bluuebunny/crossref_metadata_2025 huggingface dataset:
CD49d Is The Strongest Flow Cytometry-Based Predictor Of Overall Survival In Chronic Lymphocytic Leukemia
Authors: Bulian Pietro , Shanafelt Tait D , Fegan Chris , Zucchetto Antonella , Cro Lilla , Nuckel Holger , Baldini Luca , Kurtova Antonina , Ferrajoli Alessandra , Burger Jan A. , Gaidano Gianluca , Del Poeta Giovanni , Pepper Chris , Rossi Davide , Gattei Valter
Published: October 2019

Purpose

Although CD49d is an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL), definitive validation evidence is lacking. A worldwide multi-center analysis was performed using published and unpublished CLL series to evaluate the impact of CD49d as overall survival (OS) and treatment free survival (TFS) predictor.

Patients and Methods

A training/validation strategy was chosen to find the optimal CD49d cut-off. The hazard ratio (HR) for death and treatment imposed by CD49d was estimated by pooled analysis of 2,972 CLL, and Cox analysis stratified by center and stage was used to adjust for confounding variables. The importance of CD49d over other flow cytometry-based prognosticators (CD38, ZAP-70) was ranked by recursive partitioning.

Results

Patients with ≥30% of neoplastic cells expressing CD49d were considered CD49d+. The decrease in OS at 5 and 10-years among CD49d+ cases was 7% and 23% (decrease in TFS 26% and 25% respectively). The pooled HR of CD49d for OS was 2.5 (2.3 for TFS) in univariate analysis. This HR remained significant and of similar magnitude (HR=2.0) in a Cox model adjusted for clinical and biological prognosticators. Hierarchical trees including all cases, or restricted to early stage or patients ≤65 years, always selected CD49d as the most important flow-cytometry-based biomarker, with negligible additional prognostic information added by CD38 or ZAP-70. Consistently, by bivariate analysis, CD49d reliably identified patients' subsets with poorer outcome independent of CD38 and ZAP-70.

Conclusions

In this analysis of ∼3000 patients, CD49d emerged as the strongest flow cytometry-based predictor of OS and TFS in CLL.

Disclosures:

Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding. Burger:Pharmacyclics: Research Funding.
First US Phase I Clinical Trial Of Globin Gene Transfer For The Treatment Of Beta-Thalassemia Major
Authors: Boulad Farid , Riviere Isabelle , Wang Xiuyan , Bartido Shirley , Prockop Susan E. , Barone Rita , Moi Paolo , Maggio Aurelio , Sadelain Michel
Published: October 2019

To date, the only curative therapeutic approach for beta-thalassemia major has been allogeneic stem cell transplantation (SCT) for patients with HLA-matched siblings. For the majority of patients who do not have a matched sibling, allogeneic SCT is associated with major risks of morbidity and mortality. The stable transfer of a functional globin gene into the patient’s own hematopoietic progenitor cells (HPCs) yields a perfectly matched graft that does not require immunosuppression to engraft. We previously demonstrated successful globin gene therapy in murine thalassemia models, using a lentiviral vector that encodes the human ß-globin promoter and arrayed regulatory elements uniquely combined to achieve high level and erythroid-specific globin expression. In vivo in thalassemic mice, the vector termed TNS9.3.55, increased hemoglobin levels by an average 4-6 g/dL per vector copy. We obtained in 2012 the first US Food and Drug Administration (FDA) approval to proceed to a clinical study in adult subjects with beta-thalassemia major (NCT01639690). We have to date enrolled 5 patients and recently treated the first three, administering the transduced HPCs after non-myeloablative conditioning. Engraftment data are available for the first two patients. Patient 3 was recently infused with CD34+ cells and is at this time too early to evaluate.

