bluuebunny/crossref_metadata_2025 huggingface dataset:
Tumor-Specific Idiotype Vaccines in the Treatment of Patients With B-Cell Lymphoma — Long-Term Results of a Clinical Trial
Authors: Hsu Frank J. , Caspar Clemens B. , Czerwinski Debra , Kwak Larry W. , Liles Tina Marie , Syrengelas Athanasia , Taidi-Laskowski Behnaz , Levy Ronald
Published: October 2019

The surface Ig on each B-cell lymphoma has unique portions (idiotypes), which can be recognized by the immune system. In this study, we immunized patients against the Ig expressed by their tumor and observed their clinical outcomes. After standard chemotherapy, 41 patients with non-Hodgkin's B-cell lymphoma received a series of injections with a vaccine consisting of tumor Ig protein coupled to keyhole limpet hemocyanin and emulsified in an immunologic adjuvant. Subjects were observed for toxicity, immune responses, and tumor status. The median duration of follow-up of all patients is 7.3 years from diagnosis and 5.3 years from the last chemotherapy given before vaccine treatment. Twenty patients (49%) generated specific immune responses against the idiotypes of their tumor Ig. Two patients who had residual disease experienced complete tumor regression in association with the development of these immune responses. The median duration of freedom from disease progression and overall survival of all 20 patients mounting an anti-idiotype immune response are significantly prolonged compared to the patients who did not mount an immune response. Thirty-two patients were in their first remission and nine were in subsequent remissions before beginning vaccine treatments. Analysis of the 32 first remission patients also shows an improved clinical outcome for those patients who mounted a specific immune response compared to those who did not (freedom from progression, 7.9 years v 1.3 years P = .0001; median survival from time of last chemotherapy not yet reached v 7 years, P = .04). This study confirms an earlier report that patients with B-cell lymphoma can be induced to make a specific immune response against the Ig expressed by their own tumor. It further shows that the ability to make such an immune response is correlated with a more favorable clinical outcome. Prospective controlled trials will be needed to prove a causal relationship between anti-idiotype immunity and improved clinical outcome.
Strategies for Hematopoietic Stem Cell Gene Therapy: Insights From Computer Simulation Studies
Authors: Abkowitz Janis L. , Catlin Sandra N. , Guttorp Peter
Published: October 2019

We simulated gene therapy using parameters derived from the analysis of autologous transplantation studies in glucose-6-phosphate dehydrogenase heterozygous cats to determine how hematopoietic stem cell (HSC) biology might influence outcomes. Simulation illustrates that a successful experiment can result by chance and may not be the repeated outcome of a specific protocol design or technical approach. As importantly, in many simulated gene therapy experiments where 1, 2, or 6 of 30 transplanted HSC were labeled, there was significant variation in the contribution from marked clones over time. Variability was minimized in simulations in which large numbers of HSC were transplanted. Strategies that may permit consistent clinically successful results are presented. Taken together, these simulation studies demonstrate that the in vivo behavior of HSC must be considered when optimizing approaches to gene therapy in large animals, and perhaps by extension, in humans.
Mediterranean Anemia
Authors: MINNICH VIRGINIA , NA-NAKORN SUPA , CHONGCHAREONSUK SOODSARKON , KOCHASENI SU-ED
Published: October 2019

1. Thirty-two patients with Mediterranean anemia were studied in Thailand. The diagnotic criteria are presented. All cases fulfilled these requirements.

2. In addition to the classical blood findings, the following observations are presented: (a) fetal hemoglobin, (b) erythrocytic inclusion bodies, (c) liver function tests.

3. Eighty-four per cent of the patients studied were found to have abnormally high amounts of fetal hemoglobin.

4. The inclusion bodies observed in four cases (case histories included) are presented and discussed.

5. Tests for liver function, plasma protein fractions, cephalin cholesterol flocculation, bromsulfalein retention, and bilirubin are reported for twenty patients.

6. A consideration of the effects of splenectomy on the clinical and hematologic status in Mediterranena anemia is presented.
Splenic Neutropenia in the Felty Syndrome
Authors: HUTCHISON H. E. , ALEXANDER W. D.
Published: October 2019

A 59 year old woman with chronic rheumatoid arthritis of twenty years' duration developed chronic granulocytopenia and excessive susceptibility to infection. Mild anemia and symptomless thrombocytopenia were also present. Eight marrow punctures over two and one half years all showed what appeared to be maturation arrest affecting the neutrophils so that segmented polymorphs alone were reduced in number. Throughout this period no form of therapy, including a five day course of ACTH, was effective in promoting normal granulopoiesis; only at the end of this period did the spleen become palpable. Splenectomy resulted in complete symptomatic cure which has been maintained for one year but hematologic cure was not fully obtained. It is believed that this case of splenic neutropenia is really an exaggerated form of Felty's syndrome for the literature shows that malignant neutropenia is not rare in this disease. Furthermore analysis of the literature on splenic neutropenia reveals other cases of an essentially similar nature and the suggestion is put forward that Felty's syndrome may be a fairly common cause of what has been regarded as primary splenic neutropenia. The possible integration of these findings with the immunologic leukopenic mechanism of Moeschlin is briefly discussed.
Separation of Individuals of Any Blood Group into Secretors and Non-Secretors by Use of a Plant Agglutinin (Lectin)
Authors: BOYD WILLIAM C. , SHAPLEIGH ELIZABETH
Published: October 2019

