bluuebunny/crossref_metadata_2025 huggingface dataset:
Nanoparticle-Encapsulated Allogeneic T Cells Mitigate Graft-Versus-Host Disease but Retain Graft-Versus-Leukemia Activity
Authors: Zhang Lingling , Zhao Shuting , Devine Steven M. , He Xiaoming , Yu Jianhua
Published: October 2019

Allogeneic hematopoietic stem cell transplantation (HSCT) has curative potential for hematological malignancies, but is often associated with life-threatening complications including graft-versus-host disease (GVHD). The graft-versus-leukemia (GVL) activity which accompanies HSCT is responsible for eradication of tumor cells and prevention of relapse. GVHD and GVL are usually associated with each other and the separation of the two activities occurs in limited circumstances. In this study, we aimed to mitigate GVHD but retain GVL through transplantation of allogeneic T cells encapsulated with bio-degradable nanoparticle materials.

For the above purpose, donor T cells were encapsulated with chitosan and alginate through layer-by-layer coating using electrostatic deposition. Encapsulated donor T cells were characterized in vitro, and their ability to inhibit GVHD and retain GVL was determined in vivo after being transplanted together with non-encapsulated donor bone marrow (BM) cells in a C57BL/6 → BALB/c HSCT mouse model.

We found 85.7% of donor T cells were successfully encapsulated by the above method (Fig 1A). In vitro studies showed that the encapsulation did not change the phenotype of T cells as defined through the following parameters: size, viability, proliferation, antibody binding, cytokine secretion, and cytotoxicity of T cells (Fig. 1B and data not shown). Mice transplanted with encapsulated allogeneic T cells exhibited less severe acute GVHD and prolonged survival (Fig. 1 C-E). The mice showed a lower GVHD score, less liver damage, a smaller CD8/CD4 T cell ratio, and a higher number of donor BM-derived cells following transplantation with encapsulated donor T cells (Fig. 1 C-E and data not shown). When this GVHD model was combined with implantation of A20 lymphoma cells, GVL of encapsulated T cells was not compromised, while GVHD was still suppressed and the mouse survival also prolonged (Figure 2).

In summary, nanoencapsulation of T cells with bio-degradable materials attenuated the severity of GVHD but retained GVL, presenting a novel and potentially safer and effective approach of allogeneic HSCT for future clinical application.

Figure 1 Figure 1. Figure 2 Figure 2.

Disclosures

No relevant conflicts of interest to declare.
T-Cell Therapy for Multiple Myeloma Using NY-ESO-1+ T-Cell Antigen Presenting Cells (T-APC) Combined with Adoptive Cellular Transfer (ACT) to Augment Immunotherapy
Authors: Patel Krina K. , Olivares Simon , Hurton Lenka V. , Huls Helen , Qazilbash Muzaffar H. , Champlin Richard E. , Cooper Laurence JN
Published: October 2019

T cells have been previously genetically manipulated to act as a cellular vaccine to present viral antigens such as MP1 from influenza A. We have now genetically modified T cells as antigen presenting cells (T-APC) to express the tumor-associated antigen (TAA) NY-ESO-1 (Figure 1). This TAA is reportedly found in the majority of patients with high risk multiple myeloma, as well as other malignancies, but expression is absent from normal tissues. Therefore, immunologic targeting of NY-ESO-1 may be a potential treatment strategy for myeloma that is resistant to conventional therapies. One approach to improve therapeutic outcome is by adoptive transfer of T cells rendered specific for a TAA preferentially expressed on tumor cells. T-cell specificity can be redirected to intracellular TAAs by enforced expression of a characterized T-cell receptor (TCR) or chimeric antigen receptor (CAR) that recognizes processed antigen in the context of restricting human leukocyte antigen (HLA). However, persistence of these infused genetically modified cells, which is directly related to a favorable clinical response, may be variable. Therefore, T-APC may have a dual function of increasing persistence of adoptively transferred CAR+ and/or TCR+ T cells as well as inducing long term immunity through direct and cross priming of endogenous immune cells. To enhance these presentation functions, a new co-stimulatory molecule that tethers IL-15 to the cell surface (membrane-bound IL-15, mIL15) was expressed on NY-ESO-1+T-APC using the Sleeping Beauty (SB) gene transfer system. NY-ESO-1-specific HLA A2+ T cells expressing TCR and CAR could be selectively propagated ex vivo on autologous mIL15+NY-ESO-1+T-APC. The T-APC were also able to selectively propagate NY-ESO-1-specific T cells from autologous peripheral blood which can be described by binding of specific pentamer (Figure 2). Our data demonstrate that it is feasible to generate T-APC that can activate T cells engineered to have a defined specificity for NY-ESO-1 as well as grow out NY-ESO-1-specific T cells. The SB system is in place for genetic modification of clinical-grade T cells to express CAR, TCR, NY-ESO-1, and mIL15. Thus, we are proceeding to a human trial to infuse T-APC with and without genetically modified T cells in patients with NY-ESO-1+ tumors such as myeloma.

Figure 1 Figure 1.

Figure 2 Figure 2.

Disclosures

Cooper: InCellerate: Equity Ownership; Sangamo: Patents & Royalties; Targazyme: Consultancy; GE Healthcare: Consultancy; Ferring Pharmaceuticals: Consultancy; Fate Therapeutics: Consultancy; Janssen Pharma: Consultancy; BMS: Consultancy; Miltenyi: Honoraria.
Relationship between Gene Polymorphism of Recipient/ Donor Immune Pathway and Invasive Fungal Infection after Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Shi Jimin , Xiao Haowen , Luo Yi , Tan Yamin , He Jingsong , Xie Wanzhuo , Ye Xiujin , Cai Zhen , Lin Maofang , Huang He
Published: October 2019

Invasive fungal infections are a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation(allo-HSCT). During the past 2 decades, changes in transplantation practices and strategies to diagnose and treat IFI have likely impacted the epidemiology of IFI. Polymorphism of genes, which take part in the allogeneic immune response after allo-HSCT, lead to allograft immunoreactivity differences between individuals. It also may affect IFI after transplantation.

