of human T cell leukemia virus type I in rabbits
Authors: Uemura Y , Kotani S , Yoshimoto S , Fujishita M , Yamashita M , Ohtsuki Y , Taguchi H , Miyoshi I
Published: October 2019

We studied the mode of natural transmission of human T-cell leukemia virus type I (HTLV-I) in rabbits. Four virus-infected rabbits (2 males and 2 females) were individually mated with 4 noninfected rabbits. Two virus-infected females mated with noninfected males gave birth to 7 offspring, and 2 noninfected females mated with infected males delivered 5 offspring. Four of the seven offspring born to the virus- infected mothers seroconverted for HTLV-I when aged 6 to 13 weeks with antibody titers of 1:40 to 1:160. None of the five offspring born to the noninfected mothers became seropositive during the observation period of 6 months, however. Peripheral lymphocytes were cultured with T cell growth factor, and HTLV-I-carrying lymphoid cell lines were established from the four seroconverted rabbits. All four cell lines were of T cells positive for Ia antigens. In addition, none of five newborn rabbits killed immediately after birth to a virus-infected rabbit was infected with HTLV-I. These findings provide an experimental support for the milkborne transmission of HTLV-I from mother to child in humans and indicate that the virus is tropic for T cells in rabbits as well.
Ki-M8 monoclonal antibody reactive with an intracytoplasmic antigen of monocyte/macrophage lineage
Authors: Radzun HJ , Kreipe H , Bodewadt S , Hansmann ML , Barth J , Parwaresch MR
Published: October 2019

A monoclonal antibody (MoAb), Ki-M8, that reacts specifically with cells of the monocyte/macrophage system is described. On light and electron microscopic immunohistochemistry, Ki-M8 recognizes intracytoplasmatically localized antigens of mol wt 30,000 and 32,000, increasingly expressed during differentiation of monocytes into macrophages. Ki-M8 antigen is detectable on almost all known tissue macrophages and monocyte/macrophage-related cell lines after appropriate stimulation. In functional terms Ki-M8 significantly impairs the generation of oxygen radicals during an induced respiratory burst. Applied to acute nonlymphoblastic leukemias, a clear-cut differentiation of the monocytic phenotype and differentiation is possible on the basis of Ki-M8 immunoreactivity. Ki-M8 represents a reagent specific for the monocyte/macrophage system with regard to antigen distribution in normal and neoplastic cells as well as with regard to its influence on a typical monocyte/macrophage-related function.
Fate of the DNA in plasmid-containing Escherichia coli minicells ingested by human neutrophils
Authors: Fox HB , De Togni P , McMahon G , Levy SB , Robinson JS , Karnovsky MJ , Babior BM
Published: October 2019

Escherichia coli minicells containing the plasmid pSC101 (approximately 10 kb) or pBR322 (approximately 4 kb) were opsonized and incubated with human neutrophils. The neutrophils responded to the minicells as they would to native E coli: they ingested the minicells, discharged their granule contents into the minicell-containing phagosomes, and expressed a respiratory burst. After one hour of incubation, the fate of the ingested plasmid DNA was examined. No DNA degradation was detected by trichloroacetic acid precipitation or agarose gel electrophoresis. Moreover, when pBR322 recovered from ingested minicells was transformed into E coli, no mutations in either of the antibiotic resistance genes carried by the plasmid were detected out of many thousand transformants screened. These findings confirm the surprisingly limited effect of neutrophils on ingested DNA.
Expression of the M-CSF (CSF-1) gene by human monocytes
Authors: Rambaldi A , Young DC , Griffin JD
Published: October 2019

Monocyte colony-stimulating factor (M-CSF, CSF-1) is a macrophage lineage-specific growth factor. Northern blot analysis using a human M- CSF cDNA probe and a specific bioassay for human M-CSF were used to investigate the cellular sources of M-CSF. Expression of the M-CSF gene was induced in blood mononuclear cells stimulated by phorbol myristate acetate (PMA) or gamma-interferon. When mononuclear cells were fractionated into highly purified populations of T cells and monocytes, M-CSF transcripts were detected predominantly in monocytes. Further, monocytes stimulated with PMA released an M-CSF-like activity. These results demonstrate that M-CSF can be produced by activated cells of the macrophage lineage.
Differences in nonspecific esterase from normal and leukemic monocytes
Authors: Cohn PD , Emanuel PD , Bozdech MJ
Published: October 2019

