bluuebunny/crossref_metadata_2025 huggingface dataset:
Association Between C-Reactive Protein in the First 1-3 Days Post-Transplant and Allogeneic Immune Reactions in Pediatric Haploidentical Stem Cell Transplantation
Authors: Chen Yao , Liu Kai-Yan , Huang Xiao-Jun , Huan Chen , Wang Feng-Rong , Wang Jing-Zhi , Zhang Xiao-Hui , Chen Yu-Hong , Wang Yu , Han Wei , Chen-Hua Yan , Zhang Yuan-Yuan , Xu Lan-Ping
Published: October 2019

Background: C-reactive protein (CRP) has been shown to be a reliable biomarker of innate immunity. The purpose of the current study was to evaluate whether CRP levels in the first 1-3 days could predict allogeneic immune reactions including engraftment syndrome (ES) or acute graft-versus-host disease (GVHD) under haploidentical stem cell transplantation (SCT) in children.

Patients and methods: The study population comprised 175 consecutive pediatric patients (age no more than 18 years) who received haploidentical SCT from Oct, 2009 to Dec, 2014. All the children followed Beijing protocol in our institute as previously described. Allogeneic immune reactions include engraftment syndrome and acute GVHD. ROC analysis was performed to identify cutoff CRP value in the first 1-3 days post-transplant that best identified patients with allogeneic immune reactions. Patients were classified into 2 groups as high-CRP group (≥cut-off value) and low-CRP group (<cut off value).

Results:Totally, 129 patients (73.7%) were alive and without primary disease.The median age of recipients was 14 years (range: 2-18 years). The primary diseases were as follows: ALL in 95 (54.3%), AML in 62 (35.4%), hybrid acute leukemia in 3 (1.7%), CML in 10 (5.7%), and MDS in 5 (2.9%). Of the 175 patients, 68 (38.9%) developed ES on a median of day 10 (range: 7-16). 51(29.1%) out of 175 children developed 2-4 grade acute GVHD on a median of day 30 (range: 22-89) including 15 (8.6%) patients of severe acute GVHD on a median of day 29 (range: 22-85). Higher incidence of ES, 2-4 grade GVHD and 3-4 grade GVHD were totally observed in high-CRP group, compared to the low-CRP. A multivariate analysis demonstrated that high-level CRP(≥20.1 mg/L)in day 1-3 post-transplant was a risk factor for the development of allogeneic immune reactions. High CRP group was associated with an increased occurrence of ES (HR=2.046; 95%CI=1.204-3.477; P=0.008), II-IV aGVHD (HR=2.203; 95%CI=1.192-4.073; P=0.001) and severe GVHD (HR=6.371; 95%CI=1.798-22.581; P=0.004).

Conclusions: Our data suggested higher CRP level during the first 1-3 days post-transplant could be a predictor of allogeneic immune reactions in pediatric haploidentical SCT.

Disclosures

No relevant conflicts of interest to declare.
High-Efficiency Transduction of Primary Human Hematopoietic Stem/Progenitor Cells By AAV6 Vectors: strategies for Overcoming Donor-Variation and Implications in Genome Editing
Authors: Ling Chen , Bhukhai Kanit , Yin Zifei , Tan Mengqun , Yoder Mervin , Leboulch Philippe , Payen Emmanuel , Srivastava Arun
Published: October 2019

We have reported that of the 10 commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem/progenitor cells (HSPCs). However, the transduction efficiency of the wild-type (WT) AAV6 vector varies greatly in HSPCs from different donors. Here we report two distinct strategies to further increase the transduction efficiency in HSPCs from donors that are transduced poorly with the WT AAV6 vectors. The first strategy involved modification of the viral capsid proteins where specific surface-exposed tyrosine (Y) and threonine (T) residues were mutagenized to generate a triple-mutant (Y705+Y731F+T492V) AAV6 vector. The second strategy involved the use of ex vivo transduction at high cell density, which revealed a novel mechanism, which we have termed, 'cross-transduction'. The combined use of these strategies resulted in transduction efficiency exceeding ~90% in HSPCs. Our studies have significant implications in the optimal use of capsid-optimized AAV6 vectors in genome editing in HSPCs.

Disclosures

Leboulch: bluebird bio: Patents & Royalties. Payen:bluebird bio: Patents & Royalties. Srivastava:AGTC; Genzyme: Patents & Royalties.
Hemophilia Lend-Lease Program in Italy Successfully Meets Albania Factor Needs
Authors: Aledort Louis M. , De Luca Michele , Jose Caldes Pinilla Maria , Zanobini Alberto , Carli Simona , Godo Anila , Manastirliu Ogerta , Kolaj Gentian , Caja Tatiana , Neziri Vera
Published: October 2019

Many countries can not provide yet an adequate haemophilia care for their patients. Albania is among the countries that are trying now to improve its healthcare system but still do not have fully implemented it. As to the Haemophilia treatment the need of FVIII for patients exceeds the available resources thus jeopardizing the possibilities of having I place prophilaxis and/or major chirurgic treatments.

Tuscany is one of the region in Italy where the rate of Albanian inhabitant people is higher. Among them many are also blood donors and the links between Italians and the Albanian community are quite strong as well as the Tuscany commitment toward Albania in the general health system (many are the programs put in place by the International Health Cooperation of Tuscany).

In this context in 2013 a joint program between Hospital Mother Teresa in Tirana and Hospital Meyer in Florence was undersigned for the supply of 3 MIU of FVIII at 9 cents/I.U. from donors of the region each year for three years.

