bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium-dependent cysteine protease activity in the sera of patients with thrombotic thrombocytopenic purpura
Authors: Murphy WG , Moore JC , Kelton JG
Published: October 2019

Plasma and serum from patients with thrombotic thrombocytopenic purpura (TTP) can cause activation and aggregation of normal human platelets in vitro. It is possible that this platelet-activating factor contributes to the disease. In this report we describe studies designed to identify the platelet-activating factor in TTP. Platelet activation by sera from 15 patients with TTP was inhibited by leupeptin, iodoacetamide, and antipain but not by phenylmethylsulphonylfluoride, epsilon-aminocaproic acid, soybean trypsin inhibitor, aprotinin, and D-phenylanyl-1-prolyl-1- arginine chloromethyl ketone. These studies suggested that the platelet- activating factor in TTP serum was a cysteine protease. We confirmed that a calcium-dependent cysteine protease (CDP) was present in the sera of each of the 15 patients when we used an assay based on the ability of CDP to proteolyse platelet membrane glycoprotein 1b (GP1b) and hence to abolish the ability of CDP-treated normal platelets to agglutinate in the presence of ristocetin and von Willebrand factor. This proteolytic activity was inhibited by EDTA, leupeptin, antipain, iodoacetamide, and by N-ethyl-maleamide (NEM) but not by the serine protease inhibitors. Activity was detected in 15 of 15 patients with TTP tested before therapy was begun. In contrast, no activity was detected in the serum of any of five of the TTP patients tested in remission or in any of the sera from 36 patients with thrombocytopenia and 423 nonthrombocytopenic controls. To look for in vivo CDP activity in patients with TTP, we studied platelets from two patients with acute TTP (drawn into acid-citrate-dextrose, NEM, and leupeptin). These platelets showed a loss of GP1b from the platelet surface. Both patients were also studied in remission: GP1b on the platelet surface had returned to normal. These studies provide evidence that CDP is present in the sera of patients with TTP, that it is specific to this disease, and that is is active in vivo as well as in vitro. We postulate that a disorder of CDP homeostasis plays a major role in the pathophysiology of TTP.
Molecular analysis of Burkitt's leukemia in two hemophilic brothers with AIDS
Authors: Rechavi G , Ben-Bassat I , Berkowicz M , Martinowitz U , Brok-Simoni F , Neumann Y , Vansover A , Gotlieb-Stematsky T , Ramot B
Published: October 2019

In two hemophilic brothers infected by the human immunodeficiency virus (HIV), Burkitt's leukemia developed within 1 year. Both patients were treated by aggressive chemotherapy, and both are still in complete remission for 23 and 14 months, respectively. Sera from both brothers contained anti-HIV antibodies. However, DNA extracted from the tumor cells, when analyzed by Southern blot using a cloned HIV probe, did not reveal HIV-related sequences. Hybridization experiments with an Epstein- Barr virus (EBV) probe revealed the presence of EBV-specific sequences in the tumors' DNA. In both patients' tumors rearranged c-myc genes were found. The rearrangements occurred in both genes 3′ to the third exon of c-myc, thereby suggesting that a variant chromosomal translocation took place in both cases. Indeed, karyotype analysis of the malignant cells of one of the patients revealed the variant t(2:8) translocation. In contrast to the majority of Burkitt's tumors carrying this translocation, which are kappa light-chain producers, cells of our patient expressed lambda chains. Furthermore, in both cases the lymphoblasts carried IgG on the surface, again an unusual finding in Burkitt's tumors. Finally, because both patients had an identical HLA phenotype, the role of genetic factors in the development of such tumors should be considered.
Monoclonal antibody--specific immobilization of platelet antigens (MAIPA): a new tool for the identification of platelet-reactive antibodies
Authors: Kiefel V , Santoso S , Weisheit M , Mueller-Eckhardt C
Published: October 2019

The analysis of sera containing different platelet-reactive antibodies, eg, autoantibodies, platelet-specific alloantibodies like anti-PIA1, - PIA2, -Baka, and HLA antibodies, is still difficult. Recently, monoclonal antibodies against major platelet membrane constituents (glycoproteins IIb/IIIa and Ib and HLA class I molecule) have become available. In this report we describe a new assay that takes advantage of these highly specific reagents to investigate selectively platelet reactive antibodies against epitopes on different glycoproteins. The reliability and specificity of this assay is demonstrated with known platelet-reactive autoantibodies and alloantibodies (anti-PIA1, -Baka, - Pen). The discovery of a PIA2 antibody in a serum of a polytransfused patient underscores the efficiency of this technique. Possible applications of this assay are discussed in detail.
Human granulocyte-macrophage progenitors and their sensitivity to cytotoxins: analysis by limiting dilution
Authors: Barber KE , Crosier PS , Gillis S , Watson JD
Published: October 2019

Limiting dilution analysis of granulocyte-macrophage progenitor cells was performed by using adherent and T cell-depleted normal human bone marrow and the recombinant human growth factors, granulocyte colony- stimulating factor (G-CSF) and granulocyte-macrophage colony- stimulating factor (GM-CSF). Estimated frequencies for progenitor cells responding to G-CSF were one in 489 for colonies scored at day 7, and one in 1,015 for day 14 colonies. For GM-CSF the frequencies were one in 1,407 (day 7) and one in 574 (day 14). The effects of tumor necrosis factor (TNF) and lymphotoxin (LT) on the frequency of progenitors responding to either G-CSF or GM-CSF was determined. Both TNF and LT inhibited the response of cells to G-CSF, and in these cultures the frequency of progenitor cells that responded to G-CSF was reduced to less than one in 100,000 cells. In contrast, the frequency of cells able to form colonies in cultures stimulated with GM-CSF was unaltered by either cytotoxin. This differential sensitivity to cytotoxins suggests that either G-CSF and GM-CSF are acting on separate granulocyte progenitor populations or that TNF and LT selectively influence the biochemical pathways associated with the activation of receptors for G-CSF.
Hematopoetic precursors respond to a unique B lymphocyte-derived factor in vivo
Authors: Niskanen E , Gorman J , Isakson PC
Published: October 2019