Nonspecific esterase (NSE) is used to identify normal and leukemic mononuclear phagocytes cytochemically. It can be demonstrated by the hydrolysis of alpha-naphthyl acetate or butyrate. NSE from leukapheresed leukemic monocytes were extracted with several types of detergent and grouped into two isoelectric point (pl) categories based on the ease of solubility: pl 5.7–6.3 (8 bands) greater than or equal to pl 6.6–7.6 (6 bands). Normal monocytes yielded only the pl 5.7–6.3 isozymes. Isozymes from both leukemic and normal monocytes were inhibited similarly by sodium fluoride, pH less than 4.7, and a serine active site inhibitor. All isozymes were bound by Sepharose- concanavalin A (Con-A) and displayed similar substrate preferences and pH v activity slopes. Under the conditions of detergent solubilization, the smallest mol wt species retaining enzymatic activity was 50 +/- 5 kd. Despite these similarities, the isozymes of pl 5.7 through 6.3 and pl greater than 6.3 exhibited different degrees or types of inhibition by phenylmethylsulfonyl fluoride (PMSF), resistance to heat and antigenic character. Thus, the esterase isozymes represent two families of glycoproteins, both of them probable cell surface enzymes, resembling classical liver carboxyl or B-esterases (EC 3.1.1 1).
Plasma cyclic nucleotide levels in acute leukemia patients
Authors: Peracchi M , Toschi V , Bamonti-Catena F , Lombardi L , Bareggi B , Cortelezzi A , Colombi M , Maiolo AT , Polli EE
Published: October 2019

To verify the clinical usefulness of extracellular cyclic nucleotide determination as a tumor marker, plasma cyclic AMP (cAMP) and cyclic GMP (cGMP) levels were measured in 70 normal subjects and 173 acute leukemia patients studied in different stages of their disease. Mean plasma cAMP levels were similar in leukemic and normal subjects, although in 48 patients in the active stage of the disease, first diagnosis, or relapse, the cAMP values were below the normal range, and most of these patients failed to respond to chemotherapy. Plasma cGMP levels were markedly elevated in untreated patients, normalized in all patients who attained complete remission, and increased promptly to pretreatment values in patients who relapsed, suggesting that their determination may be useful to monitor the patients' response to treatment.
Hematologic effects of immunotherapy with lymphokine-activated killer cells and recombinant interleukin-2 in cancer patients
Authors: Ettinghausen SE , Moore JG , White DE , Platanias L , Young NS , Rosenberg SA
Published: October 2019

Immunotherapy with interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells generated from autologous lymphocytes has produced significant tumor regressions in patients with advanced cancer. In the current study, we reviewed the hematologic effects associated with this therapy in our initial 42 patients. Eighty-eight percent of the treated patients developed anemia that required greater than or equal to 4 units of red cell transfusions, and 43% received at least 8 units. Only a blood loss of 2 to 3 units could be attributed to repeated phlebotomy, cytophereses, and hemodilution. IL-2 administration also resulted in thrombocytopenia as well as lymphopenia and eosinophilia. Forty-three percent of patients developed platelet counts of less than or equal to 50,000/microL, and 36% of the total group required platelet transfusions. Mild neutropenia and a rebound lymphocytosis followed discontinuation of IL-2 treatment. To explore the possible mechanisms for these hematologic effects, standard hematopoietic colony assays were conducted on serial blood samples from five patients. IL-2 produced a significant decline in circulating erythroid (BFU-E) and granulocytic/macrophage (CFU-C) progenitors, which rebounded after the discontinuation of IL-2 therapy. Infusion of IL-2 also resulted in measurable serum levels of gamma-interferon. Some of the hematologic effects of immunotherapy with LAK cells and IL-2 may be the result of IL-2-mediated suppression of hematopoiesis.
The effects of GM-CSF and G-CSF in promoting growth of clonogenic cells in acute myeloblastic leukemia
Authors: Vellenga E , Young DC , Wagner K , Wiper D , Ostapovicz D , Griffin JD
Published: October 2019