Albania recognizes that hemophilia treatment is a urgent part of their healthcare plan and expects to be able soon to provide more funds to continue this program. Albania has been offered more FVIII from Italian donors for at least a 5 years period in the framework of an Italian recent law that is encouraging the use of FVIII in excess to Italian needs for other countries and with the support of Kedrion , who Is the company fractionating the plasma from Tuscany donors into finished product. Kedrion not only committed to pay for the shipping of goods from Italy to Albania but it is also acting as a partner and liaison between Tuscany and the country

Industry should take note that with local philanthropy and financial support from the biologic industry one can achieve both improving healthcare and not just donating product but working with developing countries to make them support their population.

Disclosures

Aledort: Kedrion: Other: chair scientific advisory committee for kedrion.
Runx1 Deficiency and MDS-Associated U2af1 Mutation Cooperate for Leukemia Development in a New Mouse Model
Authors: Zhen Tao , Liang Fei Dennis , Zhao Ling , Lopez Guadalupe , Varmus Harold , Liu Pu Paul
Published: October 2019

The pathogenesis of hematopoietic malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), is very complicated with mutiple genetic alterations required for full-blown disease. Mutations of splicing factor genes, including U2AF1, are found in overal half of MDS patients and 5~10% de novo AML patients. U2AF1 mutations, occuring in roughly 10% of patients with MDS and 3% in de novo AML, are heterozygous and localized almost exclusively in two codons, S34 and Q157, suggesting they are gain-of-function mutations. In addition, our recent studies suggest retention of the wild type U2AF1 allele is required for cell viability (Fei et al., BioRxiv, 2016). Interestingly, RUNX1 is the most commonly co-mutated gene in MDS patients with U2AF1 mutations, and U2AF1 and RUNX1 are also co-mutated in some AML patients. These findings suggest RUNX1 deficiency and U2AF1 mutation cooperate in the pathogenesis of MDS and AML.

To test this hypothesis, we crossed Cre-based conditional Runx1 knockout mice (Runx1f/f) with mice carrying a newly developed Cre-based conditional U2af1-S34F mutation (U2af1+/S34F) to generate Runx1f/f, Mx1-Cre, U2af1+/S34Fmice, which express U2af1-S34F and deficient-Runx1 after inducing Cre from the Mx1 promoter with poly (I:C). We found that U2af1-S34F attenuated the Runx1-/--induced increase of myeloid cells in spleen and bone marrow, as well as the increase of progenitor (lineage-/Sca1-/C-kit+) and stem cells (lineage-/Sca1-/C-kit+). Competitive repopulation assays showed that combining the U2af1 mutation and Runx1 deficiency impaired the transplantation capacity of bone marrow progenitor cells, especially for the myeloid lineage. We further aged these mice to examine the cooperative effects of U2af1-S34F and Runx1 deficiency in disease progression, upon the treatment of a one time, low-dose N-ethyl-N-nitrosourea. Mx1-Cre, U2af1+/S34F mice didn't show survival defect when compared to control mice, while the Runx1f/f, Mx1-Cre mice developed MDS, as previously reported. Interestingly, of fourteen Runx1f/f, Mx1-Cre, U2af1+/S34F mice, two developed AML, and the rest developed MDS. Moreover, transplanted spleen and bone marrow cells from one of four MDS Runx1f/f, Mx1-Cre, U2af1+/S34F mice also developed AML in the recipients. Although Runx1f/f, Mx1-Cre mice also had MDS, their MDS cells did not develop into leukemia after transplantation. Our data therefore suggest that Runx1 deficiency and MDS-associated U2af1 mutation can cooperate in the genesis of AML from MDS.

Disclosures

No relevant conflicts of interest to declare.
Genomic Analysis of Plasma DNA in Prenatal Medicine and Beyond
Authors: Lo Yuk Ming Dennis
Published: October 2019

The discovery of cell-free fetal DNA in maternal plasma has enabled the development of noninvasive prenatal testing (NIPT). With the increased use of NIPT globally, there have been a number of reports of plasma DNA aberrations that have orignated from maternal malignancies, rather than signifying genomic aberrations in the fetus. In this regard, it would be highly desirable to develop a technology for tracing the tissue of origin of genomic aberrations observed in plasma. Hence, we have developed a technology called plasma DNA tissue mapping which allows this to be achieved. Through the development of over 5800 methylation markers, we are able to deduce the tissue of origin of plasma DNA molecules. We have demonstrated the utility of this approach by tracing genomic aberrations observed in the plasma of a pregnant woman during the course of NIPT to a B-cell lymphoma. We believe that this technology would be broad applications in medical research and diagnostics.

Disclosures

Lo: Cirina Limited: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Xcelom Limited: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Illumina: Patents & Royalties; Sequenom: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
Platelet Toll-Like Receptors: Bridging Inflammation and Immunity
Authors: Kubes Paul
Published: October 2019

Since the discovery that there is a series of pattern recognition receptors that allow the immune system to detect pathogens, there has been a lot of work to elucidate the signaling molecules that contribute to this detection system. Although the cell types involved would seem to be just as important, our understanding of which cells are critical remains less well explored. Using intravital imaging to visualize the different cell types, we were surprised to find that in almost all conditions that we examined, platelets were rapidly recruited to afflicted tissues. Under some conditions, platelets bound vascular macrophage including Kupffer cells and helped contain bacteria. These were instantaneous responses. At later times, platelets bound neutrophils and induced the production of neutrophil extracellular traps (NETs) that helped to catch bacteria as well as viruses but did induce some local vascular injury. In some scenarios, platelets bound endothelial cells and whether this was to wall off and contain infections or a hijacking of platelets by bacteria to induce vascular dysfunction and poor perfusion remains unclear. Visualizing platelets in sterile injury also revealed important contributions to helping recruit other immune cells that help to heal. The role of the platelet as an effector in infections and inflammation will be discussed.