In this study we detected a factor that stimulates the proliferation of bone marrow-derived hematopoietic precursors in diffusion chambers implanted in mice. This factor, called diffusible colony-stimulating factor (D-CSF), was found in medium conditioned in the presence of spleen and peripheral blood cells from mice with B cell leukemia (BCL1). After the administration of D-CSF, the number of colonies formed in the plasma clot inside the chamber (CFU-DG) was increased, as were the number of hematopoietic precursors (CFU-MIX, CFU-S, CFU-C, and BFU-E) as judged by a subculture of diffusion chamber contents. Depletion of macrophages and T cells from the spleen cell suspension did not decrease the production of D-CSF, thereby indicating that it was derived from B cells. Neoplastic BCL1 cells appear to be the source because D-CSF could not be detected in medium conditioned with normal B cells. BCL1-conditioned medium (CM) did not enhance CFU-MIX, BFU-E, and CFU-C colony formation in vitro, which suggested that D-CSF is different from multi-CSF, EPA, or CSF. The addition of BCL1 CM to multi- CSF-, erythroid potentiating activity (EPA), and CSF (EL-4CM)- containing cultures had no effect on CFU-MIX, BFU-E, and CFU-C colony formation, thus indicating the absence of a synergistic or inhibitory activity. On the other hand, EL-4 CM, which stimulates CFU-MIX, BFU-E, and CFU-C in vitro, had no effect on CFU-DG in vivo. Biochemical characterization of BCL1 CM revealed that D-CSF is relatively heat stable and loses its bioactivity with protease treatments. It binds to lentil-lectin, according to gel-filtration chromatography has a relative molecular weight of approximately 43,000, and on reverse-phase high-performance liquid chromatography elutes with acetonitrile. These data also indicate that transformed B cells may serve as a source for hematopoietic regulators that act on hematopoietic precursors in vivo.
Kinetic evaluation of the pool sizes and proliferative response of neutrophils in bacterially challenged aging mice
Authors: Rothstein G , Christensen RD , Nielsen BR
Published: October 2019

Clinical observations during infection suggest that in aged patients, the kinetic or proliferative responses of neutrophils to infection may be deranged. To test this hypothesis, the neutrophil responses of 6- month-old and 30-month-old mice were compared. After intrapulmonary injection of Escherichia coli, young mice exhibited neutrophilia and diminution of the neutrophil storage pool (NSP) by a mean of 6.4 x 10(6) neutrophils/two femurs. This was accompanied by an increase in the pool of CFU-GM from a control value of 1.1 x 10(5) cells/two femurs (range 0.7 to 1.4) to 1.5 x 10(5) (1.1 to 1.9) (P less than .05) and the thymidine suicide (relative proliferative rate) of CFU-GM rose from 27% (19 to 42) to 51% (31 to 61) (P less than .05). Furthermore, the CFU-GM of infected young mice displayed enhanced differentiation to the neutrophil series. In contrast, old mice exhibited a greater mean diminution of the NSP: 12.8 x 10(6) neutrophils. Also, old mice experienced a reduction in CFU-GM from 2.3 x 10(5) (1.0 to 3.9) (controls) to 1.3 x 10(5) (1.2 to 1.5)/two femurs (P less than .05), a reduction in the proliferation of CFU-GM and reduced differentiation of CFU-GM to neutrophils. These experiments establish that the neutrophil response of infected old mice is disordered, with exaggerated depletion of the NSP and lack of stimulus-driven granulocytopoiesis as reflected by a paradoxical reduction in the number and proliferative rate of precursors. This defect may be compounded by decreased differentiation of precursors to neutrophils.
Experimental transmission and pathogenesis of immunodeficiency syndrome in cats
Authors: Hoover EA , Mullins JI , Quackenbush SL , Gasper PW
Published: October 2019

We describe the identification, experimental transmission, and pathogenesis of a naturally occurring powerfully immunosuppressive isolate of feline leukemia virus (designated here as FeLV-FAIDS) which induces fatal acquired immunodeficiency syndrome (AIDS) in 100% (25 of 25) of persistently viremic experimentally infected specific pathogen- free (SPF) cats after predictable survival periods ranging from less than 3 months (acute immunodeficiency syndrome) to greater than one year (chronic immunodeficiency syndrome), depending on the age of the cat at time of virus exposure. The pathogenesis of FeLV-FAIDS-induced feline immunodeficiency disease is characterized by: a prodromal period of largely asymptomatic viremia; progressive weight loss, lymphoid hyperplasia associated with viral replication in lymphoid follicles, lymphoid depletion associated with extinction of viral replication in lymphoid follicles, intractable diarrhea associated with necrosis of intestinal crypt epithelium, lymphopenia, suppressed lymphocyte blastogenesis, impaired cutaneous allograft rejection, hypogammaglobulinemia, and opportunistic infections such as bacterial respiratory disease and necrotizing stomatitis. The clinical onset of immunodeficiency syndrome correlates with the replication of a specific FeLV-FAIDS viral variant, detected principally as unintegrated viral DNA, in bone marrow, lymphoid tissues, and intestine. Two of seven cats with chronic immunodeficiency disease that survived greater than 1 year after inoculation developed lymphoma affecting the marrow, intestine, spleen, and mesenteric nodes. Experimentally induced feline immunodeficiency syndrome, therefore, is a rapid and consistent in vivo model for prospective studies of the viral genetic determinants, pathogenesis, prevention, and therapy of retrovirus-induced immunodeficiency disease.
Interleukin 2-activated killer cells in patients following transplants of soybean lectin-separated and E rosette-depleted bone marrow
Authors: Keever CA , Welte K , Small T , Levick J , Sullivan M , Hauch M , Evans RL , O'Reilly RJ
Published: October 2019