Methods: We analyzed allo-HSCT recipients at our center during the period January 2001 through March 2009. We analysis gene polymorphism of innate immune gene TLRs among 240 pairs of specimens of recipients and donors, in order to find out IFI individual difference and high risk genotype of IFI after transplantation.

Results: There were 99 patients (41.2%) occured IFI after allo-HSCT. Hematological malignant diseases, serious acute GVHD, and extensive chronic GVHD were risk factor for IFI after allo-HSCT. Univariate analysis of the relationship between risk factors of IFI after all0-HSCT and the TLRs gene polymorphism, we found that in the 10 SNP loci in 5 TLRs , two polymorphisms of TLR8 gene, (+1 A/G, rs3764880; +354 C/T, rs2159377) effect the incidence of IFI after allo-HSCT. Other gene polymorphism of TLRs (TLR1, TLR2, TLR3, TLR9) had no significant effects on the risk of IFI after allo-HSCT.

Conclusion: The study of the relationship between the gene polymorphism of natural immune molecule TLRs and the risk factors after allo-HSCT showed that TLR8 genotype of donor stem cell significantly influenced the incidence of IFI after allo-HSCT at the first time. It provide the evidence to establishment a fungal infection index system, which based on risk stratification of genetic background before transplantation of donors and recipients stem cell, and provide basis for selection of unrelated donor and prevention and treatment of IFI

Disclosures

No relevant conflicts of interest to declare.
Research on the Key T Cells and Cytokines of aGVHD after Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Tan Yamin , Luo Yi , Zhao Yanmin , Liao Weichao , Fu Huarui , Shi Jimin , Cai Zhen , Huang He
Published: October 2019

Objective: Acute graft versus host disease(aGVHD) is due to one of the most important causes of treatment related mortality after allo-HSCT. T cells play the key roles in the development of aGVHD after allo-HSCT, and the different subsets play different roles. Research on the reconstruction of T cell immune function and its cytokines in the occurrence of aGVHD has become one of the hottest topic in aGVHD research. We investigated the key T lymphocyte subsets and its cytokines, to explore the relationship between the characteristics of T cell subsets and the process of aGVHD. We desire to find some biomarkers and establish early diagnosis index for aGVHD, furthermore find an important target for immunotherapy. Ultimately the strategy could reduce aGVHD related mortality and improve long-term disease-free survival in transplantation patients.

Methods: 64 patients were included in the study, from May 2013 to January 2014. Blood samples of these patients were monitoring every week. Treg, Th1 and Th17 cells number were detected by flow cytometry. Serum levels of IL-2, IL-6, IL-10, IL-17, TNF-a and IFN-γwere detected by flow cytometry (CBA). ELISA technique was used for the detection of serum TGF- β level.

Results: 36 patients (56.3%) suffered from II-IV aGVHD, and 10 cases (15.6%) among them was diagnosed of III°-IV° aGVHD. Before the onset of aGVHD, Treg, Th1, Th17 cells levels had no statistical difference between aGVHD patients and non aGVHD patients. In aGVHD patient, serum levels of IL-2 (P=0.0417) and TGF- β (P=0.0168) elevated in the early stage of aGVHD. IL-2 (P=0.0231), IL-10 (P=0.0089),TGF-β (P=0.0138) serum levels after aGVHD was significantly higher than that in non aGVHD patients, and Treg cellsnumber was significantly lower than that of patients without aGVHD (P=0.0150). Using ROC and logistic regression analysis, we found a composite biomarker panel (IL-2, IL-10 and Treg cell, which could optimally discriminated patients with and without aGVHD, the formula is: 1.656+0.103×IL-2|0.052 ×IL-10|0.037×Treg. ROC curve of composite panel is 0.89 (95% CI, 0.78-0.99, P=0.0002), which is larger than that of any single index.

Conclusion: Increased IL-2, IL-10, TGF-β serum levels in early stage after allo-HSCT can help to establish the clinical diagnosis of aGVHD, and which is expected to provide a clinical basis for further target immunotherapy. Treg cell level was negatively related to the onset of aGVHD. Furthermore, we found a composite biomarker panel (IL-2, IL-10 and Treg cell), which could optimally discriminated patients with and without aGVHD. It could provide a reliable basis for early diagnosis of aGVHD.

Disclosures

No relevant conflicts of interest to declare.
Donor Cell Leukemia: A Prospective Study of Its Identification and Treatment
Authors: Ruiz-Delgado Guillermo J , Gonzalez-Ramirez Monica P. , Martagon-Herrera Nora A , Garces-Eisele Javier , Ruiz-Arguelles Alejandro , Gonzalez-Cortes Angelica , Ruiz-Arguelles Guillermo J.
Published: October 2019