It has been found that an anti-H agglutinin, prepared by simple saline extraction of seeds of Ulex europeus, is suitable for the routine separation of individuals into secretors and non-secretors, merely by testing the ability of the undiluted saliva of the subjects to inhibit the agglutination of group O erythrocytes by the undiluted Ulex extract.
Case Report: The Successful Therapy with Streptokinase-Streptodornase of Ankle Ulcers Associated with Mediterranean Anemia
Authors: COOPER CHARLES D. , WACKER WARREN E. C.
Published: October 2019

1. A successful method of treatment of chronic leg ulcers associated with Mediterranean anemia, employing streptokinase-streptodornase in carboxymethylcellulose jelly, is presented.
The Occurrence of Leukemia
Authors: COOKE JEAN V.
Published: October 2019

Since leukemia is always fatal, the mortality rates are a reasonably accurate reflection of the occurrence of the disease at various ages. Certain features about the occurrence of leukemia were analyzed from the Vital Statistics of the United States over a twenty year period with special reference to age. A comparison of the occurrence (mortality) during this time has shown a considerable and progressive increase in incidence in persons over 50 years of age from 1930 to 1949. In ages under 50 years the increase was apparently slight but not remarkable.

In addition a comparison of the clinical occurrence of patients with the chronic forms of leukemia has shown that, in recent years, the proportion of patients over 50 years of age is twice as great as in similar groups of patients observed some twenty years previously.

The evidence strongly suggests that the occurrence of leukemia in persons over 50 years of age is actually increasing.
A Dynamic State in Stored Erythrocytes as Reflected by the Undulating Nature of in Vitro Measurements
Authors: THIELE ELIZABETH H. , PENNELL ROBERT B.
Published: October 2019

The fluctuating capacity of stored erythrocytes to release gas when suspended in the presence of dextrose in Krebs-Ringer bicarbonate solution in an atmosphere of 5 per cent CO2-95 per cent N2 is demonstrated.

A not dissimilar fluctuation of the cholinesterase activity of the stored cells is reported.

Possible seasonal variation in the state of red cells is suggested.

Some implications of these observations for erythrocyte storage are suggested.
A Quantitative Comparison of the Nucleated Cells in the Right and Left Humeral Bone Marrow of the Guinea Pig
Authors: HARRIS H. S. , HERDAN G. , ANCILL R. J. , YOFFEY J. M.
Published: October 2019

In seventeen young male guinea pigs a quantitative study was made of the nucleated cells of the bone marrow in the right and left humerus. Analysis of the data shows a close correspondence between the two sides in regard to the main cell groups.
The Fecal Excretion of Urobilinogen of Normal and Lead Poisoned Guinea Pigs
Authors: BAIKIE A. G.
Published: October 2019

1. The method of Watson for the estimation of fecal urobilinogen has been modified for use in guinea pigs.

2. Estimations on three or four day fecal collections give less variable results than estimations on single day collections. Loss of urobilinogen consequent upon storage to the third or fourth day is small.

3. For normal guinea pigs a mean daily excretion of 0.23 mg. with a standard deviation of 0.10 was found.

4. Serial estimations were carried out of the urobilinogen excretion of guinea pigs before and during experimental poisoning with lead nitrate. During lead poisoning there developed a statistically significant increased excretion of urobilinogen. This increased excretion was shown, moreover, to be correlated with the fall in hemoglobin levels which occurred in the poisoned animals.

5. It is concluded that these results provide confirmatory evidence for the view that hemolysis plays an important part in the anemia of chronic lead poisoning.
Relationship of Erythrocyte Cholinesterase Activity to Cell Metabolism
Authors: THIELE ELIZABETH H. , PENNELL ROBERT B.
Published: October 2019

Production by normal human erythrocytes of a substrate for cholinesterase has been demonstrated.

At body temperature, the formation and hydrolysis of the cholinesterase substrate is delicately controlled.

At lowered temperatures, 0 to -1.5 C., production of cholinesterase substrate greatly exceeds its hydrolysis.

The quantity of cholinesterase substrate capable of exerting its influence governs the quantity of dextrose converted to lactic acid by the erythrocyte under anaerobic conditions.

The observations corroborate and extend the suggestion of Lindvig, Greig, and Peterson that cholinesterase is part of a cellular system governing erythrocyte permeability.

The balance between production and hydrolysis of cholinesterase substrate at low temperature exhibits the same undulating fluctuations during red cell storage that have been noted for other erythrocyte metabolic functions.
Serum Polysaccharide and Fever in Thrombocytopenic Bleeding of Leukemia
Authors: FREEMAN GUSTAVE , BUCKLEY EDWARD S.
Published: October 2019

Evidence indicates that bleeding is associated with fever in critically thrombocytopenic leukemic children. It appears that fever, generally the result of secondary infection, is associated with elevations of protein-bound serum polysaccharide. Polysaccharide and platelets are quantitatively independent but both excess polysaccharide and critical deficiency of platelets have direct relationships to the hemorrhagic diathesis in leukemic children. Implications are discussed.
Hematologic Findings in the Total Body X-irradiated Hamster
Authors: FULTON GEORGE P. , JOFTES DAVID L. , KAGAN ROMA , LUTZ BRENTON R.
Published: October 2019

Hematologic determinations were made on cardiac blood from normal hamsters and from four groups of hamsters at daily intervals after total body irradiation with 600 r, 1000 r, 1200 r and 1500 r. Pancytopenia, lowered hematocrit, and decreased hemoglobin occurred. The clotting times were extended in the 1200 r and 1500 r groups, and this defect was correlated temporally with thrombocytopenia and disrupted mast cells. The sedimentation rates were increased terminally in the lethally-irradiated hamsters. The significance of these findings and the relationships to visual observations on the peripheral circulation such as leukocytic pavements on capillary and venular endothelium, platelet thromboembolic phenomena, erythrocytic extravasations, and circulating red cell aggregates are discussed.
Idiopathic Myoglobinuria in Man
Authors: SPAET THEODORE H. , ROSENTHAL MARTIN C. , DAMESHEK WILLIAM
Published: October 2019