A small subset of leukemic cells from most patients with acute myeloblastic leukemia (AML) have properties of stem cells and can be assayed by colony formation in agar or methylcellulose. Colony formation generally requires the addition of exogenous growth factors, but the exact factors required are incompletely defined. The AML colony- promoting activities of two recombinant human colony-stimulating factors (GM-CSF and G-CSF) were investigated by using blasts from 48 patients with AML. In nine cases, no colonies formed with either CSF. In seven cases colonies formed only in response to G-CSF and in 11 cases only in response to GM-CSF. In 21 cases colonies formed in response to either GM-CSF or G-CSF, and in 12 of these cases there was an additive effect between the two CSFs in determining maximum colony size. For cases responding to both GM- and G-CSF, the total number of colonies formed in response to the combination of both CSFs was almost always less than additive compared with the number of colonies formed in response to the individual CSFs. Further, the AML-CFU responding to either GM-CSF or G-CSF could not be distinguished by surface markers or by the cytochemical staining pattern of the colonies. These results suggest that there is considerable overlap between the GM-CSF- and G- CSF-responsive AML-CFU subpopulations in most cases. For five of seven cases, the combination of GM-CSF and G-CSF could replace a leukocyte feeder layer in providing maximum growth stimulation. These results indicate that GM-CSF and G-CSF are active growth factors for AML cells and are frequently additive in promoting maximum colony size.
Low molecular weight B cell growth factor induces proliferation of human B cell precursor acute lymphoblastic leukemias
Authors: Wormann B , Mehta SR , Maizel AL , LeBien TW
Published: October 2019

Experiments were conducted to determine the effect of low mol wt B cell growth factor (L-BCGF) on B cell precursor acute lymphoblastic leukemia (ALL). L-BCGF induced a significant increase in 3H-TdR incorporation in 28 of 37 bone marrow aspirates from patients with B cell precursor ALL, with stimulation indices ranging from 2 to 129. Fluorescence-activated cell sorting confirmed that in five of seven patients the common acute lymphoblastic leukemia antigen (CALLA)/CD10 positive leukemic cells were responding directly to L-BCGF. L-BCGF was capable of inducing, in some patients, an increase in absolute viable cells and could also induce colony formation in vitro. The response of B cell precursor ALL was not attributable to beta IL 1, IL 2, or gamma interferon. These results indicate that the majority of B cell precursor ALL undergo a proliferative response to L-BCGF, suggesting a regulatory role for this lymphokine in the growth of B cell precursors.
Development of the human coagulation system in the full-term infant
Authors: Andrew M , Paes B , Milner R , Johnston M , Mitchell L , Tollefsen DM , Powers P
Published: October 2019

The investigation of many hemostatic defects in the newborn is limited by the lack of normal reference values. This study was designed to determine the postnatal development of the human coagulation system in the healthy full-term infant. Consecutive mothers of healthy full-term infants born at St JosePh′s Hospital in the city of Hamilton were approached for consent. One hundred eighteen full-term infants (37 to 42 week's gestational age) were entered into the study. Demographic information and a 2-mL blood sample were obtained in the postnatal period on days 1, 5, 30, 90, and 180. Between 40 and 79 full-term infants were studied on each day for each of the coagulation tests. Plasma was fractionated and stored at -70 degrees C for batch assaying of the following tests: prothrombin time, activated partial thromboplastin time, thrombin clotting time, and factor assays (biologic): fibrinogen, II, V, VII, VIII, IX, X, XI, XII, and high- molecular weight kininogen. Factor XIII subunits A and S, von Willebrand factor, and the inhibitors antithrombin III, alpha 2- antiplasmin, alpha 2-macroglobulin, alpha 1-antitrypsin, C1 esterase inhibitor, protein C, and protein S were measured immunologically. Plasminogen, prekallikrein, and heparin cofactor II were measured by using chromogenic substrates. The large number of infants studied at each time point allowed us to determine the following: the range of normal for each test at five time points in the postnatal period; that coagulation tests vary with the postnatal age of the infant; that different coagulation factors show different postnatal patterns of maturation; and that near-adult values are achieved for most components by 6 months of life. In summary, this large cohort of infants studied consecutively in the postnatal period allowed us to determine the normal development of the human coagulation system in the full-term infant.
Growth factor requirements of childhood acute leukemia: establishment of GM-CSF-dependent cell lines
Authors: Lange B , Valtieri M , Santoli D , Caracciolo D , Mavilio F , Gemperlein I , Griffin C , Emanuel B , Finan J , Nowell P
Published: October 2019