During the early period following bone marrow transplantation before the immune system has reached full functional maturity, unprimed, nonspecific lytic systems may play a critical role as antiviral or antitumor effectors. The reconstitution of cells with this potential is of particular importance in recipients of bone marrow that has been depleted of mature T lymphocytes to prevent graft v host disease (GVHD). We examined the recovery of natural killer (NK) cells and interleukin 2 (IL 2)-augmented lymphokine-activated killer cells (LAK) in 48 patients at various intervals following transplantation of bone marrow depleted of mature cellular elements by treatment with soybean agglutinin and sheep RBCs (SBA-E- BMT). We found normal levels of both NK and LAK activity as early as 3 weeks following SBA-E- BMT. When compared with cells from controls, NK and LAK precursors from transplant recipients appeared to be activated in vivo in that freshly isolated peripheral blood mononuclear cells (PBMCs) from patients had an elevated cytolytic activity toward NK-insensitive targets and a more rapid response to activation by IL 2. In patients as well as controls, both LAK precursors and LAK effectors lacked antigens present on mature T lymphocytes (CD3, CD4, or CD8) but expressed antigens present on NK cells (CD2, CD16, and NKH1A). The LAK cells did not lyse either donor or host peripheral blood T cell targets. The activity of NK effectors but not LAK precursors survived the in vivo total body irradiation used for pretransplant conditioning in three patients studied. LAK precursors could be demonstrated as early as 18 days following transplant at a time when the bone marrow contained primarily donor- derived cells. Little or no LAK activity could be generated from cells of the SBA-E- BM graft itself, suggesting that LAK precursors differentiate rapidly from more primitive progenitors in the marrow graft. Thus, our data indicate that the NK and LAK lytic systems are among the earliest activities to recover during immune reconstitution following T cell-depleted BMTs.
Platelet-activating factor both stimulates and "primes" human polymorphonuclear leukocyte actin filament assembly
Authors: Shalit M , Dabiri GA , Southwick FS
Published: October 2019

The phospholipid inflammatory mediator, platelet-activating factor (PAF), can stimulate polymorphonuclear leukocyte (PMN) chemotaxis. Conversion of cytoplasmic actin from monomers to filaments is associated with PMN motile functions. Using the fluorescent actin filament stain nitrobenzodiaxole phallicidin, we have investigated PAF's effects on human PMN actin polymerization. Concentrations of PAF between 1 x 10(-11) to 1 x 10(-6) mol/L induced actin filament (F- actin) assembly. An optimal concentration of PAF (1–5 x 10(-8) mol/L) induced a significantly lower rise in relative F-actin content (1.72 +/- 0.07 SEM) than an optimal concentration (5 x 10(-7) mol/L) of the chemotactic peptide FMLP (2.21 +/- 0.06). Unlike FMLP (F-actin content: 1.25 +/- 0.04 at five seconds), PAF stimulation was associated with a delay of more than five seconds (1.04 +/- 0.01 at five seconds) before an increase in F-actin could be detected. F-actin concentration reached maximum levels by 30 to 60 seconds. Prolonged stimulation (20 minutes) with PAF was associated with two phases of polymerization and depolymerization. Like FMLP, the initiation of actin filament assembly by PAF required receptor occupancy, this reaction being totally blocked by the PAF receptor inhibitor, SKI 63–441. As evidenced by the lack of inhibition by nordihydroguaiaretic acid (5 to 20 mumol/L), the production of leukotriene B4 was not required for the PAF-induced changes in F-actin. Like FMLP, PAF's ability to stimulate PMN actin polymerization was inhibited by pertussis toxin (.05 to 2.5 micrograms/mL) but not impaired by the addition of EGTA and/or the calcium ionophore A23187. Preincubation with 1 x 10(-11) to 1 x 10(-8) mol/L PAF for 2 to 60 minutes enhanced the rise in F-actin content induced by low concentrations of FMLP (5 x 10(-12) to 1 x 10(-10) mol/L) indicating that this phospholipid was capable of “priming” the PMN actin polymerization response.
Rearrangement and expression of T cell antigen receptor genes in B cell chronic lymphocytic leukemia
Authors: Norton JD , Pattinson J , Hoffbrand AV , Jani H , Yaxley JC , Leber BF
Published: October 2019

Fifty-nine patients with B cell chronic lymphocytic leukemia (B-CLL) were screened for clonal rearrangement of T cell receptor (TCR) beta and gamma chain genes. Four were found with rearranged TCR beta genes, but none had detectable rearrangement of TCR gamma genes. One typical patient with B-CLL had a TCR beta gene structure consistent with a variable-diversity-joining rearrangement into the C beta 2 gene on one allele. An apparently identical rearrangement pattern was seen in a second patient, which suggested that there may be a restriction on the repertoire of possible TCR beta gene recombinations in mature B cells. Two further patients had a simple deletion of sequences, consistent with a diversity-joining rearrangement into C beta 2 on one allele. All four patients had rearrangements of immunoglobulin heavy- and light- chain genes typical of mature B cell malignancies. However, on review of clinical, morphological, and immunophenotype data, two had features consistent with B cell prolymphocytic leukemia or B lymphoma, and a third had progressed to a prolymphocytic transformation. Low-level expression of a predominantly 1.0- to 1.2-kilobase germ line TCR beta gene transcript was detected in several B-CLLs and at a comparable level in the four with rearranged TCR beta genes. This, together with the low frequency of TCR gene rearrangement, suggests that most B-CLL cases arise at a developmental stage when factors required for TCR gene activity are not operative.
Structure and expression of genes of GM-CSF and G-CSF in blast cells from patients with acute myeloblastic leukemia
Authors: Cheng GY , Kelleher CA , Miyauchi J , Wang C , Wong G , Clark SC , McCulloch EA , Minden MD
Published: October 2019