A case of myoglobinuria of idiopathic origin is described. A diagnostic triad of (1) muscular pseudoparalysis, (2) a highly colored urine containing a benzidine positive pigment, and (3) plasma free from visible discoloration was present. Identification of the urinary pigment as myoglobin was established by spectrophotometry, and the presence of accompanying metmyoglobinuria was detected. Exercise of the lower extremities was the precipitating factor during each attack of myoglobinuria. Attempts to induce attacks by means other than exercise were unsuccessful, and no amelioration of myoglobinuria could be produced by treatment with ascorbic acid or cortisone. The myoglobinopathies are discussed together with the present concept of the physiology of myoglobin. The differentiation of myoglobinurias from hemoglobinuria and porphyrias is readily accomplished by the laboratory procedures indicated above.
PU.1/Pip and Basic Helix Loop Helix Zipper Transcription Factors Interact With Binding Sites in the CD20 Promoter to Help Confer Lineage- and Stage-Specific Expression of CD20 in B Lymphocytes
Authors: Himmelmann Andreas , Riva Agostino , Wilson Gaye Lynn , Lucas Brian P. , Thevenin Claire , Kehrl John H.
Published: October 2019

CD20 is a B-lineage–specific gene expressed at the pre–B-cell stage of B-cell development that disappears on differentiation to plasma cells. As such, it serves as an excellent paradigm for the study of lineage and developmental stage-specific gene expression. Using in vivo footprinting we identified two sites in the promoter at −45 and −160 that were occupied only in CD20+ B cells. The −45 site is an E box that binds basic helix-loop-helix-zipper proteins whereas the −160 site is a composite PU.1 and Pip binding site. Transfection studies with reporter constructs and various expression vectors verified the importance of these sites. The composite PU.1 and Pip site likely accounts for both lineage and stage-specific expression of CD20 whereas the CD20 E box binding proteins enhance overall promoter activity and may link the promoter to a distant enhancer.
Early Onset of Immunoglobulin Heavy Chain Gene Rearrangements in Normal Human Bone Marrow CD34+ Cells
Authors: Davi Frédéric , Faili Ahmad , Gritti Catherine , Blanc Catherine , Laurent Caroline , Sutton Laurent , Schmitt Christian , Merle-Béral Hélène
Published: October 2019

To characterize early B-cell precursors in humans, we correlated immunoglobulin heavy chain (IgH) gene rearrangement status with the CD34, CD19, and CD10 cell surface markers. Highly purified adult bone marrow (BM) cell fractions were obtained by two successive rounds of flow cytometric cell sorting, and IgH rearrangements were analyzed by polymerase chain reaction (PCR) amplification. Complete VDJH rearrangements were observed in the CD34+ CD19+ fraction, but not in the more immature CD34+ CD19− fraction. About one quarter of these rearrangements had an open reading frame, thus potentially permitting the synthesis of a μ chain. Partial DJH rearrangements were detected in both CD34+ CD19+ and CD34+ CD19− subsets, although they were less abundant in the latter. When triple labeling was used to better characterize the CD34+ CD19− population, DJH rearrangements were found to be present in the CD34+ CD10+ CD19− fraction, but not in the more primitive CD34+ CD10− CD19−. These results indicate that IgH gene rearrangements occur in CD34+ BM cells and that they initiate in immature progenitors expressing the CD10, but not yet the CD19 surface antigen. Finally, the presence of IgH gene rearrangements in CD34+ BM cells provides a useful marker of clonality to evaluate the possible involvement of these cells in various B-cell lymphoid malignancies.
Elevated Serum CD44 Level Is Associated With Unfavorable Outcome in Non-Hodgkin's Lymphoma
Authors: Ristamäki Raija , Joensuu Heikki , Lappalainen Kimmo , Teerenhovi Lasse , Jalkanen Sirpa
Published: October 2019

CD44 molecule is a cell surface glycoprotein involved in many cell-cell and cell-matrix interactions. Circulating serum CD44 (s-CD44) levels have been found to change in parallel with response to therapy, but little is known about the predictive or prognostic significance of s-CD44. In the present study, we measured s-CD44 levels in sera taken before treatment from 194 patients with non-Hodgkin's lymphoma using a chemiluminescence-enzyme immunoassay method. All except 1 patient were regularly followed-up after therapy at least for 60 months (range, 33 to 143 months). The median pretreatment s-CD44 level was 440 ng/mL (range, 13 to 1,220 ng/mL). Only 32% of the 92 patients with an International Prognostic Index (IPI) score of 0 or 1 had an s-CD44 concentration higher than the median as compared with 67% of the patients with an IPI score ≥2 (P < .0001). Patients with lower than the median s-CD44 achieved more often a complete remission to therapy (P = .0002) and had better survival (P = .007) than those with higher s-CD44 levels. However, in a multivariate analysis, only the IPI score had independent prognostic value (P < .001). The findings were similar if only the patients with diffuse large-cell lymphoma (n = 51) were included in the analysis, but among patients with low-grade lymphoma, the median s-CD44 level was not significantly associated with the IPI or survival. In conclusion, a high s-CD44 level at diagnosis is associated with a high IPI score, poor response to treatment, and unfavorable outcome in non-Hodgkin's lymphoma.
The Role of Mig, the Monokine Induced by Interferon-γ, and IP-10, the Interferon-γ–Inducible Protein-10, in Tissue Necrosis and Vascular Damage Associated With Epstein-Barr Virus-Positive Lymphoproliferative Disease
Authors: Teruya-Feldstein Julie , Jaffe Elaine S. , Burd Parris R. , Kanegane Hirokazu , Kingma Douglas W. , Wilson Wyndham H. , Longo Dan L. , Tosato Giovanna
Published: October 2019