Disclosures

No relevant conflicts of interest to declare.
Observations on the Changes in the Kinetics of Red Cell Proliferation Following Irradiation and Hypertransfusion
Authors: STOHLMAN FREDERICK
Published: October 2019

Data are presented on the changing pattern of the Fe59 uptake and emergence curve following irradiation and hypertransfusion. The data obtained appear to be inconsistent with the notion that population pressure influences the release of red cells from the marrow. The implications of these data on the understanding of the kinetics of red cell production are discussed.
RENAL FUNCTION DURING CHRONIC ANEMIA IN MAN
Authors: BRADLEY STANLEY E. , BRADLEY GERALDINE P.
Published: October 2019

1. Renal function has been studied quantitatively in 15 patients with chronic anemia, 8 of whom were proved to have pernicious anemia. In 7 the anemia was secondary to chronic blood loss, iron deficiency, paroxysmal nocturnal hemoglobinuria, and leukemia. The effective renal plasma flow and glomerular filtration rate were measured by clearance technics; and tubular function, by saturation methods (diodrast Tm and glucose Tm).

2. The effective renal plasma flow, the glomerular filtration rate, and the filtration fraction (percentage of plasma filtered at the glomerulus) were reduced slightly below the normal values in most subjects. The effective renal whole blood flow was always greatly reduced, by 46 per cent on the average in males and by 31.8 per cent in females.

3. Since arterial pressure was not significantly depressed it was concluded that renal vasoconstriction occurs in chronic anemia, possibly as a homeostatic device for the diversion of blood to tissues more sensitive to oxygen lack. The relatively small reduction of filtration fraction implies afferent and efferent arteriolar vasoconstriction with dominance by the afferent arterioles. These changes were shown to be reversible, a return to normal values paralleling the return of the blood picture to normal.

4. Diodrast Tm was reduced significantly in 9 of 10 patients while the values of glucose Tm were normal in 6 of 7 patients. The normal values for glucose Tm indicated continued operation of all glomeruli and implied the absence of shunting or of cessation of blood flow in any significant portion of the kidney. The fall in diodrast Tm, which appeared to be reversible in 2 of 4 individuals, was interpreted as evidence of intracellular dysfunction rather than destruction or inactivation of nephrons.
MONOCYTIC LEUKEMIA A CASE REPORT ILLUSTRATING VARIATIONS IN THE CLINICAL PICTURE
Authors: RAPPOPORT ARTHUR E. , KUGEL VICTOR H.
Published: October 2019

Monocytic leukemia is an entity that has engendered a variety of divergent clinical and histologic opinions. A case is presented in which prolonged and intensive clinical and laboratory observations demonstrate the erratic course of this disease and illustrate the erroneous conclusions that may be derived from inconstant, momentary observations of this dynamic process. Study of the "natural history" of monocytic leukemia yields observations which tend to reconcile many of the hitherto conflicting opinions regarding this disease.

The origin and characteristics of the monocytic cell are discussed, and its probable derivation from mesenchyma is emphasized.
HEMATOLOGIC OBSERVATIONS IN A CASE OF KALA-AZAR
Authors: RACHMILEWITZ M. , BRAUN K. , DE VRIES A.
Published: October 2019

The hematologic findings in a case of kala-azar under the influence of specific treatment are described. The type of the anemia, the hyperplastic bone marrow, the increased urobilinogen output before treatment, and the subsequent changes following treatment strongly suggest increased red cell destruction (most probably by phagocytosis) as the cause of the anemia.
CONGENITAL HEMOLYTIC ICTERUS IN A NEGRO FAMILY
Authors: GOODMAN E. G. , CATES B. R.
Published: October 2019

1. A Negro family is reported in which 3 sisters were found to have congenital hemolytic icterus.

2. All members of the family observed showed strong Negroid characteristics. There was nothing to suggest an admixture of white blood.

3. The literature was reviewed. Including the 3 reported in this paper, 8 cases have been reported. The disease may be more common in the female as the reported cases show 7 females to 1 male.

4. Congenital hemolytic icterus, on rare occasions, occurs in the Negro race and recognizing it as such is helpful since splenectomy is of distinct value.
THE VALUE AND THE LIMITATIONS OF THE COAGULATION TIME IN THE STUDY OF THE HEMORRHAGIC DISEASES
Authors: QUICK ARMAND J. , C. RENE HONORATO , STEFANINI MARIO
Published: October 2019

The coagulation time is a measurement of the intrinsic power of the blood to convert fibrinogen to fibrin. It is an empirical test no matter how performed, and therefore in order to be reliable requires that the test be done on venous blood under strictly controlled conditions. A recommended procedure is outlined in detail.

The coagulation time is prolonged in hemophilia, hypoprothrombinemia, afibrinogenemia and heparinemia. In hemophilia, the coagulation time theoretically is a measure of the severity of the disease but practically is of limited value since the coagulation time may be within normal limits in some patients; the prothrombin consumed in the coagulation of hemophilic blood is therefore a better guide for diagnosis. The coagulation time in hypoprothrombinemia is relatively little prolonged until a drastic reduction occurs. The test is therefore of no value for establishing a hemorrhagic condition in hypoprothrombinemia. In afibrinogenemia the blood is incoagulable. A small amount of fibrinogen restores the coagulation time to normal.

The presence of heparin increases the coagulation time. The test is therefore useful in controlling the therapeutic action of this drug.

The senior author, in making a survey of the literature on hemorrhagic diseases in preparation of his monograph, was impressed by the significant and diverse contributions which Dr. George R. Minot made to this field of medicine. We feel honored to contribute this study to the collection of papers offered as a fitting tribute to Dr. Minot, who has so successfully and productively combined science and clinical medicine.
THE EFFECT OF INTRAVENOUS INJECTION OF TRYPSIN INHIBITOR ON THE COAGULATION OF BLOOD
Authors: TAGNON H. J. , SOULIER J. P.
Published: October 2019

1. The intravenous injection of a soya bean preparation containing a trypsin inhibitor into 2 dogs and 3 rabbits produced the following effects: prolongation of the clotting time and of the prothrombin time, and increase in the antiproteolytic activity of the blood plasma or serum.