The hematopoietic growth factors granulocyte/macrophage colony- stimulating factor (GM-CSF) and G-CSF, available as recombinant products, stimulate the growth in culture of blasts from patients with acute myeloblastic leukemia (AML). We used cDNA probes for each gene to study the genomic organization in blast cells of 22 patients and expression in the blast cells of 18 patients. Alteration in the structure of G-CSF (two instances) and GM-CSF (two instances) was found. In two patients in whom it was possible to study DNA from bone marrow obtained at remission, the new bands detected in the leukemic cells were not found. Fifteen of 18 patients showed no RNA expression of either growth factor. Both patients with GM-CSF abnormalities as seen by Southern analysis expressed an abnormally large GM-CSF message but no G-CSF messages. One patient with an abnormal Southern pattern with G-CSF expressed normal-sized G-CSF and GM-CSF messages. The biologic significance of these findings remains to be determined. Nonetheless, the abnormal Southern patterns may prove to be useful clonal markers in the study of AML.
Pharmacokinetics and thrombolytic properties of deletion mutants of human tissue-type plasminogen activator in rabbits
Authors: Collen D , Stassen JM , Larsen G
Published: October 2019

The following mutants of human tissue-type plasminogen activator (t-PA) were constructed by deletion mutagenesis of t-PA cDNA, expressed in Chinese hamster ovary cells and purified to homogeneity: (a) t-PA-delta FE:t-PA lacking both the fibronectin fingerlike (F) domain and the epidermal growth factor (E) domain, (b) t-PA-delta FE1X:t-PA-delta FE with the glycosylated 117Asn mutagenized to Gln, and (c) t-PA-delta FE3X:t-PA-delta FE with the three known glycosylated Asn residues replaced by Gln. The mutant and natural t-PA (Mel-t-PA obtained from melanoma cell culture) were infused intravenously for four hours into rabbits with jugular vein thrombosis at doses ranging between 0.12 and 0.75 mg/kg. Fifty percent thrombolysis, determined by interpolation, was obtained with 0.4 mg/kg Mel-t-PA, 0.37 mg/kg t-PA-delta FE, 0.2 mg/kg t-PA-delta FE1X, and 0.40 mg/kg t-PA-delta FE3X. These infusion rates resulted in plateau levels of t-PA antigen in plasma of 0.055, 2.1, 0.6, and 0.5 micrograms/mL, respectively. At 50% lysis, the residual fibrinogen 30 minutes after the end of the infusion was 100%, 81%, 100% and 85% of baseline, and the residual alpha 2-antiplasmin was 82%, 55%, 85%, and 90%, respectively. These results indicate that t-PA- delta FE1X and t-PA-delta FE3X have a specific thrombolytic activity and fibrin specificity comparable to that of Mel-t-PA. t-PA-delta FE has a comparable specific thrombolytic activity but a lower fibrin specificity than Mel-t-PA. After the end of the infusion, t-PA-related antigen disappeared from plasma with an initial t1/2 of four minutes for Mel-t-PA, 25 minutes for t-PA-delta FE, 42 minutes for t-PA-delta FE1X, and 14 minutes for t-PA-delta FE3X. It is concluded that t-PA can be modified by deletion mutagenesis to yield variants with a markedly longer half-life in the blood. Some of these variants have a specific thrombolytic activity and fibrin specificity similar to that of natural t-PA. These variants may be useful to identify the structures in t-PA responsible for its clearance, specific thrombolytic activity, and fibrin specificity in vivo.
Mild and severe beta-thalassemia among homozygotes from Turkey: identification of the types by hybridization of amplified DNA with synthetic probes
Authors: Diaz-Chico JC , Yang KG , Stoming TA , Efremov DG , Kutlar A , Kutlar F , Aksoy M , Altay C , Gurgey A , Kilinc Y
Published: October 2019

Through the procedure of gene amplification combined with hybridization to synthetic 19 base pair (bp) oligonucleotide probes, it has been possible to identify nine different mutations in the DNA of 47 subjects from Turkey and Northern Cyprus with a beta-thalassemia homozygosity. The IVS-I nucleotide (nt) 110 G----A and the IVS-I nt 6 T----C substitutions and the frameshift at codon 8 were most frequently observed. Direct correlations were made between these data and clinical observations; mild disease was associated with homozygosity for IVS-I nt 6 T----C, for frameshift at codon 8, for the C----G substitution at nt -87, and for IVS-I nt 5 G----T, and for a double heterozygosity for some of these conditions. Moderate disease, observed in some of the patients, could be explained by combinations of specific mutations. All mutations were associated with specific haplotypes, while in some the observed beta zero-thalassemia was of the mild type due to a considerable production of Hb F.
Relationship between an activated N-ras oncogene and chromosomal abnormality during leukemic progression from myelodysplastic syndrome
Authors: Hirai H , Okada M , Mizoguchi H , Mano H , Kobayashi Y , Nishida J , Takaku F
Published: October 2019