Eight permanent cell lines were established from cells of 50 consecutive patients with childhood acute leukemia. Three cell lines required growth factor-containing conditioned media. Analysis using blocking antisera and recombinant granulocytic macrophage (GM) colony- stimulating factor (CSF) identified GM-CSF as a growth factor required to establish the latter three cell lines and necessary for their continuous proliferation in chemically defined medium. Two of the GM- CSF-dependent cell lines were derived from patients with undifferentiated T- and a biphenotypic B-myelomonocytic leukemia, which suggests that GM-CSF might maintain proliferation of leukemias originating from immature progenitor cells. Cytogenetic analysis indicated that all established leukemic cell lines were aneuploid, with six lines containing chromosomal alterations related to those observed in the leukemic cells of the patient. Two patients did not have an abnormal clone identified in the marrow but did yield an aneuploid cell line. These studies indicate that GM-CSF-dependent leukemic cell lines can be established in a fraction of childhood leukemia. These cell lines lend themselves to studies aimed at the evaluation in vitro of the role of growth factors in controlling proliferation and differentiation of leukemic cells.
Fibrinogen-independent aggregation and deaggregation of human platelets: studies in two afibrinogenemic patients
Authors: Cattaneo M , Kinlough-Rathbone RL , Lecchi A , Bevilacqua C , Packham MA , Mustard JF
Published: October 2019

Platelets from two afibrinogenemic patients were used to determine whether fibrinogen is essential for platelet aggregation and to examine whether released fibrinogen contributes to the stabilization of platelet aggregates when platelets have been induced to aggregate and release their granule contents by stimulation with thrombin. The addition of adenosine diphosphate (ADP) to platelet-rich plasma (PRP) or to suspensions of washed platelets from the afibrinogenemic patients caused the formation of small aggregates, which was either not inhibited or only slightly inhibited by the F(ab')2 fragments of an antibody to fibrinogen but was inhibited by an antibody (10E5) to glycoprotein IIb/IIIa. Thus there is a component of ADP-induced platelet aggregation that is not dependent on fibrinogen or other plasma proteins but is dependent on glycoprotein IIb/IIIa. There was little difference in the extent of aggregation and the release of granule contents of normal and afibrinogenemic platelets in response to the release-inducing agents collagen, platelet-activating factor (PAF), sodium arachidonate, or thrombin. With normal or afibrinogenemic platelets, aggregation by thrombin (0.2 U/mL or higher) was not inhibited by the F(ab')2 fragments of an antibody to human fibrinogen. Deaggregation by combinations of inhibitors of platelets aggregated by 1 U/mL thrombin showed no difference between platelets from afibrinogenemic and control subjects, indicating that released fibrinogen does not make a major contribution to the stabilization of platelet aggregates formed by thrombin stimulation.
An evaluation of crossmatching, HLA, and ABO matching for platelet transfusions to refractory patients
Authors: Heal JM , Blumberg N , Masel D
Published: October 2019

Refractoriness occurs in many patients receiving multiple platelet transfusions. We used a sensitive ELISA assay to assess the utility of crossmatching HLA-A,B matched single donor platelets in 51 consecutive, typical refractory patients. Of the 222 transfusions evaluated at 1 to 4 hours posttransfusion, only 17 of 54 (31%) with positive crossmatches had corrected platelet count increments of greater than or equal to 7,500/microL. In contrast, 95 of 168 (57%) of those with negative crossmatches had such increments (P less than .001). Regardless of the results of the crossmatch, HLA-A,B, and ABO matching had independent influences on transfusion outcome. The median corrected 1- to 4-hour increment for crossmatch negative transfusions was 13,300/microL for A/BU grade matches, 9,700 for BX, and 7,800 for C. Increments were 10,000/microL for ABO-identical transfusions and 5,900 for transfusions of platelets ABO incompatible with the recipient's plasma antibodies. When the donor platelets were ABO compatible, but the donor plasma contained ABO antibodies to the recipient's platelets, the increment was intermediate (8,200/microL). The most important factor in predicting platelet survival was the crossmatch, followed by HLA-A,B and ABO, each having independent predictive value. These data demonstrate that the predictive value of a negative crossmatch may be considerably less than that reported in previous studies with stable, less ill patients. In typical refractory patients, there appear to be mechanisms of platelet destruction that are related to HLA-A,B and ABO but are not detected with current crossmatch methods. We hypothesize that soluble plasma HLA-A,B and ABO antigens contribute to the destruction of donor and sometimes recipient platelets by an immune complex or other “innocent bystander” mechanism. With our crossmatching technique, HLA-A,B and ABO match grades remain relevant to platelet transfusion therapy in some refractory patients.
Immunologic aberrations, HIV seropositivity and seroconversion rates in patients with hemophilia B
Authors: Brettler DB , Brewster F , Levine PH , Forsberg A , Baker S , Sullivan JL
Published: October 2019