The mechanisms of tissue necrosis and vascular damage characteristics of certain Epstein-Barr virus (EBV)-associated lymphoproliferative disorders are unknown. The CXC chemokines interferon-γ–inducible protein-10 (IP-10) and the monokine induced by interferon-γ (Mig) caused tissue necrosis and vascular damage in Burkitt's lymphoma tumors established in nude mice. We report higher levels of IP-10 and Mig gene expression in tissues with necrosis and vascular damage from EBV-positive lymphomatoid granulomatosis and nasal or nasal-type T/natural killer (NK)-cell lymphomas compared with tissues with lymphoid hyperplasia, which lacked tissue necrosis and vascular damage. By immunohistochemistry, Mig and IP-10 proteins localized with similar patterns in viable tissue surrounding dead tissue, mostly within endothelial cells, monocyte/macrophages, and lymphocytes. Circulating levels of IP-10 were abnormally elevated in patients with EBV-positive lymphomatoid granulomatosis and nasal or nasal-type T/NK-cell lymphomas. These experiments provide the first description of the presence of Mig in any human normal or diseased tissue and the first description of IP-10 in certain lymphoproliferative lesions. These data suggest that Mig and IP-10 play an important role in the pathogenesis of tissue necrosis and vascular damage associated with certain EBV-positive lymphoproliferative processes.
Biochemical and Molecular Characterization of Hereditary Myeloperoxidase Deficiency
Authors: Romano Maurizio , Dri Pietro , Dadalt Liviana , Patriarca Pierluigi , Baralle Francisco E.
Published: October 2019

Hereditary myeloperoxidase (MPO) deficiency is a neutrophil disorder characterized by the lack of peroxidase activity. Cytochemical, biochemical, spectroscopic, immunocytochemical, and genetic studies were carried out on a 5-year-old MPO-deficient subject and on her parents. The father was also MPO-deficient, whereas the mother had 24% of normal MPO activity. Although the typical absorption spectrum of MPO was absent in both the father and daughter, the father's neutrophils, and not those of the daughter, contained material antigenically related to MPO. In the MPO gene of the father, two mutations were found, each located in a different allele: a T → C transition, causing the nonconservative replacement M251T and a 14-base deletion within exon 9. The M251T substitution occurred in the carboxy-terminal region of the light chain that is included in the heme pocket. The daughter inherited the 14-base deletion from her father. The study of the MPO mRNAs present in liquid cultures of granulocyte precursors surprisingly showed that the same genetic defect, ie, the 14-base deletion, seemed to exhibit different mRNA phenotypes in the father and the daughter. In fact, mRNA derived from the 14-base–deleted allele was not found in the father and an aberrantly spliced MPO mRNA with a 77-base deletion of exon 9, which includes the 14-base deletion and leads to the generation of a premature stop codon, was found in the daughter. The possibility that Δ77 mRNA could derive from other mutations linked to the Δ14 allele was dismissed because no sequence differences were found in the region (exons and exon-intron junctions). Our data indicate that the alteration of the mRNA context caused by the 14-base deletion provide a basis for the 77-base deletion in the mRNA processing. Since the granulocyte precursors from the liquid cultures of the father were more differentiated than those from the daughter, the observed different behavior of the 14-base–deleted allele in the father and daughter may be the result of a differentiation-stage dependent control of altered spliced mRNA, which may be tolerated during the early stages of differentiation but degraded at later stages. In the liquid cultures of the daughter's cells, in addition to the mRNA with the 77-base deletion, a mRNA with the wild type sequence was also found. This mRNA was inherited from the mother, since no mutations were found in her MPO cDNA and MPO gene. The MPO defect might be caused by a regulatory mutation that induces the MPO gene switch off at an early stage of granulocyte differentiation.
Tumor Necrosis Factor α-Induced Eosinophil Accumulation in Rat Skin Is Dependent on α4 Integrin/Vascular Cell Adhesion Molecule-1 Adhesion Pathways
Authors: Sanz Maria-Jesus , Hartnell Adele , Chisholm Patricia , Williams Cindy , Davies Dawn , Weg Vivian B. , Feldmann Marc , Bolanowski Mark A. , Lobb Roy R. , Nourshargh Sussan
Published: October 2019

Tumor necrosis factor α (TNFα) is a cytokine implicated in the pathogenesis of numerous chronic and acute inflammatory conditions. In the present study, we have characterized the ability of TNFα in inducing eosinophil accumulation in rat skin and have shown the inhibitory effects of anti-α4 integrin and anti–vascular cell adhesion molecule-1 (VCAM-1) antibodies on this response. The intradermal injection of recombinant human TNFα induced a slowly developing, dose-dependent accumulation of 111In-eosinophils in rat skin that was maximal at the dose of 10−11 mol/site. Coadministration of TNFα with the soluble TNFα receptor (p55)-IgG fusion protein (TNFR-IgG) totally inhibited the 111In-eosinophil accumulation induced by the cytokine. The TNFα-induced 111In-eosinophil accumulation was not affected after pretreatment of rats with the platelet-activating factor (PAF) receptor antagonist UK-74,505 or the antihuman interleukin-8 monoclonal antibody (MoAb) DM/C7. In contrast, the intravenous administration of an anti-α4 integrin MoAb, HP2/1 (3.5 mg/kg), or an anti–VCAM-1 MoAb, 5F10 (2 mg/kg), greatly inhibited the 111In-eosinophil accumulation induced by TNFα (the responses detected at 10−11 mol/site were inhibited by 78% and 50%, respectively). These results show that TNFα is an effective inducer of eosinophil accumulation in vivo, with this response being dependent on an interaction between α4 integrins and VCAM-1.
Intracellular Pool of Vascular Endothelial Growth Factor in Human Neutrophils
Authors: Gaudry Murielle , Brégerie Olivier , Andrieu Valérie , El Benna Jamel , Pocidalo Marie-Anne , Hakim Jacques
Published: October 2019