Leukemia relapses occurring in donor cells, so called donor cell leukemias (DCL) after allogeneic hematopoietic stem cell transplantation have been reported in several cases and still are considered as rare diseases. Cytogenetic analysis, flow cytometry and molecular testing have been used to confirm this event in the cases so far reported. The etiology of DCL is unclear, and the reported literature does not suggest a common mechanism. The incidence of this condition is largely unknown, as well as the results of its treatment. We have prospectively searched for DCL in a 12-year period, in a single institution. In a group of 106 consecutive patients allografted because of leukemia; we have identified 7 cases of DCL; six of them were allografted because of relapsed acute lymphoblastic leukemia (ALL) and one because of paroxysmal nocturnal hemoglobinuria / aplastic anemia; these figures suggest that the real incidence of DCL has been underestimated in previous studies. All the patients were allografted from HLA-identical siblings, employing a reduced-intensity conditioning regimen. The cases of DCL appeared one to 40 months (median 10) after the allograft; the number of blast cells when the leukemic activity ensued was above 50% in all cases, whereas the chimerism studies revealed more than 90% cells of donor origin. The origin of the leukemia cells was shown by microsatellites in all cases and in three with a sex mismatch it was confirmed by the enumeration of XX / XY cells in the leukemic cells. The six patients with lymphoblastic DCL were treated prospectively with a pediatric-inspired combined chemotherapy schedule designed for “de novo” ALL patients. A complete response was obtained in 3/6 patients with lymphoblastic DCL these patients being alive in a complete remission at 11, 12 and 98 months after the diagnosis of DCL. The patient with hairy cell DCL has had a benign course, not needing any treatment. In the whole group the median survival (SV) has not been reached, the overall SV being 57% at 98 months. The long-term DCL survivors remain full chimeras and did not need a second transplant. It is concluded that the prevalence of DCL may be higher if it is prospectively looked for, and that acceptable therapeutic results are obtained if patients are treated as “de novo” leukemias employing combined chemotherapy.

Disclosures

No relevant conflicts of interest to declare.
Chemotherapy Versus Hypomethylating Agents for the Treatment of Relapsed Acute Myeloid Leukemia and Myelodysplastic Syndrome Following Allogeneic Stem Cell Transplant: A Retrospective Review
Authors: Motabi Ibraheem H , Liu Jingxia , Abboud Camille N. , Cashen Amanda F. , Stockler-Goldstein Keith E. , Uy Geoffrey L. , Vij Ravi , Westervelt Peter , DiPersio John F.
Published: October 2019

Allogeneic stem cell transplant (allo-SCT) is a potentially curative option for high risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Disease relapse after transplant is the major cause of treatment failure and is associated with a poor outcome. Intensive chemotherapy followed by donor lymphocytes infusion (DLI) or a second SCT may result in a durable response in some patients, but is associated with increased toxicity. More recently, a less aggressive therapy with hypomethylating agents (HA) has been reported to have activity in treatment of post-SCT relapse. We compared the treatment outcomes of intensive chemotherapy with that of HA. The primary end points were overall response rate (ORR) and overall survival (OS). Secondary endpoints were complete remission (CR) rate and progression free survival (PFS). A total of 100 patients with AML in morphological evidence of relapse were included: 73 patients received chemotherapy and 27 patients received a HA. Fifty-six percent of patients in the chemotherapy group and 33% of patients in the HA group received at least one DLI after treatment. Treatment with chemotherapy resulted in a higher ORR (51% vs. 18.5%, p = 0.004) and a higher CR rate (40% vs. 7%, p= 0.002). The median OS (6 months vs. 4 months, p = 0.024) and PFS (8 months vs. 2.3 months, p = 0.053) were longer in chemotherapy group. The overall survival (OS) at one year was 32% in the chemotherapy group compared with 4% in the HA group (p = 0.024). Similar benefit of chemotherapy over HA was maintained in all treatment outcomes after controlling for the use of DLI. The use of chemotherapy followed by DLI may offer the greatest benefit ( ORR of 68%; 1-year OS of 44%; and median OS of 18 months). Conclusion: our results suggest that intensive chemotherapy may result in a better response rate and a better overall survival compared with hypomethylating therapy in patients with morphological relapse of AML after SCT. We propose prospective studies before the use of hypomethylating agents in this setting for medically fit patients.

Disclosures

Off Label Use: Rituximab is a genetically engineered monoclonal antibody that targets a specific protein, known as CD20 found on the surface of normal and malignant B-lymphocytes.Use of Rituximab treatment for other than FDA approved indications include a rare condition of acquired factor VIII inhibitors in individuals without inherited hemophilia as an autoimmune phenomenon that may lead to life-threatening bleeding. Due to the low incidence rate of acquired inhibitors, published data consists of only case reports, and reviews.. Vij:celgene: Honoraria, Research Funding.
Cpeb4-Mediated Translational Regulatory Circuitry Controls Terminal Erythroid Differentiation
Authors: Hu Wenqian , Yuan Bingbing , Lodish Harvey F.
Published: October 2019

While we have considerable understanding of the transcriptional networks controlling mammalian cell differentiation, our knowledge of post-transcriptional regulatory events is very limited. Using differentiation of primary erythroid cells as a model, we show that the sequence-specific mRNA-binding protein Cpeb4 is induced by the erythroid important transcription factors Gata1 and Tal1, is strongly upregulated during terminal erythroid development, and is essential for terminal erythropoiesis. By binding directly to the translation initiation factor eIF3 complex, Cpeb4 represses the translation of a large set of mRNAs, most of which are normally downregulated during terminal erythroid development. Cpeb4 also binds to its own mRNA to repress its translation, and ectopic expression of Cpeb4 blocks erythroid differentiation. Thus transcriptional induction and translational repression combine to form a negative feedback loop to control Cpeb4 protein levels within a specific range that is required for terminal erythropoiesis. Our study provides an example of how translational control is integrated with transcriptional regulation to precisely control gene expression during mammalian cell differentiation.