The relationship between chromosomal abnormality and oncogene activation was investigated during leukemic progression in two patients with myelodysplastic syndrome (MDS). Both patients had partial or complete deletion of chromosome 5 in metaphase cells obtained throughout the progression to leukemia. Analysis with specific oligonucleotide probes revealed that bone marrow cells containing an activated N-ras oncogene proliferated in a dominant manner during the process of leukemic conversion in both patients. These observations suggest that the chromosomal abnormality may precede activation of the N-ras gene in these patients, and that both the chromosomal abnormality and the activated N-ras oncogene contribute to the development of leukemia.
Increases in neutrophil counts by purified human urinary colony- stimulating factor in chronic neutropenia of childhood
Authors: Komiyama A , Ishiguro A , Kubo T , Matsuoka T , Yasukohchi S , Yasui K , Yanagisawa M , Yamada S , Yamazaki M , Akabane T
Published: October 2019

Clinical effects of purified human urinary colony-stimulating factor (CSFHU) were studied in nine patients with chronic neutropenia of childhood who ranged in age from 1 to 18 years. The patients were given CSFHU at a dose of 6 x 10(6) U/m2/d for seven consecutive days, and their peripheral neutrophil counts were serially followed for 6 to 12 months. In addition, changes in bone marrow morphology and granulocyte- macrophage colony-forming cells (GM-CFUs) were studied. Transient increases in neutrophil counts occurred during the first 4 weeks in all patients except one. Repeated increases were seen in a cyclic fashion in five patients. Three of the patients recovered from the neutropenia after several cycles of fluctuation of neutrophil counts; the counts (per microliter) reached 5,880, 5,640, and 2,000 as compared with pretreatment levels (mean +/- 2 SD) of 80 +/- 164, 182 +/- 250, and 2 +/- 14, respectively. The percentage (mean +/- SE) of marrow neutrophilic cells increased from 30.4% +/- 7.0% to 34.9% +/- 6.9% (P less than .05) and the ratio of the later neutrophil precursors to the earlier ones from 1.02 +/- 0.27 to 1.42 +/- 0.36 (P less than .02) 2 weeks after CSFHU infusion. There were no changes in GM-CFU numbers. These results indicate that CSFHU can increase neutrophil counts by increasing the number and maturity of the marrow neutrophil precursors in some types of childhood chronic neutropenia.
Thrombospondin mediates the cytoadherence of Plasmodium falciparum- infected red cells to vascular endothelium in shear flow conditions
Authors: Rock EP , Roth EF Jr , Rojas-Corona RR , Sherwood JA , Nagel RL , Howard RJ , Kaul DK
Published: October 2019

Cerebral malaria is thought to involve specific attachment of Plasmodium falciparum-infected knobby red cells to venular endothelium. The nature of surface ligands on host endothelial cells that may mediate cytoadherence is poorly understood. We have investigated the effects of soluble thrombospondin, rabbit antiserum raised against thrombospondin, and human immune serum on cytoadherence of parasitized erythrocytes in ex vivo mesocecum vasculature. Preincubation of infected red cells with soluble thrombospondin or human immune serum inhibits binding of infected red cells to rat venular endothelium. Infusion of the microcirculatory preparation with rabbit antithrombospondin antibodies before perfusion of parasitized erythrocytes also resulted in decreased cytoadherence. In addition, incubation of infected cells with human immune sera obtained from malaria patients significantly inhibited the observed cytoadherence. Our results indicate that thrombospondin mediates binding of infected red cells to venular endothelium and may thus be involved in the pathogenesis of cerebral malaria.
The polyadenylation site mutation in the alpha-globin gene cluster
Authors: Thein SL , Wallace RB , Pressley L , Clegg JB , Weatherall DJ , Higgs DR
Published: October 2019

In a previous study, we described a form of nondeletion alpha- thalassemia (alpha T Saudi alpha) found in subjects of Saudi Arabian origin. In the current study, using synthetic oligoprobe hybridization and restriction enzyme analysis, we have demonstrated that the molecular basis of alpha T Saudi alpha is due solely to a single base mutation (AATAAA----AATAAG) in the polyadenylation signal of the alpha 2 gene and that the frameshift mutation in codon 14 of the linked alpha 1 gene is the result of a cloning artefact. The alpha 2 polyadenylation signal mutation occurs in other Middle Eastern and the Mediterranean populations and is responsible for the clinical phenotype of Hb H disease in some Saudi Arabian individuals with five alpha genes (alpha T Saudi alpha/(alpha alpha alpha)T Saudi). Evidence suggests that the (alpha alpha alpha)T Saudi haplotype has arisen as a result of a recombination between two misaligned chromosomes bearing the alpha T Saudi alpha defect.
The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action
Authors: Broze GJ Jr , Warren LA , Novotny WF , Higuchi DA , Girard JJ , Miletich JP
Published: October 2019