Vascular endothelial growth factor (VEGF ), an endothelial cell mitogen, is a potent angiogenic factor produced by several cell types. Whether human neutrophils are potential producers of VEGF has not yet been described. The present work shows that phorbol-12-myristate 13-acetate (PMA), fMet-Leu-Phe, and tumor necrosis factor-α (TNF-α) triggered a time-dependent secretion of VEGF by human neutrophils. Cells incubated with 50 ng/mL of PMA released significant amounts of VEGF after 15 minutes. Because the extracellular content of VEGF in human neutrophils supernatants remained constant over a period of 2 to 24 hours and because PMA is a potent inducer of human neutrophil degranulation, the PMA-induced secretion of VEGF may be due to a pre-existing intracellular pool of this molecule. This hypothesis was reinforced by the absence of cycloheximide effect on the PMA-induced secretion of VEGF. The existence of an intracellular pool of VEGF was confirmed by measuring the intracellular content of VEGF in resting neutrophils. A dosedependent inhibition of PMA-induced VEGF secretion was observed when the cells were incubated in the presence of pentoxifylline, a methylxanthine known to inhibit neutrophil degranulation. To confirm the implication of neutrophil degranulation in VEGF release, the effects of two inducers of physiologic degranulation, fMet-Leu-Phe and TNF-α, were determined. Both agonists induced a release of VEGF in the absence of cytochalasin B, confirming the involvement of neutrophil degranulation and suggesting the intracellular localization of VEGF in the specific granule fraction. In addition, the kinetics of fMet-Leu-Phe– and TNF-α–induced secretion of lactoferrin were similar to those of VEGF release induced by these two both agonists. The subcellular fractionation of human neutrophils showed a granule-specific distribution of the intracellular pool of VEGF in resting neutrophils. The finding that human neutrophils contain an intracellular pool of VEGF, secreted in the extracellular space under PMA-, fMet-Leu-Phe–, and TNF-α–induced degranulation, suggests a role for human neutrophils as cellular effectors of physiologic as well as pathologic angiogenesis.
Interleukin-10 Upregulates Tumor Necrosis Factor Receptor Type-II (p75) Gene Expression in Endotoxin-Stimulated Human Monocytes
Authors: Dickensheets Harold L. , Freeman Sherry L. , Smith Michael F. , Donnelly Raymond P.
Published: October 2019

Interferon-γ (IFN-γ) upregulates expression of certain genes in monocytes, including cell-surface molecules such as HLA class II, B7, and ICAM-1. IFN-γ also potentiates production of cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-12. Conversely, IL-10 downregulates expression of many of these same genes and often antagonizes the effects of IFN-γ. IL-10 is known to inhibit TNF-α production in lipopolysaccharide (LPS)-stimulated monocytes; however, the effects of IL-10 on TNF receptor (TNF-R) expression are not well defined. We examined the effects of IL-10 on production of both membrane-associated (m) and soluble (s) TNF-R type II (sTNF-RII) by purified human CD14+ monocytes. We also compared the effects of IFN-γ and IL-10 on production of TNF-α and sTNF-RII by these cells. Monocytes constitutively expressed low levels of TNF-RII mRNA and mTNF-RII protein. LPS stimulation induced rapid, but transient loss (shedding) of mTNF-RII molecules and a delayed, but marked increase in TNF-RII mRNA levels. IL-10 increased expression of both mTNF-RII and sTNF-RII by LPS-stimulated monocytes, whereas IFN-γ decreased their expression. The increased levels of sTNF-RII in cultures of IL-10–treated monocytes correlated directly with increased levels of TNF-RII mRNA and inversely with the levels of TNF-α mRNA. The ability of IL-10 to upregulate TNF-RII gene expression was transcriptionally mediated because actinomycin D blocked this effect. Furthermore, IL-10 treatment did not alter the half-life of TNF-RII mRNA transcripts in LPS-stimulated monocytes. To further examine the mechanism by which IL-10 potentiates TNF-RII gene expression, a 1.8-kb fragment of the human TNF-RII promoter cloned into a luciferase expression vector (pGL2-basic) was transfected into the IL-10–responsive macrophage cell line, RAW264.7. Although IL-10 alone induced only minimal promoter activity in these cells, it markedly increased the LPS-induced response, providing further evidence that the ability of IL-10 to amplify TNF-RII gene expression is transcriptionally controlled. Together, these findings demonstrate that IL-10 coordinately downregulates expression of TNF-α and upregulates expression of TNF-RII, particularly the soluble form of this receptor, in monocytes.
Band 3 Peptides Block the Adherence of Sickle Cells to Endothelial Cells In Vitro
Authors: Thevenin Bernard J.-M. , Crandall Ian , Ballas Samir K. , Sherman Irwin W. , Shohet Stephen B.
Published: October 2019