Disclosures

No relevant conflicts of interest to declare.
Dietary Supplementation with Ipriflavone Decreases Liver Iron Loading in Wild Type Mice
Authors: Patchen Bonnie , Gaun Vera , Cheng Aaron , Fraenkel Paula G.
Published: October 2019

Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release by causing internalization and degradation of the iron exporter ferroportin. Because its levels are inappropriately low in patients with iron overload syndromes, Hepcidin is a potential drug target. We previously conducted a chemical screen in human hepatocytes (HepG2 cells) to identify small molecules that upregulate Hepcidin transcript levels. One of the small molecules that we identified was ipriflavone, a synthetic isoflavone, which has been used to treat osteoporosis in postmenopausal women. To evaluate ipriflavone’s effect on iron homeostasis in a mammalian model, we placed groups of 5-week old C57BL/6 male mice on a soy-free, iron-sufficient diet, AIN-93G containing 220 mg/kg iron and 0, 250, 500 or 750 mg ipriflavone per kg of food (n=4 or 5 per group) for 50 days, then sacrificed the animals for analysis of changes in gene expression by quantitative realtime RT-PCR, liver and spleen iron content, blood indices, and intestinal ferroportin expression. While producing less than a two-fold increase in liver hepcidin transcript levels, ipriflavone supplementation was associated with a significant decrease in liver iron content (mean±SE): 39.22±2.06 µg iron/g tissue, p=0.0033, and 44.10±2.58 µg iron/g tissue, p=0.0124, at 500 and 750 mg ipriflavone per kg of food, respectively, vs 57.77±3.39 µg iron/g tissue in mice that did not receive ipriflavone. Ferroportin expression detected in intestinal epithelial cells by immunohistochemistry was notably decreased in mice receiving ipriflavone: 66.6% and 80% of the animals receiving 500 and 750 mg/kg groups, respectively, exhibited decreased ferroportin staining versus 40% of the group not receiving ipriflavone. There was no significant difference in hemoglobin, hematocrit, or spleen iron among the groups. In conclusion, we have shown that orally administered ipriflavone is effective in decreasing liver iron content and intestinal ferroportin expression in vivo. Future experiments will evaluate ipriflavone’s effects on iron homeostasis and hematopoiesis in genetic models of iron overload disorders.

Disclosures

No relevant conflicts of interest to declare.
Retrospective Evaluation of Patients Referred for Hemochromatosis Genetic Testing
Authors: Lanktree Matthew B. , Lanktree Bruce B. , Waye John S. , Pare Guillaume , Sadikovic Bekim , Crowther Mark A.
Published: October 2019

Background: The common genetic test for hemochromatosis involves the genotyping of two polymorphisms in the HFE gene (C282Y and H63D). Current guidelines suggest testing of patients with ferritin greater than 300 µg/L and transferrin saturation greater than 45%. Testing of first degree relatives of confirmed HFE positive cases is also endorsed. However, the current test has poor sensitivity and specificity for iron overload causing end organ damage. We sought to evaluate biochemical parameters of patients referred for HFEtesting and identify those who received phlebotomy.

Methods: After receiving ethics approval, electronic charts of patients referred to Hamilton Health Sciences, Hamilton, Ontario, Canada for HFE genetic testing between January 1, 2012 and December 31, 2012 were reviewed.

Results: HFE genetic testing in 642 patients yielded 100 (15.6%) positive C282Y homozygote or C282Y/H63D compound heterozygote results. In patients with biochemical markers of iron overload available (n=160), patients with a risk HFE genotype had significantly higher iron saturation, serum iron, and hemoglobin (P<0.001), but did not have higher ferritin or liver enzymes. Only fifty percent of patients referred had biochemical evidence of iron overload (transferrin saturation [TS] > 45% and ferritin > 300). Of patients who required phlebotomy, only 27 of 40 (67.5%) were HFEpositive.

Conclusion: Many factors affect the decision to phlebotomize a patient with suspected iron overload. However, it appears that most of the current HFE genetic testing being performed did not alter patient management.

Disclosures

Crowther: Asahi Kasai: Membership on an entity's Board of Directors or advisory committees; Bayer: Speakers Bureau; Celgene: Speakers Bureau; Shire: Speakers Bureau; Boehriniger Ingelheim: Consultancy; CSL Behring: Speakers Bureau; Leo Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Portola: Membership on an entity's Board of Directors or advisory committees; Viropharma: Membership on an entity's Board of Directors or advisory committees.
Clinical and Laboratory Characterization of the c.*233G>C Beta Globin Gene Substitution: Evidence Against a Thalassemic Phenotype
Authors: Smith Debra , Park Jason , Mitui Midori , Luu Hung , Timmons Charles
Published: October 2019

Purpose:

The c.*233G>C nucleotide substitution in the HBB gene was recently proposed as a novel β thalassemia allele based on genetic analysis of 11 thalassemic individuals. Based on this single report, the c.*233G>C allele has been provisionally included as a β (+) thal allele in a commonly used β globin database (HbVar).

The c.*233G>C is frequently seen in HBB gene sequencing for hemoglobinopathies. A clinical review was performed to determine the phenotype and hematologic characteristics of the c.*233G>C nucleotide substitution.

Methods:

At a tertiary-care pediatric institution, a retrospective review was performed of all HBB gene sequencing tests over a 30-month period. A chart review was performed for all cases with the c.*233G>C variant.

Results:

Of a total of 104 HBB cases, the c.*233G>C variant was identified in 18 individuals; 17 were heterozygotes and 1 was a homozygote (Patient 17) (Table 1). HBB gene sequencing was performed as part of the work-up for suspected variant hemoglobin (Hb) but can detect non-coding alterations including β thalassemia alleles as well.