Blood coagulation is initiated when plasma factor VII(a) binds to its essential cofactor tissue factor (TF) and proteolytically activates factors X and IX. Progressive inhibition of TF activity occurs upon its addition to plasma. This process is reversible and requires the presence of VII(a), catalytically active Xa, Ca2+, and another component that appears to be associated with the lipoproteins in plasma, a lipoprotein-associated coagulation inhibitor (LACI). A protein, LACI(HG2), possessing the same inhibitory properties as LACI, has recently been isolated from the conditioned media of cultured human liver cells (HepG2). Rabbit antisera raised against a synthetic peptide based on the N-terminal sequence of LACI(HG2) and purified IgG from a rabbit immunized with intact LACI(HG2) inhibit the LACI activity in human serum. In a reaction mixture containing VIIa, Xa, Ca2+, and purified LACI(HG2), the apparent half-life (t1/2) for TF activity was 20 seconds. The presence of heparin accelerated the initial rate of inhibition threefold. Antithrombin III alpha alone had no effect, but antithrombin III alpha with heparin abrogated the TF inhibition. LACI(HG2) also inhibited Xa with an apparent t1/2 of 50 seconds. Heparin enhanced the rate of Xa inhibition 2.5-fold, whereas phospholipids and Ca2+ slowed the reaction 2.5-fold. Xa inhibition was demonstrable with both chromogenic substrate (S-2222) and bioassays, but no complex between Xa and LACI(HG2) could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Nondenaturing PAGE, however, showed that LACI(HG2) bound to Xa but not to X or Xa inactivated by diisopropyl fluorophosphate. Thus, LACI(HG2) appears to bind to Xa at or near its active site. Bovine factor Xa lacking its gamma-carboxyglutamic acid-containing domain, BXa(-GD), through treatment with alpha-chymotrypsin, was used to further investigate the Xa requirement for VIIa/TF inhibition by LACI(HG2). LACI(HG2) bound to BXa(-GD) and inhibited its catalytic activity against a small molecular substrate (Spectrozyme Xa), though at a rate approximately sevenfold slower than native BXa. Preincubation of LACI(HG2) with saturating concentrations of BXa(-GD) markedly retarded the subsequent inhibition of BXa. The VII(a)/TF complex was not inhibited by LACI(HG2) in the presence of BXa(-GD), and further, preincubation of LACI(HG2) with BXa(-GD) slowed the inhibition of VIIa/TF after the addition of native Xa. The results are consistent with the hypothesis that inhibition of VII(a)/TF involves the formation of a VIIa-TF-XA-LACI complex that requires the GD of XA.(ABSTRACT TRUNCATED AT 400 WORDS).
T-cell leukemias with rearrangement of the gamma but not beta T-cell receptor genes
Authors: Foroni L , Matutes E , Foldi J , Morilla R , Rabbitts T , Luzzatto L , Catovsky D
Published: October 2019

beta and gamma T cell receptor (TCR) gene configuration was studied in 12 patients with large granular lymphocyte T cell leukemia (LGL- leukemia). Both genes were found rearranged in ten cases. In the remaining two patients TCR beta was found in germline configuration. In one of them rearrangement of T cell-rearranging gene gamma (TRG gamma) and a gamma mRNA were demonstrated. We suggest that in this patient the leukemic T cells arose from one of the rare T cells bearing a gamma- delta rather than an alpha-beta TCR heterodimeric molecule. In the other patient several discrete TRG gamma rearrangements were detected. Because her leukemic cells were shown to be monoclonal on the grounds of their karyotype, we suggest that her leukemia originated before any rearrangement had taken place. The combined use of TCR beta and TRG gamma probes provides new information on the origin and clonal expansion of lymphoid cells in LGL-leukemia.
Clinical factors influencing the efficacy of pooled platelet transfusions
Authors: Bishop JF , McGrath K , Wolf MM , Matthews JP , De Luise T , Holdsworth R , Yuen K , Veale M , Whiteside MG , Cooper IA
Published: October 2019

To determine the relative importance of clinical factors on the efficacy of platelet transfusions, 941 pooled platelet transfusions from HLA-unmatched donors were studied prospectively in 133 patients with bone marrow failure. Multiple linear regression analyses identified the major factors influencing one-hour-corrected increments (CI) as prior splenectomy, bone marrow transplantation, disseminated intravascular coagulation, concurrent intravenous amphotericin B, splenomegaly, and HLA antibody grade. The relative impact of these factors on CI has been quantitated by using a formula developed from these data. A linear relationship was demonstrated between increasing percentage of HLA antibody grade and decreasing CI. A number of other factors were less important in the linear regression model than the aforementioned major factors. These included platelet-specific antibodies, concurrent antibacterial antibiotics, clinical bleeding grade, and temperature. Factors that did not influence CI included the number of prior platelet transfusions, prior granulocyte transfusions, prior red cell transfusions, infection, age, blood group, diagnosis, sex, pretransfusion platelet count, prior pregnancies, and concurrent antineoplastic drugs. This study identified major clinical factors that significantly influenced CI and were major causes of refractoriness to pooled platelet transfusions.
Factor V is activated and cleaved by platelet calpain: comparison with thrombin proteolysis
Authors: Bradford HN , Annamalai A , Doshi K , Colman RW
Published: October 2019

Platelets are known to process human factor V during secretion and/or membrane binding. We studied the functional and structural changes produced in human factor V by purified human platelet calpain (calcium- activated thiol protease) and compared the alterations with those induced by thrombin. A maximum increase in coagulant activity of 2.5- fold was observed when factor V (1 U/mL, 33 nmol/L) was incubated with calpain (0.03 U/mL, 2.7 nmol/L) in comparison with a 8.8-fold increment for alpha-thrombin (0.7 U/mL, 8 nmol/L) at 25 degrees C. Thrombin additions to reactions initiated by calpain resulted in further activation comparable to that of thrombin alone, whereas the subsequent addition of calpain had no effect on the extent or pattern of the activation of factor V by thrombin. The cleavage pattern of factor V produced by these two enzymes are distinctly different. Although thrombin activation eventually results in four final components designated C1 (150 kd), D (105 kd), E (71 kd), and F1F2 (71 to 74 kd), calpain yields initial components of 200 kd and 160 kd within one minute. Further digestion of the 200 kd species by calpain gives rise first to a polypeptide of 160 kd that is converted to a 140 kd and a 120 kd species by two minutes with an increase in coagulant activity. Immunoblotting of these fragments with the monoclonal antibody (MoAb) B10 directed to factor V and the thrombin-generated C1 fragment yields results demonstrating a common epitope in these calpain-generated components of 200, 160, 140 and 120 kd. The degradation of the initial 160 kd polypeptide gives rise to polypeptides of 100 and 65 kd, both undetectable on immunoblotting with MoAb B10. The 130, 87, 58, and 48 kd components are of less certain origin. Thus, platelet calpain generates a complex but reproducible cleavage pattern different from thrombin that may explain the partial activation observed. Nevertheless, calpain processing may play a role in early hemostatic reactions involving platelets before the appearance of the first thrombin molecule.
Clinical and biologic characterization of T-cell neoplasias with rearrangements of chromosome 7 band q34
Authors: Smith SD , Morgan R , Gemmell R , Amylon MD , Link MP , Linker C , Hecht BK , Warnke R , Glader BE , Hecht F
Published: October 2019