Malaria-parasitized erythrocytes have increased endothelial adherence due to exposure of previously buried intramembranous sites of band 3. Because sickle erythrocytes also show increased adhesiveness and because the membrane portion of band 3 is aggregated in both types of cells, we examined the role of band 3 in sickle cell adhesiveness. Synthetic peptides derived from the second and third exofacial, interhelical regions of band 3 completely inhibited the abnormal adherence of sickle cells to an endothelial monolayer in a static assay. This effect was observed independently of plasma factors, required micromolar levels of peptide, was sequence-specific, and was found with both L- and D-isomers. The active peptides also inhibited the increased adherence induced by low-dose calcium loading of normal red blood cells. Finally, a monoclonal antibody against an active peptide specifically immunostained a fraction of sickle cells. These findings implicate a role for band 3 in at least one type of sickle cell adhesiveness via the exposure of normally cryptic membrane sites.
Altered Tyrosine Phosphorylation Via the Very Late Antigen (VLA)/β1 Integrin Stimulation Is Associated With Impaired T-Cell Signaling Through VLA-4 After Allogeneic Bone Marrow Transplantation
Authors: Sato Toshiya , Ohashi Yoshiyuki , Tachibana Kouichi , Soiffer Robert J. , Ritz Jerome , Morimoto Chikao
Published: October 2019

Our previous study showed that the cross-linking of very late antigen (VLA)/β1 with anti-CD29 monoclonal antibody (MoAb), or interactions with extracellular matrix (ECM) proteins through VLA/β1, failed to induce T-cell costimulation via the CD3/T cell receptor (TCR) pathway for over 1 year after allogeneic bone marrow transplantation (allo-BMT), although normal CD29 and CD3 expression was observed after 3 months following allo-BMT. Molecular analysis revealed altered tyrosine phosphorylation of cellular proteins by the solid-phase cross-linking of VLA/β1 molecules in T cells from patients after allo-BMT. In T cells from early allo-BMT patients (<4 months), various sizes of highly tyrosine phosphorylated proteins were observed as high background even without the stimulation through VLA/β1 integrin. The high tyrosine phosphorylation pattern gradually disappeared and it was finally returned to normal tyrosine phosphorylation patterns by 2 years after BMT. Interestingly, poor expression of focal adhesion kinase (pp125FAK ), a VLA/β1-mediated signaling molecule, was observed within 1 year after BMT. These results suggest that these molecular defects appear to be implicated in the impaired VLA/β1-mediated signaling in T cells from patients after allo-BMT, and it could explain, in part, the persistent immunoincompetent state after allo-BMT at least 1 year.
STAT5A-Deficient Mice Demonstrate a Defect in Granulocyte-Macrophage Colony-Stimulating Factor–Induced Proliferation and Gene Expression
Authors: Feldman Gerald M. , Rosenthal Louis A. , Liu Xiuwen , Hayes Mark P. , Wynshaw-Boris Anthony , Leonard Warren J. , Hennighausen Lothar , Finbloom David S.
Published: October 2019

Responses of cells to cytokines typically involve the activation of a family of latent DNA binding proteins, referred to as signal transducers and activators of transcription (STAT) proteins, which are critical for the expression of early response genes. Of the seven known STAT proteins, STAT5 (originally called mammary gland factor) has been shown to be activated by several cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5, which are known to play important roles in growth and differentiation of hematopoietic precursors. In this report we have used mice that are deficient in STAT5A (one of two homologues of STAT5) to study the role of STAT5A in GM-CSF stimulation of cells. When bone marrow–derived macrophages were generated by differentiation with macrophage-CSF (M-CSF), exposure of cells from wild-type mice to GM-CSF resulted in a typical pattern of assembly of DNA binding proteins specific for the gamma activation sequence (GAS) element within the β-casein promoter. However, in cells from the STAT5A null mouse one of the shifted bands was absent. Immunoblotting analysis in the null mice showed that lack of STAT5A protein resulted in no alteration in activation of STAT5B by tyrosine phosphorylation. Proliferation experiments revealed that, when exposed to increasing concentrations of GM-CSF, cells derived from the null mice grew considerably more slowly than cells derived from the wild-type mice. Moreover, expression of GM-CSF–dependent genes, CIS and A1, was markedly inhibited in cells derived from null mice as compared with those of wild-type mice. The decreased expression observed with A1, a bcl-2 like gene, may account in part for the suppression of growth in cells from the null mice. These data suggest that the presence of STAT5A during the GM-CSF–induced assembly of STAT5 dimers is critical for the formation of competent transcription factors that are required for both gene expression and cell proliferation.
The Suicide Substrate Reaction Between Plasminogen Activator Inhibitor 1 and Thrombin Is Regulated by the Cofactors Vitronectin and Heparin
Authors: van Meijer Marja , Smilde Annelies , Tans Guido , Nesheim Michael E. , Pannekoek Hans , Horrevoets Anton J.G.
Published: October 2019

The interaction of thrombin with plasminogen activator inhibitor 1 (PAI-1) is shown to result in the simultaneous formation of both cleaved PAI-1 and a sodium dodecyl sulfate-stable thrombin-PAI-1 complex. The kinetics of this reaction can be described by a “suicide substrate” mechanism that includes a branched reaction pathway, which terminates in either the stable inhibitor-enzyme complex or the cleaved inhibitor plus free enzyme. Because of the branched pathway, approximately three moles of PAI-1 are needed to completely inhibit one mole of thrombin. Heparin and vitronectin enhance the rate of inhibition from 9.8 × 102 L mol−1 s−1 to 6.2 × 104 L mol−1 s−1 and 2.1 × 105 L mol−1 s−1, respectively, under optimal conditions. In addition to enhancing the rate of inhibition, both cofactors increase the apparent stoichiometry of the PAI-1–thrombin interaction, with cofactor concentration dependencies similar to the inhibition reaction. Thus, at 37°C approximately six cleavage reactions occur per inhibition reaction. Therefore, thrombin will efficiently inactivate PAI-1 in the presence of either vitronectin or heparin, unless a sufficient excess of the inhibitor is present. These results show that physiological cofactors are able to switch a protease-serpin inhibition reaction to a substrate reaction, depending on the local concentrations of each of the components.
A Novel Degradation Pathway of Tissue Factor Pathway Inhibitor: Incorporation Into Fibrin Clot and Degradation by Thrombin
Authors: Ohkura Naoki , Enjyoji Kei-ichi , Kamikubo Yu-ichi , Kato Hisao
Published: October 2019