2. Identical results were obtained in one experiment in which a crystallin trypsin inhibitor from soya bean was used.

3. The significance of these results is briefly discussed.
CYTOLOGY OF RABBIT THYMUS AND REGENERATION OF ITS THYMOCYTES AFTER IRRADIATION; WITH SOME NOTES ON THE HUMAN THYMUS
Authors: DOWNEY HAL , ENGSTROM RUBY
Published: October 2019

Thymocytes are genuine lymphocytes. The largest lymphocytes of lymph nodes do not occur in the thymus; the smallest ones with pacychromatic nuclei and scanty cytoplasm are more numerous in the thymus. Some lymphoblasts similar to myeloblasts occur in the rabbit thymus.

All three types of granulocytes develop from lymphocytes and to some extent from the mesenchymatous reticulum in the rabbit. In the rabbit the heterophil and eosinophil granules are acidophilic when first formed, while in the human thymus many have a basophilic quota as in the marrow. The reticulum is largely of epithelial origin and retains its epithelial characteristics in cytoplasm and nucleus. In the rabbit some mesenchymatous tissue is blended with the reticulum and may give origin to lymphocytes and granulocytes. The epithelium may form macrophages but does not differentiate to lymphocytes and other types of cells. In imprints from the rabbit the epithelial cells usually can be distinguished from those of the mesenchymatous reticulum. During regeneration following irradiation the latter tissue can be seen to form some lymphocytes locally; other lymphocytes enter the organ through the lymph vessels.

Imprints stained with May-Grünwald and Giemsa are of great value for detailed cytology of the thymus and for determining cell relationships. Sections of thymus fixed in Helly’s fluid, stained with methyl green and pyronin and dehydrated in dioxan are excellent for lymphocytes and the transitional stages resulting from their development from fixed mesenchymatous tissue during regeneration of the organ following exposure to x-rays.
THE EVENTS IN THE HEMOLYTIC CRISIS OF HEREDITARY SPHEROCYTOSIS, WITH PARTICULAR REFERENCE TO THE RETICULOCYTOPENIA, PANCYTOPENIA AND AN ABNORMAL SPLENIC MECHANISM
Authors: DAMESHEK WILLIAM , BLOOM MARVIN L.
Published: October 2019

The course of hereditary spherocytosis (congenital or familial hemolytic anemia) is subject to major or minor exacerbations or crises. Pancytopenia, reticulocytopenia, and extreme spherocytosis characterize the major crises, during which hypersplenic effects appear to play a major role. These are characterized by the combination of (1) an unusual degree of hemolysis with (2) inhibitory effects upon maturation and delivery of bone marrow cells. At the height of the crisis, an extreme degree of maturation arrest in erythropoiesis is present. Splenectomy, which is often urgently necessary, results in a very rapid increase in all the cellular elements of the blood, confirming the phagocytic and inhibitory effects of the abnormal spleen. Following splenectomy, no further crises occur. The presence of successive cases in the same family suggest the possible role of infection as a precipitating or trigger agent in initiating an abnormal splenic mechanism leading to crisis. The cause of the hereditary spherocytosis is commented upon and evidence bearing upon an autopsecific mechanism is discussed.
THE OCCURRENCE OF NORMOBLASTS IN THE PERIPHERAL BLOOD IN CONGESTIVE HEART FAILURE: AN INDICATION OF UNFAVORABLE PROGNOSIS
Authors: GROEN J. , GODFRIED E. G.
Published: October 2019

The authors recommend the search for normoblasts in the blood of patients with severe heart failure. When normoblasts are found, a marked interference with the oxygenation of the blood, either by pulmonary infarcts or thrombi inside the heart, is most likely to be present. It seems justifiable to consider the prognosis as very grave in these cases. This rule proved to hold even in those cases where, concomitant with an improvement in the heart failure, the normoblasts disappeared temporarily from the peripheral blood.
FEMORAL BONE MARROW BIOPSY IN THE ALBINO RAT
Authors: CAMERON D. G. , WATSON G. M.
Published: October 2019

A technic for repeated femoral bone marrow biopsy in the rat is detailed.

Differential counts confirmed the distribution of the cellular elements as described by other authors.
The Coagulation of the Blood. Investigations on a New Clotting Factor. BY PAUL A. OWREN. Oslo, J. Christian Gundersen, 1947
Authors: Taylor F. H. L.
Published: October 2019

An error was made in Dr. F. H. L. Taylor’s review of Dr. Owren’s book, "The Coagulation of the Blood," in the February issue of Blood (3: 229, 1948). The formulae as printed were incomplete and should have read:

See PDF for Equation

See PDF for Equation

See PDF for Equation
IS LEUKEMIA A DISEASE OF THE RETICULO-ENDOTHELIAL SYSTEM?
Authors: WISEMAN BRUCE K.
Published: October 2019

1. Evidence is given from case study of reticulo-endothelial disease supporting the concept that monocytic leukemia is one form of reticulo-endotheliosis.

2. On the basis of varied types of cell reactions seen in monocytic leukemia, it is suggested that all forms of leukemia may be hematologic varieties of reticulo-endotheliosis.
THE COINCIDENCE OF MEDITERRANEAN ANEMIA AND PERNICIOUS ANEMIA IN A YOUNG SICILIAN
Authors: CROSBY WILLIAM H. , SACKS HARRY J.
Published: October 2019

1. Coincidental Mediterranean anemia and pernicious anemia were found in a 26 year old soldier of Sicilian parentage.

2. The diagnosis of pernicious anemia was made on the finding of achlorhydria after histamine, glossitis, megaloblastic bone marrow and macrocytic anemia which responded to liver extract on two occasions.

3. The diagnosis of mild Mediterranean anemia was made by finding the target-oval-cell trait in the patient and in five members of his family.