Red cell indices in our series (Table 1) do not demonstrate the characteristics of heterozygous β thalassemia (microcytosis, mildly elevated RBC count, and mild anemia). MCV and RBC count data available for 9 individuals showed MCV values in the normocytic or macrocytic range and RBC counts within the normal range for all 9 patients. Of 13 patients with recorded Hb values, only one patient (Patient 14) was anemic but was found to have iron deficiency.

The patients in our series did not have elevated Hb A2, which in patients with β thalassemia is typically above the normal range (>4%); the 9 patients who could be evaluated for Hb A2 had values ranging from 2.4% to 3.7%. The remaining individuals were either too young to be evaluated for Hb A2 or possessed a variant Hb that co-eluted with HbA2.

The 13 patients in our series who were heterozygous for a Hb variant showed no excess in the variant Hb relative to Hb A. In patients with compound heterozygosity with a β thalassemia allele, the amount of the variant hemoglobin typically exceeds the amount of Hb A.

Conclusions:

Red cell indices and Hb proportions in this series of 18 individuals with c.*233G>C substitution are not consistent with a thalassemic phenotype. Our analysis supports a hypothesis that c.*233G>C substitution is not a β (+) thalassemia allele but is rather a fairly common benign polymorphism. This information has important implications in the diagnosis and genetic counseling of individuals with the c.*233G>C substitution.

References:

1. Sirdah et al. The spectrum of β-thalassemia mutations in Gaza Strip, Palestine. Blood Cells, Molecules and Diseases. 2013; 50: 247-251.

2. HbVar: A Database of Human Hemoglobin Variants and Thalassemias. http://globin.cse.psu.edu/hbvar/menu.html.

Disclosures

No relevant conflicts of interest to declare.
Regulation of Na+/Mg2+ Exchange in Sickle Erythrocytes By Endothelin-1
Authors: Rivera Pablo A. , Inostroza Yaritza , Romero Jose R. , Rivera Alicia
Published: October 2019

Excess levels of endothelin-1 (ET-1), erythrocyte sickling and chronic inflammation have been proposed as important contributors to the pathophysiology of sickle cell disease (SCD). We have shown that ET-1 receptor antagonists improve hematological parameters by reducing Gardos channel activity in two transgenic mouse models of SCD while reducing oxidant stress by decreasing circulating levels of protein disulfide isomerase. Magnesium (Mg2+) deficiency, mediated in part via increased erythrocyte Na+/Mg2+ exchanger activity, has been demonstrated to contribute to erythrocyte dehydration, K+ loss and sickling in SCD. However, the relationship between ET-1 and the Na+/Mg2+ exchanger in SCD remains unclear. We measured Na+/Mg2+ exchange activity in ex vivo red cells and observed increased activity following in vitro incubation of human (2.2 ± 0.2 to 3.2 ± 0.1 mmol/1013 cell x h, P<0.03, n=5) and mouse red blood cells with ET-1 (P<0.001, n=5); events that were significantly blocked by pre-incubation of cells with 1 μM BQ788, a selective inhibitor of ET-1 type B receptors. In addition, in vitro deoxygenation of sickle red cells led to increased exchanger activity that was inhibited by impramine, a Na+/Mg2+ exchange inhibitor, and associated with reduced deoxygenation-stimulated sickle cell dehydration. These results suggest an important role for ET-1 and cellular magnesium homeostasis in sickle cell disease. To this end, we studied Na+/Mg2+ exchange activity in ex vivo erythrocytes from three transgenic sickle mouse models and observed increased activity in these cells when compared to red cells from either Hb A transgenic or C57BL/J6 wild-type mice (P<0.03, n=4). We then tested the in vivo effects of ET-1 receptor antagonists on erythrocyte Na+/Mg2+ exchange activity in the BERK mouse, a transgenic model of SCD. We blocked ET-1 receptors type A and B by in vivo treatment with BQ-788 and BQ-123 (360mg/Kg/Day) for 14 days and observed lower erythrocyte exchanger activity when compared to cells from vehicle treated BERK mice (P<0.02, n=6). Thus our results suggest that ET-1 receptor blockade represents an important therapeutic approach to control erythrocyte volume and magnesium homeostasis that may lead to improved inflammatory and vascular complications observed in SCD. Supported by NIH R01HL090632 to AR.

Disclosures

No relevant conflicts of interest to declare.
A Novel 2 Amino Acids Deletion in the Leucine Zipper Domain of C/EBPε Leads to Neutrophil-Specific Granule Deficiency (SGD)
Authors: Akagi Tadayuki , Wada Taizo , Muraoka Masahiro , Toma Tomoko , Kaji Kenzo , Agematsu Kazunaga , Koeffler H. Phillip , Yokota Takashi , Yachie Akihiro
Published: October 2019

Neutrophil-specific granule deficiency (SGD) is a rare congenital disorder. Neutrophils of SGD patients show abnormal bilobed nuclei and lack secondary and tertiary granule gene expression. Since neutrophils of SGD patients are deficient for bactericidal activity, the patients display immunodeficiency and suffer from frequent and severe infections. The transcription factor CCAAT/enhancer binding protein epsilon (C/EBPε) is one of the major regulators of granulopoiesis and is known to be the responsible gene for the disease. At least two homozygous germline mutations (5bp deletion and A-nucleotide insertion) of C/EBPε, which are functionally defective, have been reported.