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor with three tandem inhibitory domains (K1, K2, and K3) that regulates the initial reactions of the extrinsic blood coagulation pathway through K1 and K2. In the present study, the effect of thrombin on TFPI in a purified system was first examined using recombinant TFPI from Chinese hamster ovary (CHO) cells. TFPI was inactivated by thrombin with cleavage of three peptide bonds, Lys 254-Thr 255 in the C-terminal basic region, Arg 107-Gly 108 (reactive site toward factor Xa in K2), and Lys 86-Thr 87 between K1 and K2. Then, degradation of radiolabeled TFPI by thrombin was examined in two systems: (1) mixed with plasma and then tissue factor (TF ) and calcium ion, and (2) mixed with fibrinogen and then thrombin. TFPI degradation was detected in serum from normal plasma and more extensively from antithrombin (AT)-depleted plasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Significant radioactivity was found in the clot after coagulation of the plasma, which decreased after 20 hours' incubation. These changes were more prominent in AT-depleted plasma than in normal plasma. When TFPI lacking the C-terminal basic region was used instead of full-length TFPI, most of the radioactivity was found in serum rather than in fibrin clots. Incorporation of TFPI into the fibrin clot was prevented by a synthetic C-terminal peptide of TFPI. Similar results were obtained after mixing radiolabeled TFPI with fibrinogen and then thrombin in the presence of calcium ion or EDTA. These results demonstrate a novel degradation pathway of TFPI, ie, incorporation into fibrin via the C-terminal basic region and degradation by thrombin (possibly fibrin-bound thrombin).
Human Dendritic Cells Require Exogenous Interleukin-12–Inducing Factors to Direct the Development of Naive T-Helper Cells Toward the Th1 Phenotype
Authors: Hilkens Catharien M.U. , Kalinski Pawel , de Boer Mark , Kapsenberg Martien L.
Published: October 2019

Dendritic cells (DC) are important initiators of specific primary immune responses because they are the only APC that can efficiently activate naive Th cells. DC have the capacity to produce interleukin-12 (IL-12), a cytokine that plays a pivotal role in the development of Th1-mediated cellular immune responses. The present study focuses on the conditions under which human DC produce bioactive IL-12 p70 and, consequently, direct the development of naive T helper (Th) cells toward the Th1 phenotype. Bacteria or bacterial compounds such as Staphylococcus aureus Cowan strain I (SAC) or lipopolysaccharide (LPS) induced substantial IL-12 levels in DC, which could be further upregulated by interferon-γ (IFN-γ), whereas induction of IL-12 production via CD40 ligation required IFN-γ as an obligatory, complementary signal. Also, activated naive Th cells were poor inducers of IL-12 production, unless exogenous IFN-γ was present, whereas activated memory Th cells were effective inducers of IL-12 production and did not require exogenous IFN-γ. Next, the cytokine profiles of matured Th cells that were primed by DC under different conditions were examined. DC promoted the development of naive Th cells into memory Th0 cells that produced both the type 1 cytokine IFN-γ and the type 2 cytokine IL-4. In contrast, after activation with SAC, DC efficiently directed the development of Th1 cells through the release of IL-12. An APC-independent Th cell maturation model, using either recombinant IL-12 or supernatants of SAC-activated DC and neutralizing anti-IL-12 antibodies, confirmed that DC-derived IL-12 was the major Th1 skewing factor. Together, these data indicate that the contact between DC and naive Th cells during the initiation of specific immune responses does not result in the efficient induction of IL-12 production and that, consequently, exogenous IL-12–inducing factors are required to promote primary Th1-mediated cellular immune responses.
The P-Selectin Glycoprotein Ligand-1 Is Important for Recruitment of Neutrophils Into Inflamed Mouse Peritoneum
Authors: Borges Eric , Eytner Ruth , Moll Thomas , Steegmaier Martin , Campbell Matthew A. , Ley Klaus , Mossmann Horst , Vestweber Dietmar
Published: October 2019

The P-selectin glycoprotein ligand-1 (PSGL-1) is a high-affinity ligand of P-selectin on myeloid cells and certain subsets of lymphoid cells. We generated the rat monoclonal antibody (MoAb) 2PH1 that recognizes an epitope within the first 19 amino acids at the N-terminus of the processed form of mouse PSGL-1. This antibody blocks attachment of mouse myeloid cells to P-selectin under both static and flow conditions. Intravenous administration of saturating amounts of 2PH1 reduced the number of rolling leukocytes in venules of the acutely exposed mouse cremaster muscle by 79% (±5.7%), whereas an anti–P-selectin MoAb reduced it completely. Examining the effect of the MoAb 2PH1 on the recruitment of neutrophils into chemically inflamed mouse peritoneum showed that blocking PSGL-1 inhibited neutrophil accumulation in the peritoneum by 82% (±7%) at 2 hours and by 59% (±7.9%) at 4 hours after stimulation. A similar effect was seen with the MoAb against P-selectin. Simultaneous administration of both antibodies at the 4-hour time point blocked neutrophil accumulation by 86% (±4.2%), arguing for an additional partner molecule for PSGL-1 besides P-selectin. This is the first demonstration of the importance of PSGL-1 in the recruitment of mouse neutrophils into inflamed tissue.
Myeloma Bone Marrow Plasma Cells: Evidence for Their Capacity as Antigen-Presenting Cells
Authors: Yi Qing , Dabadghao Sunil , Österborg Anders , Bergenbrant Susanne , Holm Göran
Published: October 2019