4. It is of interest that target cells were not found in the peripheral blood until treatment with liver was begun. While pernicious anemia dominated, the character of the peripheral blood picture was macrocytic. Liver therapy corrected this, whereupon "hypochromic polycythemia" characteristic of mild Mediterranean anemia was found.
IRON METABOLISM
Authors: FINCH CLEMENT A. , GIBSON JOHN G. , PEACOCK WENDELL C. , FLUHARTY REX G.
Published: October 2019

By determining the percentage utilization of intravenously administered radioiron for hemoglobin production over a period of two to three weeks, certain measurements of internal iron metabolism can be made.

With a normal rate of blood production, changes in per cent utilization reflect alteration in iron stores. Iron depletion is characterized by more rapid and more complete utilization of radioiron. States of iron excess in hemochromatosis can be identified by their profound depression of radioiron utilization.

If, on the other hand, storage iron is not greatly altered, the percentage utilization is determined by the function of the erythropoietic tissue. In myelophthisic anemias, in uremia, and in infection, a similar depression of the curve is found.

The rate of erythropoiesis may further be estimated by the slope of the utilization curve, and evidence of abnormal red cell destruction is found in early and abrupt plateau of the utilization curve.

A correlation has been made in a variety of hematologic disorders between the radioiron utilization for hemoglobin production and the clinical factors which might be expected to affect iron metabolism in these patients.
Fluorescent labeling of human platelets
Authors: Horne WC , Guilmette KM , Simons ER
Published: October 2019

Noncovalently bound fluorescent probes have been used to study changes in the platelet which may occur during platelet aggregation. Platelets were exposed to either N-phenyl-naphthylamine (NPN) or 8-anilino-1- naphthalene-sulfonic acid (ANS). Both dyes were bound by the platelet, and platelet aggregation by collagen or thrombin was unaffected by the presence of the label. No change in fluorescence intensity or wavelength of maximum intensity was observed during platelet aggregation.
Autoimmune and alloimmune phenomena in patients with aplastic anemia: cytotoxicity against autologous lymphocytes and lymphocytes from HLA identical siblings
Authors: Warren RP , Storb R , Thomas ED , Su PJ , Mickelson EM , Weiden PL
Published: October 2019

We have studied peripheral blood lymphocytes of 117 patients with severe aplastic anemia and 237 healthy individuals for reactivity against autologous lymphocytes and/or lymphocytes from HLA-identical siblings using a 51Cr release assay. Lymphocytes from 29% of the patients exhibited reactivity against their own lymphocytes, while only 3% of lymphocytes from normal individuals showed such reactivity. Lymphocytes from 49% of the patients showed reactivity against lymphocytes from their HLA-identical siblings compared to 4% of normal individuals. Correlation existed between allogeneic and autologous reactivities (p < 0.001), suggesting a common pathway for cytotoxicity. Both reactivities showed an association with extremely low granulocyte counts (p < 0.01) and an inability of the patient's mononulcear cells to stimulate in allogeneic mixed leukocyte culture (p < 0.05) as well as an inverse correlation with time from diagnosis (p < 0.001). At least two explanations exist for the transfusion-independent autologous and allogeneic cytotoxicities: (1) they could be epiphenomena secondary to the stem cell defect, such as the loss of a cell that suppresses (or regulates) naturally occurring cytotoxic cells, or (2) they may be involved in the cause of the disease.
Possible mechanisms accounting for the growth factor independence of hematopoietic progenitors from umbilical cord blood
Authors: Schibler KR , Li Y , Ohls RK , Nye NC , Durham MC , White W , Liechty KW , Le T , Christensen RD
Published: October 2019

Hematopoietic progenitors obtained from the bone marrow of healthy adults fail to undergo clonogenic maturation in vitro if a source of hematopoietic growth factors is not included in the culture dishes. In contrast, a fraction of similarly purified progenitors obtained from umbilical cord blood undergo clonogenic maturation even in the absence of added growth factors. We postulated that production of hematopoietic growth factors within the culture dishes containing the progenitors of umbilical cord blood origin might be responsible. We postulated further, that this production might be by non-progenitor cells co- plated along with the progenitors, or alternatively by CD34+ cells themselves, or by cells clonally derived from CD34+ cells. To test these possibilities we first assessed the effect of including in the cultures neutralizing antibody directed against various growth factors. Inclusion of anti-granulocyte macrophage colony-stimulating factor (GM- CSF) and anti-interleukin-3 (IL-3) (but not anti-IL-2) significantly reduced the growth factor independence of cord blood progenitors (P > .005 and P > .01). Inclusion of both anti-GM-CSF and anti-IL-3 almost completely ablated the spontaneous colony growth (P > .001). Inclusion of IL-10 also reduced, in a concentration-dependent fashion, the spontaneous generation of umbilical cord blood-derived colonies. Transcripts for GM-CSF and IL-3 were detected, by reverse transcriptase- polymerase chain reaction (RT-PCR), in the CD34+ cells from cord blood and from adult marrow. When plated without added growth factors, however, the CD34+ cells of adult marrow origin failed to produce colonies, whereas 6% of cord blood CD34+ cells similarly cultured did so. When these growth factor independent colonies were plucked from culture, transcripts for GM-CSF and IL-3 were identified in all. We conclude that production of GM-CSF and IL-3 occurs within culture dishes containing hematopoietic progenitors of umbilical cord origin, and that this explains some of their apparently unique features of in vitro growth.
Construction of a ribozyme directed against human interleukin-6 mRNA: evaluation of its catalytic activity in vitro and in vivo
Authors: Mahieu M , Deschuyteneer R , Forget D , Vandenbussche P , Content J
Published: October 2019