In T cell malignancy, rearrangements of chromosome 14 have been observed with a break in the band that contains the alpha chain gene for the T cell receptor (TCR). Because the beta chain TCR gene is in chromosome band 7q34, we searched for and report finding specific rearrangements of 7q34 exclusively in T cell malignancies. The rearrangements were reciprocal translocations between 7q34 and other points: 1p34, 9q32, 9q34, 15q22, and 19p13. The malignancies containing a 7q34 translocation were either T cell acute lymphoblastic leukemias or T cell lymphoblastic lymphomas that had similarities in clinical, enzyme, immunologic, and cellular characteristics. Hybridization using a probe to the beta-TCR gene disclosed unique rearrangements consistent with clonality in every case. A common pattern with chromosome breakpoints involving TCR genes may be emerging in T cell neoplasia.
The Corfu delta beta zero thalassemia: a small deletion acts at a distance to selectively abolish beta globin gene expression [published erratum appears in Blood 1988 May;71(5):1509]
Authors: Kulozik AE , Yarwood N , Jones RW
Published: October 2019

The Corfu delta beta zero thalassemia is characterized by the clinical picture of thalassemia intermedia. In the homozygous state there is a complete absence of hemoglobin (Hb) A and Hb A2 and a high level of Hb F. A DNA fragment containing the gamma and beta globin genes has been cosmid cloned, and the deletion breakpoint region, the beta globin gene and the promoter regions of the gamma globin genes sequenced. The deletion removes 7,201 base pairs (bp) containing part of the delta globin gene and sequences upstream. The beta globin gene contains a G--- -A mutation at IVS 1 position 5. The gamma globin gene promoters are normal. Analysis of the transcription of the mutated beta globin gene in transfected HeLa cells shows that normal message is produced at a level of approximately 20% compared with a normal gene, the remaining 80% being spliced at cryptic sites in exon 1 and intron 1. This indicates that the mutation in the beta globin gene is not the sole cause of the absence of Hb A in Corfu delta beta zero thalassemia. It is concluded that the 7.2 kilobase (kb) of deleted DNA contains sequences necessary for the normal activation of the beta globin gene. Possible mechanisms for the effect of the deletion on the expression of beta and gamma globin genes are discussed.
Role of T-cell antigens in the cytolytic activities of large granular lymphocytes (LGLs) in patients with LGL lymphocytosis
Authors: Oshimi K , Oshimi Y , Akahoshi M , Kobayashi Y , Hirai H , Takaku F , Hattori M , Asano S , Kodo H , Nishinarita S
Published: October 2019

By analyzing surface antigens and cytolytic functions of proliferating large granular lymphocytes (LGLs), three types of T cell LGL lymphocytosis were delineated. The first, most commonly encountered type exhibited CD3+4–8+16+, WT31+ phenotype, low or undetectable non- major histocompatibility complex (MHC)-restricted cytotoxicity, and moderate to strong antibody-dependent cellular cytotoxicity (ADCC) and lectin-dependent cellular cytotoxicity (LDCC). Because these LGLs carried T cell antigen receptor (Ti) recognized by WT31 monoclonal antibody (MoAb), and treatment with anti-Ti, anti-CD3 MoAbs and phytohemagglutinin elicited non-MHC-restricted cytotoxicity, they may have developed from populations of in vivo primed cytotoxic T lymphocytes with unknown antigen specificity. The second, rare type of LGL lymphocytosis exhibited CD3+4–8–16+, WT31 phenotype, and strong non- MHC-restricted, ADCC and LDCC cytotoxicities. These cells were probably derived from the lymphocytes of the same phenotype found in small numbers in normal peripheral blood. Because anti-CD3 MoAb inhibited non- MHC-restricted cytotoxicity of the LGLs, a Ti not detected by WT31 MoAb, but putatively present seemed to serve as a specific receptor for target tumor cell recognition. The third type of LGL lymphocytosis showed CD3+4+8–16+, WT31+ phenotype, and lacked cytolytic activities and parallel tubular arrays. These LGLs probably evolved from cells with the same characteristics selectively located in the germinal centers of lymphoid tissues. Taken together, in patients with LGL lymphocytosis, T cell-associated antigens expressed on LGLs were shown to be involved in the regulation of LGL-mediated cytolytic activities. In addition, studies of surface antigens and the effects of MoAbs and lectins on cytolytic activities may be useful in clarifying the normal counterpart of LGLs from which leukemic or reactively proliferating LGLs originate.
An apparently higher molecular weight gamma-chain variant in a new congenital abnormal fibrinogen Tochigi characterized by the replacement of gamma arginine-275 by cysteine
Authors: Yoshida N , Ota K , Moroi M , Matsuda M
Published: October 2019