Here, we report a novel in-frame deletion of the C/EBPεgene in a 55 years-old female patient with a life-long history of recalcitrant skin infections with ulcer and scar formation. Peripheral blood smear showed characteristic changes, including reduction of cytoplasmic granules and increased bilobed nuclei of the neutrophils, monocytosis, absence of eosinophils and increased basophils. Flow cytometric examination revealed significant reduction of CD16 and abnormal expression of CD14 on the neutrophil surface.

Sequencing of the C/EBPε gene revealed a homozygous deletion of 6 base-pairs (arginine and serine residues deletion; ΔRS) within the leucine zipper domain. Activation of luciferase driven by G-CSF receptor promoter was not induced by ΔRS; and endogenous expression of granule genes, including B9, NGAL and lactoferrin, was induced by wild-type C/EBPε but not by ΔRS in NIH3T3 cells, suggesting that transcriptional activity of ΔRS was diminished. GFP-tagged ΔRS, as well as wild-type C/EBPε, were localized to the nucleus in NIH3T3 cells, and DNA binding activity of ΔRS, which was determined by biotin-labeled DNA pulldown assay, was intact. Meanwhile, ΔRS together with Gata1 and PU.1 were not able to induce expression of eosinophil major basic protein (MBP) in NIH3T3 cells. Of note, wild-type C/EBPε associated with Gata1; however, ΔRS did not interact with Gata1, indicating that the ΔRS likely impairs protein-protein interaction of other transcription factors resulting in loss of transcriptional activation. These results further support the importance of the leucine zipper domain of C/EBPε for its essential function and indicate multiple molecular mechanisms leading to SGD.

Disclosures

No relevant conflicts of interest to declare.
Contribution of Red Blood Cells and Clot Structure to Thrombosis
Authors: Ariens Robert A S
Published: October 2019

The blood clot or thrombus is composed of red blood cells, platelets and white cells that interact with the fibrin meshwork produced by the coagulation system. The red blood cells form polyhedrocytes that seal the clot, platelets provide forces for clot contraction, and white cells contribute neutrophil extracellular traps, cytokines and complement activation. The fibrin network contributes clot elasticity and provides the proteinaceous backbone structure that stabilises the clot. Previous studies from our laboratory and others have shown that dense fibrin networks demonstrating small pores and increased resistance to fibrinolysis associate with thrombosis. However, the mechanisms underpinning this have not been fully understood. Recent studies using atomic force microscopy from our laboratory have shown that fibrinogen interacts with red cells with comparable affinity as that with platelets. A patient with mutations in the β3 integrin subunit showed no binding between red cells and fibrinogen, demonstrating that a β3-related integrin receptor is involved in the interaction. Mutations in the fibrinogen α-chain integrin binding sites (D97E and D574E) reduced frequency of red cell interactions with fibrinogen. Interestingly, a naturally occuring splice variant of the fibrinogen γ-chain that reduces binding to the platelets, fibrinogen γ', increased binding interactions between fibrinogen and red blood cells. Fibrinogen γ' is a naturally occurring splice variant of fibrinogen, in which the C-terminal AGDV residues of the more common γA-chain (85%) are replaced with a negatively charched VRPEHPAETEYDSLYPEDDL sequence of the γ' chain (15%). Fibrinogen γ' induced clustering of fibrin fibres into tightly interknit nuclei of fibrin fibres, interspersed by large pores that extend over more than 50 μm within the fibrin network structure. The effects of fibrinogen γ' on fibrin clot structure was independent of thrombin and FXIII as demonstrated using snake venom enzyme. Previously we showed impaired fibrin protofibril formation with fibrinogen γ' using atomic force microscopy. Using turbidimetric analysis of fibrin intrafibrillar structure, we show that fibrinogen γ' reduces protofibil packing per fibrin fiber. Furthermore, we find that reduced protofibril packing diminishes fibrin stiffness as analysed with magnetic tweezers both in purified systems as well as in plasma at (patho)physiological fibrinogen γ' levels that range from 3-40%, and in whole blood as analysed with thromboelastography. In conclusion, our data show that red blood cells and fibrinogen γ' play major roles in the regulation of clot structure and stability, and that these effects on clot structure are major determinants of the functional properties of the blood clot. Modulating fibrin clot structure and its interactions with blood cells may represent major new targets for the treatment of thrombosis.

Disclosures

No relevant conflicts of interest to declare.
Megaloblastic Anemia from Methophenobarbital
Authors: CALVERT R. J. , HURWORTH E. , MACBEAN A. L.
Published: October 2019

A 39-year-old spinster presented with a severe megaloblastic anemia after treatment of her epilepsy for eight years with the barbiturate, methophenobarbital, taken regularly and alone in therapeutic dose. This drug appears to have caused the anemia, which may have been conditioned by a folic-acid-poor diet. Hematologic remission followed the administration of folic acid, which she has since continued to take along with her previous dose of methophenobarbital. Disordered pigment metabolism, including methemalbuminemia, is reported and discussed.
Glucose-6-Phosphate Dehydrogenase Deficiency in Saudi Arabia: A Survey
Authors: GELPI A. P.
Published: October 2019