We have designed a ribozyme (Rz) that cleaves human interleukin-6 (IL- 6) mRNA in vivo. This Rz was tested in vitro, and was found to give expected size fragments. It was then incorporated into a mammalian expression vector containing the constitutive cytomegalovirus (CMV) immediate early promoter and transfected into human U amniotic cells (UAC). Cell clones that stably express this catalytic RNA have been obtained. Some of them displayed a marked reduction of tumor necrosis factor (TNF)-induced IL-6 production. Their reduced ability to express IL-6 was related to the amount of Rz they produced and to the extent of IL-6 mRNA cleavage as observed by a ribonuclease protection assay. These data provide a method to study further the role of IL-6 production in various biologic situations, and suggest the feasibility of developing Rzs directed against various cytokines to study their biologic role and mechanism of action.
Candidate tumor-suppressor genes MTS1 (p16INK4A) and MTS2 (p15INK4B) display frequent homozygous deletions in primary cells from T- but not from B-cell lineage acute lymphoblastic leukemias [see comments]
Authors: Hebert J , Cayuela JM , Berkeley J , Sigaux F
Published: October 2019

Using a Southern blot approach, deletions of MTS1 (multiple tumor- suppressor gene 1) and MTS2 (multiple tumor-suppressor gene 2) candidate tumor-suppressor genes have been studied in primary neoplastic cells from 55 acute lymphoblastic leukemia (ALL) patients. Homozygous MTS1 deletions were found in 20 of 24 T-ALL cases and in only 2 of 31 B-lineage cases (P < .001). The deletions involved MTS1 and MTS2 in most cases. Homozygous MTS2 deletions were observed in 16 of 24 T-ALL cases and in 1 of 31 B-lineage ALLs (P < .001), all of them displaying homozygous MTS1 deletions. In 5 cases (4 T and 1 B), deletions involved MTS1 but spared the MTS2 gene, showing that one deletion breakpoint was located between the two genes within a 25-kb region. In 1 T-ALL case, an MTS1 gene rearrangement has occurred downstream to exon 2. Possible hemizygous deletions were found in 6 cases, 4 of them of the B-cell lineage. In 7 ALL cases, cells obtained at presentation and at first relapse were studied and identical results were observed in 6 cases. In 1 B-lineage case, a germline pattern was found at presentation and a possible monoallelic MTS1/MTS2 deletion was observed at relapse. The high frequency of MTS1 and MTS2 homozygous deletions in T-ALLs supports the view that inactivation of these genes plays an important role in the pathogenesis of this type of human leukemia.
Immunohistochemical localization of hepatic nitric oxide synthase in normal and transgenic sickle cell mice: the effect of hypoxia
Authors: Osei SY , Ahima RS , Fabry ME , Nagel RL , Bank N
Published: October 2019

Nitric oxide (NO) generated from L-arginine and molecular oxygen by nitric oxide synthase (NOS) has been shown to influence hepatocellular function and pathology in response to ischemia and certain hepatotoxins. In the present study, we examined the liver of a transgenic line of sickle cell mice for hepatocellular injury and localization of two isoforms of NOS, the endothelial constitutively expressed isoform (EcNOS) and the inducible isoform (iNOS) by immunohistochemistry. Diffuse expression of EcNOS was observed in hepatocytes of control and sickle cell animals maintained under room air conditions. In contrast, iNOS was observed only in the sickle cell mice, well-localized to hepatocytes surrounding the central veins of the lobules. When normal mice were exposed to hypoxic conditions for 4 to 5 days, iNOS immunostaining appeared de novo in a patchy distribution throughout the liver lobules. In the sickle cell mice, hypoxia appeared to increase the subjective intensity of pericentral staining of iNOS. Liver histology was normal in the sickle cell mice maintained under room air conditions, but showed multifocal areas of necrosis when sickling was exacerbated by chronic hypoxic conditions. However, a pericentral zone of preserved architecture was present, corresponding to the region of iNOS staining. We postulate that pericentral induction of iNOS under ambient conditions occurs in transgenic sickle cell mice in response to particularly intense hypoxic conditions near the central veins of the liver. Increases in NO synthesis may occur in this region, which would serve to protect these cells from ischemic damage either directly or by maintaining blood flow. These findings could be relevant to liver pathophysiology in patients with sickle cell disease.
Lymphotropic herpesviruses in allogeneic bone marrow transplantation
Authors: Wang FZ , Dahl H , Linde A , Brytting M , Ehrnst A , Ljungman P
Published: October 2019

Human herpesvirus-6 (HHV-6), human herpesvirus-7 (HHV-7), Epstein-Barr virus (EBV), and human cytomegalovirus (CMV) DNA were repeatedly assayed in peripheral blood leukocytes from 37 allogeneic bone marrow transplant (BMT) patients by polymerase chain reaction. Before BMT, HHV-6 DNA was detected in 8 (22%) patients. HHV-7, EBV, and CMV DNA were detected in 21 (57%), 10 (27%), and 1 (3%) patient, respectively. After BMT, HHV-6 DNA was detected in 26 (70%), HHV-7 in 21 (57%), EBV in 28 (76%), and CMV in 21 (57%) patients. Thirty-two (87%) patients were positive with more than one virus. HHV-6, HHV-7, and EBV DNA were found earlier than CMV DNA in most patients after BMT. The proportions of HHV-6-positive samples during the first 3 months after BMT were higher in the patients with either delayed granulocyte engraftment (P = .04, Fisher's exact test) or delayed platelet engraftment (P = .001, Fisher's exact test). The HHV-6 DNA in samples from the patients with delayed engraftment was confirmed to be variant B. The detection of any lymphotropic herpesvirus was not related to the development of acute graft-versus-host disease (aGVHD). High-dose acyclovir (ACV) prophylaxis significantly (P < .01) reduced the proportion of HHV-6-positive samples and tended to lower HHV-6 DNA levels (P = .06). Our data indicate that HHV-6 variant B can inhibit marrow engraftment and that high-dose ACV may be beneficial to engraftment after BMT by preventing HHV-6 reactivation. No relation between the proportions of HHV-7-, EBV-, and CMV-positive samples in the first 3 months and engraftment or aGVHD was found.
Recombinant Human Thrombopoietin in Combination With Granulocyte Colony-Stimulating Factor Enhances Mobilization of Peripheral Blood Progenitor Cells, Increases Peripheral Blood Platelet Concentration, and Accelerates Hematopoietic Recovery Following High-Dose Chemotherapy
Authors: Somlo George , Sniecinski Irena , ter Veer Anna , Longmate Jeffrey , Knutson Gaylord , Vuk-Pavlovic Stanimir , Bhatia Ravi , Chow Warren , Leong Lucille , Morgan Robert , Margolin Kim , Raschko James , Shibata Stephen , Tetef Merry , Yen Yun , Forman Stephen , Jones Dennie , Ashby Mark , Fyfe Gwen , Hellmann Susan , Doroshow James H.
Published: October 2019