Because there have been reports that factor IX concentrate is less immunosuppressive and therefore factor IX users have less immunologic aberrations, we have studied a group of 22 patients with hemophilia B and six patients with factor VIII deficiency and high titer inhibitors with respect to lymphocyte numbers and function, human immunodeficiency virus (HIV) serology, and factor usage. This group was compared to 111 patients with hemophilia A and a group of 28 healthy male volunteer controls. When the study began in 1983, the majority of patients with hemophilia B and with higher titer factor VIII inhibitors were seronegative, 77% and 83% respectively, as compared to only 30% of patients with hemophilia A. At that time the factor IX users also had milder immune aberrations than the hemophilia A group. However, with time and increasing clotting factor concentrate usage, seroconversion and more striking abnormalities in immune function have occurred in the hemophilia B group. In a subgroup of 16 patients with hemophilia B studied twice, the incidence of seropositivity increased from 31% in 1983 to 69% in 1985. We thus conclude that factor IX concentrate in itself is not less immunosuppressive than factor VIII concentrate. Seroconversion in factor IX concentrate users appears to be lagging behind seroconversion in factor VIII concentrate users, perhaps secondary to the lower cumulative dosage of concentrate that patients with hemophilia B utilize.
Plasma and platelet HLA in normal individuals: quantitation by competitive enzyme-linked immunoassay
Authors: Kao KJ
Published: October 2019

Recent studies on platelet HLA indicate that greater than 50% of platelet HLA antigens are adsorbed on the platelet surface and may be derived from plasma. It has been speculated that platelet HLA may be directly proportional to plasma HLA concentration. To determine the quantitative correlation between plasma and platelet HLA, a precise competitive enzyme-linked immunoassay (ELISA) for measurements of soluble and cellular HLA antigens was developed by using purified HLA antigens and W6/32 anti-HLA monoclonal antibody. The useful range of the standard curve for the assay was 0.01 to 5.0 micrograms/mL. The intraassay and interassay variations were 7% and 14%, respectively. The plasma HLA concentrations measured in 61 healthy adults ranged from 0.25 to 4.1 micrograms/mL, and the mean plasma HLA concentration was 1.47 +/- 0.87 micrograms/mL (+/- SD). Platelet HLA concentrations determined in the same 61 persons ranged from 4.7 to 17.33 fg/platelet, and the mean concentration was 9.3 +/- 2.9 fg/platelet (+/- SD). Chloroquine-elutable platelet HLA concentrations were also determined in 42 of the 61 persons, with the mean value of 5.7 +/- 2.1 fg/platelet (+/- SD). The plasma HLA concentration of each individual was then correlated with the same person's total or chloroquine-elutable platelet HLA concentration. Linear regression analyses of the results revealed no significant correlation between platelet and plasma HLA concentrations. Thus, it is unlikely that chloroquine-elutable HLAs are derived from plasma. The developed solid-phase assay for HLA will be useful for further study of the quantitative significance of plasma HLA antigens in allosensitization of transfused individuals.
Equal stabilities of normal beta globin and nontranslatable beta0 -39 thalassemic transcripts in cell-free extracts
Authors: Stolle CA , Payne MS , Benz EJ Jr
Published: October 2019