A survey of red cell G-6-PD deficiency has been conducted among 1296 Saudi subjects. Thirteen per cent of randomly selected male subjects and 2.4 per cent of the females tested were found to be G-6-PD deficient. In this study, the screening test employed did not detect female (partially G-6-PD deficient) heterozygotes. The deficiency appeared to be localized to the oasis of the Eastern province, and in village studies a very high incidence was found in male children from the Qatif and Al-Hasa oases. The geographic limits of G-6-PD deficiency correspond precisely to the areas known to be hyperendemic for malaria in previous years. However, the population group affected represents a distinct minority in terms of religious and cultural tradition, and anthropometric type. In Saudi Arabia, falciparum malaria does not appear to be the only significant factor determining marked regional differences in the incidence of G-6-PD deficiency: this genetic marker is essentially confined to the Shiite muslim population. The Sunni population, regardless of its proximity to areas of endemic falciparum malaria, has a low incidence of G-6-PD deficiency.
The Normal Development of the Lymphoid Mass in the Golden Hamster and its Relationship to the Effects of Thymectomy
Authors: ADNER MARVIN M. , SHERMAN JOSEPH D. , DAMESHEK WILLIAM
Published: October 2019

A study of lymphoid organ sections and imprints of normal hamsters from fetal to adult state revealed a sequential pattern of organ structure and growth. The thymus developed an adult structure by 1 week, maximum size at 6 weeks. The mesenteric lymph node had an adult structure by 4 weeks, the spleen by 6 weeks, with an accelerated growth of both organs between 6 to 8 weeks. The growth and development of the peripheral lymphoid organs appears to be under thymic control until the attainment of a "critical peripheral lymphoid mass." This appears capable of functioning independently of thymic control. Thymectomy is effective in producing lymphoid atrophy with resultant immunosuppression and wasting disease only if performed prior to the attainment of the "critical peripheral lymphoid mass."
Effects of Endotoxin and Nitrogen Mustard on Leukocyte Kinetics
Authors: HERION JOHN C. , WALKER RICHARD I. , HERRING WILLIAM B. , PALMER JEFFRESS G.
Published: October 2019

In rabbits given a single large dose of HN2 circulating granulocytes are maintained at a normal concentration until 50-52 hours; thereafter the onset of a rapid fall in concentration coincides with maximum marrow hypocellularity. Recovery from granulocytopenia, however, does not begin until after 90 hours when considerable recovery of marrow cellularity is evident.

Bacterial endotoxin in differing doses given at various times up to 24 hours after HN2 produces the same changes in circulating granulocytes as in normal animals. The rapid decline in circulating granulocytes, heralding exhaustion of the pool of postmitotic granulocytes, occurs earlier in HN2-treated animals given bacterial endotoxin; they also recover sooner than controls from HN2-induced granulocytopenia. Endotoxin given at 51 hours after HN2 when the postmitctic pool is exhausted, increases the rate of fall in the concentration of circulating granulocytes producing an earlier appearance of maximum granulocytopenia, but recovery by these animals from HN2-induced granulocytopenia does not occur sooner than in controls. Thus the time of recovery from HN2-induced granulocytopenia appears to be related to the time of depletion of marrow stores of postmitotic cells.

Rabbits made tolerant to endotoxin and given HN2 show an earlier onset of the rapid fall in circulating granulocytes and an earlier recovery from granulocytopenia. Maximum marrow hypocellularity appears sooner after HN2 in tolerant animals, but marrow cellularity is always greater than in nontolerant rabbits. The increased cellularity of the marrow of tolerant animals may represent an increase in the size of the mitotic compartment of cells since the postmitotic compartment seems smaller than in normal animals.

The granulocytopenia produced by endotoxin results from an increased removal of granulocytes from the circulation and significant numbers of these cells do not appear to recirculate. Granulocytosis following the injection of endotoxin results from movement of cells out of the marrow and occurs only if there is a store of postmitotic granulocytes. Release of cells from the marrow following this stimulus, however, appears to be related to cell maturity; only the more mature ones enter the circulation.

Movement of mature cells out of the marrow, depleting the reservoir of these elements, may provide the stimulus for the increased cell production that appears in normal animals given endotoxin.
The effect of chemotherapy on the kinetics and proliferative capacity of normal and tumorous tissues in vivo
Authors: Rosenoff SH , Bull JM , Young RC
Published: October 2019

The proliferative state of a given tissue is a major determinant of its sensitivity to both phase-specific and cycle-specific chemotherapeutic agents. To study the extent of injury induced by antitumor agents to normal and tumorous tissues, a technique for following DNA synthesis as reflected in the incorporation of tritiated thymidine (3H-TdR) into DNA was compared to the conventional radioautographic technique of the labeling index (LI) and to the functional kinetic technique of granulocyte colony formation in vitro. Alterations in DNA synthesis induced by a single dose of cyclophosphamide in normal and tumorous tissues in vivo paralleled in many respects the changes seen when the more time-consuming techniques of the LI or granulocyte colony formation were employed. However, the recovery of granulocyte colony formation after cyclophosphamide therapy laged behind the recovery of DNA synthesis in the bone marrow, obscuring a kinetic event of potential therapeutic significance. The determination of DNA synthesis simultaneously in normal and tumorous tissues in vivo was easy to perform and supplied therapeutically pertinent results comparatively quickly.
Effect of bleeding on hematopoiesis following irradiation and marrow transplantation
Authors: Boggs SS , Boggs DR
Published: October 2019