Lineage-specific growth factors mobilize peripheral blood progenitor cells (PBPC) and accelerate hematopoietic recovery after high-dose chemotherapy. Recombinant human thrombopoietin (rhTPO) may further increase the progenitor-cell content and regenerating potential of PBPC products. We evaluated the safety and activity of rhTPO as a PBPC mobilizer in combination with granulocyte colony-stimulating factor (G-CSF) in 29 breast cancer patients treated with high-dose chemotherapy followed by PBPC reinfusion. Initially, patients received escalating single doses of rhTPO intravenously (IV) at 0.6, 1.2, or 2.4 μg/kg, on day 1. Subsequent patients received rhTPO 0.6 or 0.3 μg/kg on days −3, −1, and 1, or 0.6 μg/kg on days −1 and 1. G-CSF, 5 μg/kg IV or subcutaneously (SC) twice daily, was started on day 3 and continued through aphereses. Twenty comparable, concurrently and identically treated patients (who were eligible and would have been treated on protocol but for the lack of study opening) mobilized with G-CSF alone served as comparisons. CD34+ cell yields were substantially higher with the first apheresis following rhTPO and G-CSF versus G-CSF alone: 4.1 × 106/kg (range, 1.3 to 17.6) versus 0.8 × 106/ kg (range, 0.3 to 4.2), P = .0003. The targeted minimum yield of 3 × 106CD34+ cells/kg was procured following a single apheresis procedure in 61% of the rhTPO and G-CSF–mobilized group versus 10% of G-CSF–mobilized patients (P = .001). In rhTPO and G-CSF mobilized patients, granulocyte (day 8 v 9, P= .0001) and platelet recovery (day 9 v 10, P= .07) were accelerated, and fewer erythrocyte (3 v 4,P = .02) and platelet (4 v 5, P = .02) transfusions were needed compared with G-CSF–mobilized patients. Peripheral blood platelet counts, following rhTPO and G-CSF, were increased by greater than 100% and the platelet content of PBPC products by 60% to 110% on the first and second days of aphereses (P < .0001) with the greatest effect seen with repeated dosing of rhTPO at 0.6 μg/kg. rhTPO is safe and well tolerated as a mobilizing agent before PBPC collection. Mobilization with rhTPO and G-CSF, in comparison to a comparable, nonrandomized G-CSF–mobilized group of patients, decreases the number of apheresis procedures required, may accelerate hematopoietic recovery, and may reduce the number of transfusions required following high-dose chemotherapy for breast cancer.
Misdiagnosis of primary immune thrombocytopenia and frequency of bleeding: lessons from the McMaster ITP Registry
Authors: Arnold Donald M. , Nazy Ishac , Clare Rumi , Jaffer Anushka M. , Aubie Brandon , Li Na , Kelton John G.
Published: October 2019

Key Points

One in 7 patients suspected of having primary ITP was misdiagnosed at some point during their disease course; 56.1% had grade 2 bleeding. The McMaster ITP Registry is a useful tool to improve the diagnosis of ITP and identify unique subgroups of patients.
A cross-sectional analysis of cardiovascular disease in the hemophilia population
Authors: Sood Suman L. , Cheng Dunlei , Ragni Margaret , Kessler Craig M. , Quon Doris , Shapiro Amy D. , Key Nigel S. , Manco-Johnson Marilyn J. , Cuker Adam , Kempton Christine , Wang Tzu-Fei , Eyster M. Elaine , Kuriakose Philip , von Drygalski Annette , Gill Joan Cox , Wheeler Allison , Kouides Peter , Escobar Miguel A. , Leissinger Cindy , Galdzicka Sarah , Corson Marshall , Watson Crystal , Konkle Barbara A.
Published: October 2019

Key Points

CV risk factors are common in older men with hemophilia. Although older men with hemophilia have less CV disease than comparable unaffected men, CV events do occur and require treatment.
Trajectory and mortality of preserved ratio impaired spirometry: the Rotterdam Study
Authors: Wijnant Sara Renata Alex , De Roos Emmely , Kavousi Maryam , Stricker Bruno Hugo , Terzikhan Natalie , Lahousse Lies , Brusselle Guy G.
Published: October 2019

Preserved ratio impaired spirometry (PRISm) is a heterogeneous condition but its course and disease progression remain to be elucidated. We aimed to examine its prevalence, trajectories and prognosis in the general population.

In the Rotterdam Study (population-based prospective cohort) we examined prevalence, trajectories and prognosis of subjects with normal spirometry (controls; forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC) ≥0.7, FEV1  ≥80%), PRISm (FEV1/FVC ≥0.7, FEV1 <80%) and chronic obstructive pulmonary disease (COPD) (FEV1/FVC <0.7) at two study visits. Hazard ratios with 95% confidence intervals for mortality (until December 30, 2018) were adjusted for age, sex, body mass index, current smoking and pack-years.