Myeloma plasma cells constitute 10% to 90% of the total bone marrow cell count in patients with multiple myeloma (MM). These cells express a variety of cell surface markers, such as HLA-ABC and HLA-DR, and surface antigens that are necessary for professional antigen-presenting cells, including adhesion and costimulatory molecules. In this study, we examined the expression of major histocompatability complex (MHC) and costimulatory molecules on CD38(bright,++) plasma cells in bone marrow aspirates from eight MM patients. Small percentages of plasma cells expressed weak but detectable levels of HLA-DR, HLA-DQ, CD40, CD80, and CD86, which could be upregulated by interferon-γ (IFN-γ) and tumor necrosis factor-α. CD38++ plasma cell and CD38(dim,+) cells were sorted from freshly isolated bone marrow mononuclear cells and tested for their capacity to act as antigen-presenting cells. Indeed, both CD38++ plasma cells and CD38+ cells were able to stimulate allogeneic T cells and present the soluble antigens purified protein derivative and tetanus toxoid to autologous T cells. Recognition of the antigens led to T-cell proliferation and secretion of IFN-γ and was MHC class-I and -II restricted. Antigen processing and presentation by CD38++ and CD38+ cells were abolished by treatment of the cells with chloroquine. Hence, our study provides for the first time evidence that myeloma plasma cells may act as antigen-presenting cells. Further studies are warranted to examine in detail the molecules required for inducing T-cell stimulation.
MDM2 Protein Overexpression Promotes Proliferation and Survival of Multiple Myeloma Cells
Authors: Teoh Gerrard , Urashima Mitsuyoshi , Ogata Atsushi , Chauhan Dharminder , DeCaprio James A. , Treon Steven P. , Schlossman Robert L. , Anderson Kenneth C.
Published: October 2019

The murine double minute 2 (MDM2) protein facilitates G1 to S phase transition by activation of E2F-1 and can enhance cell survival by suppressing wild-type p53 (wtp53) function. In this study, we examined MDM2 expression and function in multiple myeloma (MM) cells. MDM2 is strongly and constitutively expressed in MM cell lines (ARH-77, RPMI 8226, and OCI-My5) and in the cells of plasma cell leukemia (PCL) patients, but is not expressed in normal bone marrow mononuclear cells (BM MNCs). Treatment of MM cells with MDM2 antisense, but not sense, nonsense, or scrambled, oligodeoxyribonucleotides (ODNs) decreased DNA synthesis and cell viability; it also induced G1 growth arrest, as evidenced by propidium iodide (PI) staining and induction of retinoblastoma protein (pRB) to E2F-1 binding. Moreover, inhibition of MDM2 using antisense ODNs also triggered MM cell apoptosis as evidenced by acridine orange–ethidium bromide staining. We next studied the association of MDM2 with wtp53 and/or mutant p53 (mtp53), E2F-1, CDK4, and p21. MDM2 constitutively binds to E2F-1 in all MM cells, to both wtp53 and mtp53, and to p21 in tumor cells lacking p53. These data suggest that MDM2 may enhance cell-cycle progression in MM cells both by activating E2F-1 and by downregulating cell-cycle inhibitory proteins (wtp53 and p21). Overexpression of MDM2 may therefore contribute to both growth and survival of MM cells, suggesting the potential utility of treatment strategies targeting MDM2 in MM.
Induction of γ-Globin by Histone Deacetylase Inhibitors
Authors: McCaffrey Patricia G. , Newsome David A. , Fibach Eitan , Yoshida Minoru , Su Michael S.-S.
Published: October 2019

The short-chain fatty acid butyrate has been shown to elevate fetal hemoglobin (HbF ) by inducing expression of the γ-globin gene. Regulation of gene expression by butyrate is thought to proceed via inhibition of the enzyme histone deacetylase, leading to elevated levels of core histone acetylation which affect chromatin structure and transcription rates. To determine whether changes in histone acetylation are critical for the regulation of the γ-globin gene, we tested three potent and specific inhibitors of histone deacetylase, the cyclic tetrapeptides trapoxin and Helminthsporium carbonum toxin (HC toxin), and the antifungal antibiotic trichostatin A for their ability to induce fetal hemoglobin expression in erythroid cells. These compounds induced fetal hemoglobin in both primary erythroid cell cultures and human erythroleukemia (K562) cells. A butyrate-responsive element spanning the duplicated CCAAT box region of the γ-globin promoter has been identified in transient transfection assays using a reporter construct in K562 cells, and we show that the same promoter region is required for response to trapoxin and trichostatin. Mutational analysis of the γ-globin promoter indicates that the distal CCAAT box and 3′ flanking sequence (CCAATAGCC) is critical for activation by butyrate, trapoxin, and trichostatin, whereas the proximal element (CCAATAGTC) plays a less important role. These results show that inhibition of histone deacetylase can lead to transcriptional activation of γ-globin promoter reporter gene constructs through proximal promoter elements, and suggest that butyrate induces γ-globin expression via such changes in histone acetylation.