Patients with beta zero thalassemia arising from premature terminator codon mutations in the gene for beta globin do not produce beta globin protein; these individuals also exhibit a decreased amount of beta globin mRNA in their erythroid cells. The absence of beta globin protein is readily explained by the inability of the beta zero-39 mRNA to be translated. The decrease in beta globin mRNA has been attributed to either decreased cytoplasmic stability of the nontranslatable decreased cytoplasmic stability of the nontranslatable mRNA or to an undefined nuclear lesion. To compare directly the relative stabilities of normal and beta zero-39 thalassemic globin transcripts, we prepared normal and thalassemic beta globin pre-mRNAs and mRNAs using cloned DNA templates and the SP6 promoter-polymerase system. The stability of the transcripts was assessed by incubation in various cell-free extracts. Our results indicate that although the stabilities of the beta globin transcripts varied considerably from one extract to another the stabilities of the beta zero-39 thalassemic pre-mRNAs and mRNAs were equal to those of normal beta globin mRNAs in every extract tested.
Detection of activated platelets in whole blood using activation- dependent monoclonal antibodies and flow cytometry
Authors: Shattil SJ , Cunningham M , Hoxie JA
Published: October 2019

Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We developed a direct test for activated platelets in whole blood using flow cytometry. Whole blood was incubated with either biotin-PAC1, a monoclonal antibody specific for the fibrinogen receptor on activated platelets, or biotin-S12, an antibody specific for an alpha-granule membrane protein that associates with the platelet surface during secretion. Platelet-bound antibodies were detected with streptavidin conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Platelets were differentiated from the larger erythrocytes and WBCs by their light- scatter profile. Alternatively, platelets could be identified with FITC- AP1, an antibody specific for platelet membrane glycoprotein Ib, and analyzed further for PAC1 or S12 binding with PE-streptavidin. No centrifugation or washing steps were required. With gel-filtered platelets, there was a direct correlation between ADP-induced biotin- PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = .99; P less than .001). Furthermore, as few as 0.8% activated platelets could be detected by flow cytometry when activated platelets were mixed with unstimulated platelets. In whole blood, unstimulated platelets demonstrated no PAC1- or S12-specific fluorescence, indicating that they did not bind these antibodies. On stimulation with agonists, however, the platelets demonstrated a dose- dependent increase in fluorescence similar to that observed for platelets in plasma or buffer. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate (TPA) that evokes full platelet aggregation and secretion induced maximal PAC1 and S12 binding. Activated platelets could also be analyzed in whole blood samples that had been fixed with paraformaldehyde. These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This technique may be useful to assess the degree of platelet activation and the efficacy of antiplatelet therapy in clinical disorders.
Anemia of the Belgrade rat: evidence for defective membrane transport of iron
Authors: Bowen BJ , Morgan EH
Published: October 2019

The mechanisms underlying the impaired utilization of transferrin-bound iron by erythroid cells in the anemia of the Belgrade laboratory rat were investigated using reticulocytes from homozygous anemic animals and transferrin labeled with 59Fe and 125I. The results were compared with those obtained using reticulocytes from phenylhydrazine-treated rats and iron-deficient rats. Each step in the iron uptake mechanism was investigated, ie, transferrin-receptor interaction, transferrin endocytosis, iron release from transferrin, and transferrin exocytosis. Although there were quantitative differences, no fundamental difference was found in any of the abovementioned aspects of cellular function when the reticulocytes from Belgrade rats were compared with those from iron-deficient animals. The basic defect in the Belgrade reticulocytes must therefore reside in subsequent steps in iron uptake, after it is released from transferrin within endocytotic vesicles, ie, in the mechanism by which it is transferred across the lining membrane of the vesicles into the cell cytosol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of reticulocyte ghosts extracts demonstrated a prominent protein band of mol wt 69,000 that was absent or present only in low concentration extracts from the other two types of reticulocytes. This may be a result of the genetic defect.
Immunological specificity and mechanism of action of IgG lupus anticoagulants
Authors: Pengo V , Thiagarajan P , Shapiro SS , Heine MJ
Published: October 2019