Of 5487 subjects (age 69.1±8.9 years; 7.1% PRISm), 1603 were re-examined after 4.5 years. Of the re-examined PRISm subjects, 15.7% transitioned to normal spirometry and 49.4% to COPD. Median lung function decline was highest in subjects with incident PRISm (FEV1−92.8 mL·year−1, interquartile range (IQR) −131.9– −65.8 mL·year−1; FVC −93.3 mL·year−1, IQR −159.8– −49.1 mL·year−1), but similar in persistent PRISm (FEV1−30.2 mL·year−1, IQR −67.9– −7.5 mL·year−1; FVC −20.1 mL·year−1, IQR −47.7–21.7 mL·year−1) and persistent controls (FEV1−39.6 mL·year−1, IQR −64.3–−12.7 mL·year−1; FVC −20.0 mL·year−1, IQR −55.4–18.8 mL·year−1). Of 5459 subjects with informed consent for follow-up, 692 (12.7%) died during 9.3 years (maximum) follow-up: 10.3% of controls, 18.7% of PRISm subjects and 20.8% of COPD subjects. Relative to controls, subjects with PRISm and COPD Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2–4 had increased all-cause mortality (PRISm: HR 1.6, 95% CI 1.2–2.0; COPD GOLD 2–4: HR 1.7, 95% CI 1.4–2.1) and cardiovascular mortality (PRISm: HR 2.8, 95% CI 1.5–5.1; COPD 2–4: HR 2.1, 95% CI 1.2–3.6). Mortality within <1 year was highest in PRISm, with patients often having cardiovascular comorbidities (heart failure or coronary heart disease; 70.0%).

PRISm is associated with increased mortality and this population encompasses at least three distinct subsets: one that develops COPD during follow-up, a second with high cardiovascular burden and early mortality, and a third with persistent PRISm and normal age-related lung function decline.
Direct and indirect costs of COPD progression and its comorbidities in a structured disease management program: results from the LQ-DMP study
Authors: Kirsch Florian , Schramm Anja , Schwarzkopf Larissa , Lutter Johanna I. , Szentes Boglárka , Huber Manuel , Leidl Reiner
Published: October 2019

Background

Evidence on the economic impact of chronic obstructive pulmonary disease (COPD) for third-party payers and society based on large real world datasets are still scarce. Therefore, the aim of this study was to estimate the economic impact of COPD severity and its comorbidities, stratified by GOLD grade, on direct and indirect costs for an unselected population enrolled in the structured German Disease Management Program (DMP) for COPD.

Methods

All individuals enrolled in the DMP COPD were included in the analysis. Patients were only excluded if they were not insured or not enrolled in the DMP COPD the complete year before the last DMP documentation (at physician visit), had a missing forced expiratory volume in 1 s (FEV1) measurement or other missing values in covariates. The final dataset included 39,307 patients in GOLD grade 1 to 4. We used multiple generalized linear models to analyze the association of COPD severity with direct and indirect costs, while adjusting for sex, age, income, smoking status, body mass index, and comorbidities.

Results

More severe COPD was significantly associated with higher healthcare utilization, work absence, and premature retirement. Adjusted annual costs for GOLD grade 1 to 4 amounted to €3809 [€3691–€3935], €4284 [€4176–€4394], €5548 [€5328–€5774], and €8309 [€7583-9065] for direct costs, and €11,784 [€11,257–€12,318], €12,985 [€12,531-13,443], €15,805 [€15,034–€16,584], and €19,402 [€17,853–€21,017] for indirect costs. Comorbidities had significant additional effects on direct and indirect costs with factors ranging from 1.19 (arthritis) to 1.51 (myocardial infarction) in direct and from 1.16 (myocardial infarction) to 1.27 (cancer) in indirect costs.

Conclusion

The findings indicate that more severe GOLD grades in an unselected COPD population enrolled in a structured DMP are associated with tremendous additional direct and indirect costs, with comorbidities significantly increase costs. In direct cost category hospitalization and in indirect cost category premature retirement were the main cost driver. From a societal perspective prevention and interventions focusing on disease control, and slowing down disease progression and strengthening the ability to work would be beneficial in order to realize cost savings in COPD.
Efficient expression vectors and host strain for the production of recombinant proteins by Yarrowia lipolytica in process conditions
Authors: Park Young-Kyoung , Vandermies Marie , Soudier Paul , Telek Samuel , Thomas Stéphane , Nicaud Jean-Marc , Fickers Patrick
Published: October 2019

Background

The oleaginous yeast Yarrowia lipolytica is increasingly used as an alternative cell factory for the production of recombinant proteins. Recently, regulated promoters from genes EYK1 and EYD1, encoding an erythrulose kinase and an erythritol dehydrogenase, respectively, have been identified and characterized in this yeast. Hybrid promoters up-regulated by polyols such as erythritol and erythrulose have been developed based on tandem copies of upstream activating sequences from EYK1 (UAS1EYK1) and XPR2 (encoding extracellular protease, UAS1XPR2) promoters.

Results

The strength of native (pEYD1) and engineered promoters (pEYK1-3AB and pHU8EYK) was compared using the extracellular lipase CalB from Candida antarctica as a model protein and a novel dedicated host strain. This latter is engineered in polyol metabolism and allows targeted chromosomal integration. In process conditions, engineered promoters pEYK1-3AB and pHU8EYK yielded 2.8 and 2.5-fold higher protein productivity, respectively, as compared to the reference pTEF promoter. We also demonstrated the possibility of multicopy integration in the newly developed host strain. In batch bioreactor, the CalB multi-copy strain RIY406 led to a 1.6 fold increased lipase productivity (45,125 U mL−1) within 24 h as compared to the mono-copy strain.

Conclusions

The expression system described herein appears promising for recombinant extracellular protein production in Y. lipolytica.