In previous studies, bleeding after irradiation did not affect the rate of regeneration of endogenous spleen colony-forming cells, but induced an early (4–6 days after irradiation) appearance of erythrocytic colonies which differentiated and disappeared by days 7–8. This “abortive” wave was associated with a similarly abortive wave of splenic 59Fe uptake. The present experiments were done to determine whether or not an abortive wave of erythropoiesis could be induced in the transplanted, exogenous stem cell system. Lethally irradiated mice were given normal bone marrow cells and one-half of the group were bled of about one-third their blood volume within 4 hr of irradiation. Groups were killed on days 3–10 after irradiation. Seventeen to twenty hours prior to killing, 59Fe was injected. Hematocrits, spleen weights, colony numbers, and per cent 59Fe uptake were determined. Hematocrits of bled mice averaged about 70% of those of cell-injected controls. Spleen weights, colony counts, and per cent 59Fe uptake per spleen began to increase about 1 day earlier in bled mice (days 4–5 as compared to days 5–6), and rates of increase were the same as those of controls. However, no abortive wave of erythropoiesis was detected. A large cell dose resulted in earlier increases in all parameters than a small dose. Thus, bleeding after injection of cells produced results similar to those obtained by increasing the cell dose. The inability of bleeding to induce an early abortive wave of erythropoiesis in transplanted as compared to endogenous colony-forming systems may reflect differences in the cell cycling characteristics of these systems.
Further studies of canine von Willebrand's disease
Authors: Dodds WJ
Published: October 2019

Additional characterization of von Willebrand's disease (VWD) in a family of German shepherd dogs is presented. Genetic studies of three generations of affected dogs indicate that about 50% of the progeny are affected if one parent has VWD and about 60% if both parents have the defect. Some of these progeny manifested an incomplete form of VWD, suggesting autosomal dominant inheritance with variable expressivity. The disease become progressively less severe with advancing age and repeated pregnancies. Ristocetin-induced platelet aggregation was significantly reduced in VWD dogs as compared with normal, thrombopathic, and hemophilic carrier dogs. Immunodiffusion and electroimmunodiffusion studies with rabbit anticanine factor VII showed the level of factor VII-related antigen to be low in VWD dogs but present in increased amounts in hemophilic dogs. VWD affected dogs had markedly delayed hemostatic plug formation, but their plugs appeared normal by light and electron microscopy. Their platelet nucleotides, ATP/ADP ration, and platelet protein content were normal. Platelet and fibrinogen survival times with [75Se] selenomethionine were also normal, although platelets from VWD dogs incorporated more radioactivity than did those from normal dogs or from dogs with incomplete VWD.
Observations on the mechanical precipitation of oxy Hb S and other mutants
Authors: Roth EF Jr , Elbaum D , Nagel RL
Published: October 2019

Oxyhemoglobin S exhibits greater mechanical instability than oxyhemoglobin A. The rate of precipitation of Hb S when agitated by vortexing depends upon the geometry of the tube, the volume of the hemoglobin solution, and the concentration of hemoglobin. The rate of precipitation is inversely related to concentration. Precipitation is inhibited by temperatures near 4 degrees C and alkylureas whose protective capacity is approximately proportional to the carbon chain length of the alkyl group. Blocking the beta93 -SH group with parahydroxymercuribenzoate has only a small enhancing effect on the precipitation rate. Other mutants such as Hb Gun Hill, Leiden, (both heat unstable), and C-HARLEM are also unstable. In the case of C- HARLEM, the precipitation rate is greater than that for Hb S. The heat- unstable mutants are not as well protected by cold temperatures or alkyl ureas. D2O has only a minor stabilizing effect on hemoglobin S, but NaCl and related salts markedly enhance precipitation at concentrations of 0.5 M. It is concluded that mechanical instability of oxyhemoglobins is a multifactorial process involving surface denaturation, pH, ionic strength, hydrophobic interactions, protein conformation, and primary protein structure. This phenomenon will require more extensive investigation.
Short communication: possible association of newly absorbed serotonin with nonmetabolic, granule-located adenine nucleotides in human blood platelets
Authors: Holmsen H , Setkowsky CA , Day HJ
Published: October 2019

[3H]-adenine-labeled human platelets in plasma were incubated with or without nonradioactive serotonin. Release reaction was then induced by ADP, epinephrine, collagen, or thrombin. Platelets that had been incubated with serotonin released four times as much serotonin as platelets incubated without serotonin. The specific radioactivities of the ATP and ADP released to plasma during release reaction induced with all four inducers were the same in both systems. This shows that when serotonin is taken up by human platelets, it enters the compartment containing nonmetabolic, granula-stored ATP, and not the compartment with metabolic extragranular ATP. These results suggest that the mechanism of serotonin storage in human platelets is similar to that in other species investigated, i.e., rabbit, guinea pig, and pig.
The effect of EDTA, cations, and various buffers on the morphology of erythrocyte membranes: an electron-microscopic study
Authors: Pinteric L , Manery JF , Chaudry IH , Madapallimattam G
Published: October 2019

Membranes of human erythrocytes were prepared by stepwise osmotic hemolysis in Ca2+-free solutions. Examination with the electron microscope after negative staining showed some short, conelike protuberances on the surface of about 20 percent of the ghosts, while 80 percent were round, intact spheres. After Ca2+ treatment, all membranes were round and intact. After exposure to ethylenediaminetetraacetic acid (EDTA) (1.0 mM, pH 7.4), the entire ghost surface was covered with long, thin extrusions called stromalytic forms (about 460 per cell). Their sizes, shapes, and fine structure are described. Exposure to ionic calcium (1.4 times 10-minus 4M) abolished the EDTA-induced stromalytic forms. A second exposure to EDTA reversed this Ca2+ effect. ATP, like EDTA, produced stromalytic forms. EDTA- induced stromalytic forms were also abolished by Zn2+, La3+, and Nd3+ at concentrations of 1–5 times 10-minus 4 M. Mg2+ at 10-minus 2 M was ineffective. Ghosts were prepared by graded lysis in various buffers. Those prepared in phosphate were the most stable and provided consistent EDTA effects and Ca2+ reversal. Ghosts in Tris-HCl showed breakdown unless salt was added. Moderately satisfactory ghosts were also obtained in Hepes-NaOH buffer and salt.