A gamma-chain variant with an apparently higher molecular weight than the normal gamma-chain was detected in a new congenital abnormal fibrinogen with impaired polymerization of the fibrin monomer and with normal release of fibrinopeptides A and B in a 51-year-old male. Purified fibrinogen analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under the reduced condition in the system of Laemmli contained two protein bands in the gamma-chain region (molecular weight, 50,500 as compared with 50,000 for the normal), both with normal crosslinking ability. The presence of two types of gamma- chains was more clearly detected when reduced and carboxymethylated fibrinogen was analyzed by SDS-PAGE or when reduced fragment D2 was analyzed on SDS-PAGE followed by Western blotting, and identified by positive staining for anti gamma-chain monoclonal antibody. Cyanogen bromide- or lysylendopeptidase-cleavage of purified gamma-chains analyzed on reverse-phase high performance liquid chromatography showed the decrease of one peptide compared with the normal and the appearance of an abnormal peptide peak. Amino acid sequence analysis demonstrated that the gamma arginine-275 of gamma-chain variant was replaced by a cysteine. These data suggest that some regions or conformations containing gamma 275 will affect the polymerization of fibrin monomers. The propositus' two daughters had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Tochigi, and the gamma-chain variant as gamma Tochigi.
Differential requirements for platelet aggregation and inhibition of adenylate cyclase by epinephrine. Studies of a familial platelet alpha 2-adrenergic receptor defect
Authors: Rao AK , Willis J , Kowalska MA , Wachtfogel YT , Colman RW
Published: October 2019

We describe a family whose members have impaired platelet aggregation and secretion responses to epinephrine with normal responses to adenosine diphosphate and collagen. Platelet alpha 2-adrenergic receptors (measured using 3H methyl-yohimbine) were diminished in the propositus (78 sites per platelet), his two sisters (70 and 27 sites per platelet), and parents (37 and 63 sites per platelet), but not in two maternal aunts (12 normal subjects, 214 +/- 18 sites per platelet; mean +/- SE). However, the inhibition of cyclic adenosine monophosphate (cAMP) levels by epinephrine in platelets exposed to 400 nmol/L PGI2 was similar in the patients and five normal subjects (epinephrine concentration for 50% inhibition, 0.04 +/- 0.01 mumol/L v 0.03 +/- 0.01 mumol/L; P greater than .05). In normal platelets, the concentration of yohimbine (0.18 mumol/L) required for half maximal inhibition of aggregation induced by 2 mumol/L epinephrine was lower than that for inhibition of its effect on adenylate cyclase (1.6 mumol/L). In quin2 loaded platelets, thrombin (0.1 U/mL) stimulated rise in cytoplasmic Ca2+ concentration, [Ca2+]i, was normal in the two patients studied. The PGI2 analog ZK 36,374 completely inhibited thrombin-induced rise in [Ca2+]i; the reversal of this inhibition by epinephrine was normal in the two patients. Thus, despite the impaired aggregation response to epinephrine, platelets from these patients have normal ability to inhibit PGI2-stimulated cAMP levels. These patients with an inherited receptor defect provide evidence that fewer platelet alpha 2-adrenergic receptors are required for epinephrine-induced inhibition of adenylate cyclase than for aggregation.
Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization
Authors: Koury ST , Bondurant MC , Koury MJ
Published: October 2019

In situ hybridization was used to localize the cells that produce erythropoietin (EP) in anemic murine kidneys. Kidneys from anemic and nonanemic mice were fixed and processed for paraffin embedding. Sections were hybridized with a 35S-labeled RNA probe complementary to mRNA coding for EP. An uncommon, but specific type of cell was intensely labeled in the cortices of anemic kidneys. The labeled cells were clearly nonglomerular and nontubular. Their location outside of the tubular basement membrane was consistent with that of a subset of interstitial cells or capillary endothelial cells.
The localization of a platelet GpIIb-IIIa-related protein in endothelial cell adhesion structures
Authors: Dejana E , Languino LR , Colella S , Corbascio GC , Plow E , Ginsberg M , Marchisio PC
Published: October 2019

Previous studies have shown that human endothelial cells (ECs) adhere to fibrinogen (fg) and fibronectin (fn) and organize their cytoskeleton on these substrata. However, the mechanism governing this chain of events is poorly known. In ECs glycoproteins immunologically and biochemically similar to the platelet membrane GpIIb-IIIa complex have been described. The functional role of this complex in ECs remains to be established. In this study we show that the antigens recognized by polyclonal antibodies raised against human platelet GpIIb-IIIa and crossreacting with the EC form have a discrete and well-organized distribution at cell adhesion structures. Indeed these antigens are located at vinculin-rich focal contacts found at the membrane insertion of microfilament bundles of the stress fiber type. They are also found at cell-to-cell contacts and, with a diffuse pattern, at the dorsal surface of ECs. GpIIb-IIIa antibodies, added to EC suspensions prior to plating, inhibit EC spreading on fg and vitronectin (vn) substrata in a concentration-dependent way. In contrast, the antibodies are very poorly active when the cells are seeded on fn-coated glass. The same antibodies, added to adherent cells, disrupt cell-to-cell contacts and cause their rounding and detachment. Overall these results indicate that EC GpIIb-IIIa complex is involved in controlling the adhesion mechanism of these cells to extracellular matrix proteins.
Introduction of interleukin-3 gene into stromal cells from the bone marrow alters hemopoietic differentiation but does not modify stem cell renewal
Authors: Zipori D , Lee F
Published: October 2019

Cloned endothelial-adipocytes from the stroma of mouse bone marrow (designated 14F1.1) induced growth of stem cells in vitro, accompanied by either pre-B lymphopoiesis or myelopoiesis. We examined the contribution of colony stimulating factors (CSF) to the process. mRNA for GM-CSF, interleukin 3 (IL3), G-CSF, and IL4 could not be detected in the stromal cells. Expression of IL3 gene, achieved by transfection of 14F1.1 cells with a plasmid carrying an IL3 cDNA, shifted the direction of differentiation but did not improve stem cell maintenance. It is proposed that novel stromal cell factors, distinct from known CSFs, regulate stem cell renewal.