Although observations have implied that lupus anticoagulants have immunologic specificity toward anionic phospholipids, this assumption has been directly demonstrated in only one patient with a monoclonal IgM paraprotein. We tested the generality of this hypothesis directly by isolating five IgG lupus anticoagulants from patients with lupuslike syndromes and/or thrombosis. IgG lupus anticoagulant fractions were isolated free of other plasma proteins and free of contaminating phospholipid by adsorption to and elution from cardiolipin-cholesterol- dicetyl phosphate liposomes, followed by chromatography on protein A- Sepharose. Cardiolipin liposomes, but not phosphatidylcholine liposomes, were capable of removing all, or nearly all, lupus anticoagulant activity from patient plasma. The affinity-purified IgG preparations reacted with cardiolipin, phosphatidylserine, phosphatidylinositol, and phosphatidic acid, but not with phosphatidylcholine or phosphatidylethanolamine, and inhibited calcium- dependent binding of prothrombin and of factor X to phosphatidylserine- coated and to cardiolipin-coated surfaces. F(ab')2 fragments retained lupus anticoagulant activity and bound to cardiolipin in an enzyme- linked immunosorbent assay (ELISA). Anticardiolipin and lupus anticoagulant activity were both present in acidic fractions on isoelectric focusing. These data strongly suggest that most, if not all, lupus anticoagulants are antibodies that have immunologic specificity towards anionic phospholipids, thereby blocking the calcium- mediated binding of vitamin K-dependent coagulation factors to coagulation-active phospholipid surfaces.
Immunophenotyping of acute myeloid leukemia using monoclonal antibodies and the alkaline phosphatase-antialkaline phosphatase technique
Authors: Hanson CA , Gajl-Peczalska KJ , Parkin JL , Brunning RD
Published: October 2019

The leukemic cells from 41 cases of acute myeloid leukemia (AML) and 17 cases of acute lymphocytic leukemia (ALL) were immunophenotyped by the alkaline phosphatase-antialkaline phosphatase (APAAP) immunocytochemical technique utilizing eight monoclonal antibodies (MoAb) reactive with cells of myeloid origin and seven MoAb reactive with lymphoid antigens. Ninety percent of the cases of AML reacted with one or more of the pan-myeloid MoAb, My7, My9, or 20.3. Reactivity of the myeloid panel of MoAb showed some correlation with the French- American-British (FAB) classification of AML. Five of six cases of acute promyelocytic leukemia (APL) were HLA-DR negative; the one HLA-DR- positive APL had a minor population of HLA-DR-negative promyelocytes. OKM5 and/or My4 reacted with 16 of 16 monocytic leukemias. No specific marker of early erythroid development was identified. AP3, a MoAb reactive with platelet glycoprotein (GPIIIa), was specific for acute megakaryoblastic leukemia. Immunocytochemistry was also helpful in classifying seven cases of AML with equivocal or negative routine cytochemistry. Two cases of AML had minor populations of blasts detected by the APAAP technique that were immunologically distinct from the major blast population; these minor populations emerged as the predominant cell type at relapse. Two cases of ALL expressed multiple myeloid and lymphoid antigens. Two other cases that morphologically were ALL reacted with only myeloid MoAb; one consisted entirely of immature basophils on ultrastructural examination. Immunophenotyping results using the APAAP technique were comparable with those obtained with flow cytometry. The APAAP technique is a reliable method for immunophenotyping leukemia that complements other methods of immunologic evaluation. The primary advantages of this method include its use with routinely prepared blood and bone marrow smears and the ability to correlate immunocytochemical reactions with morphology.
Erythropoietin production by the fetal liver in an adult environment
Authors: Flake AW , Harrison MR , Adzick NS , Zanjani ED
Published: October 2019

To gain insight into the mammalian liver to kidney erythropoietin (Ep) switch, we heterotopically transplanted livers from preswitch, switched, and postswitch fetal and newborn lambs into normal adult sheep. Recipients' serum Ep and circulating reticulocyte levels were serially determined until rejection of the graft and compared with identical samples from sham-operated control adult ewes. Transplantation of preswitch and switched fetal livers caused an impressive rise in recipients' serum Ep activity and provoked a corresponding increase in reticulocytosis. In contrast, Ep activity and reticulocyte counts did not change from preoperative levels in adult ewes transplanted with postswitch livers or in the sham-operated controls. The production of Ep by the preswitch fetal liver in the adult environment was not dependent on the presence or absence of host kidneys and was stimulated by anemic hypoxia. These results suggest that the fetal liver is capable of producing relatively large amounts of Ep activity, and the production of Ep can be maintained in the adult environment in the presence of functional adult kidneys. This argues against suppression of liver Ep production by renal Ep, or some other factor in the postnatal environment, and suggests that the liver to kidney switch of Ep production during ontogeny may represent a genetically determined event.