Patient 1 is a 23 year old female with a ß039 – IVS1,110 mutation. Patient 2 is an 18 year old female with a ß039 – IVS1,6 mutation. Both patients underwent mobilization of peripheral blood stem cells (PBSCs) with filgrastim and mobilized 25 x 10^6 and 9.9 x 10^6 CD34 cells/Kg respectively. CD34+ PBSCs were transduced with the lentiviral vector TNS9.3.55 encoding the normal human beta-globin gene. The average vector copy number (VCN) in bulk CD34+ cells for these two patients was respectively 0.39 and 0.21 copies per cell. Both patients underwent non-myeloablative cytoreduction with busulfan administered at 2 mg/Kg/dose Q12H x 4 doses (total 8 mg/Kg), followed by reinfusion of 11.8 x 10^6 and 8.4 x 10^6 CD34+ cells/Kg, respectively. Both patients tolerated cytoreduction well and recovered their blood counts. While they continue to be transfusion dependent, both patients show a gradual rise in vector copy number in peripheral blood white blood cells and neutrophils, steadily increasing by 1-2% every month, reaching an average VCN of 5-7% 3-6 months after transplantation.

In summary, patients with thalassemia major underwent safe and effective mobilization followed by excellent transduction of mobilized CD34+ cells. The transplant non-myeloablative conditioning was well tolerated, and followed by rapid engraftment and gradual rise in VCN. Continued clinical and molecular monitoring is on-going and will be presented.

Disclosures:

No relevant conflicts of interest to declare.
Characterizing the Global Transcriptional Effects of TEL-AML1-Fusion in Childhood Acute Lymphoblastic Leukemia
Authors: Teppo Susanna , Teittinen Kaisa J , Grönroos Toni , Viiri Keijo , Kaikkonen Minna , Heinäniemi Merja , Lohi Olli
Published: October 2019

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer affecting thousands of children worldwide each year. Most patients are cured but only after long chemotherapy treatment and still 15 % of them relapse. Therefore, better understanding of the molecular biology behind the disease is fundamental. TEL-AML1 fusion - comprised of two transcription factors important in hematopoiesis regulation - is the most frequent genetic variation found in 25 % of pediatric ALL-patients. The translocation occurs during pregnancy and is suggested to serve as a “first hit” enabling secondary changes that lead to cancer formation. Despite its frequency, genetic targets of this aberrant transcription factor are unclear, and its function in leukemia initiation and progression remains elusive.

Here we aim at solving the genome-wide effects of TEL-AML1 on transcription using pre-B-leukemic cell models. After a time-series induction of wild-type or DNA-binding compromised mutated TEL-AML1, or a constitutive expression of shRNA silencing the fusion, the cells were studied using global nuclear run-on sequencing (GRO-seq). GRO-seq technology can map RNA polymerase II location and identify actively transcribed coding and non-coding genes and their directionality. Thus, GRO-seq provides a snapshot of global active transcription and was used here to identify bona fide targets of the TEL-AML1-fusion protein.

In addition to transcriptional changes in known genes, we identified a notable number of novel transcripts including antisense-, lincRNA-, and microRNAs. We also characterized genomic locations where the fusion functions as a direct regulator by taking advantage of non-coding RNA transcripts known as enhancer RNAs. Furthermore, we performed transcription factor motif enrichment analysis on these active regulatory eRNA regions. The results uncover early transcriptional changes induced by TEL-AML1-fusion and may reveal critical steps in leukemia initiation and development. The findings also highlight the importance of non-coding RNAs in the pathogenesis of pediatric leukemia.

Disclosures

No relevant conflicts of interest to declare.
In Vitro Preactivation of PBMCs with PM-mb21-41BBL Particles Stimulates in Vivo Expansion of NK Cells Under Low IL-2 in NSG Mice
Authors: Oyer Jeremiah L , Pandey Veethika , Igarashi Robert Y , Colosimo Dominic A , Solh Melhem M. , Altomare Deborah A , Copik Alicja J
Published: October 2019

NK cell immunotherapy as a cancer treatment shows promise, but methods for consistent expansion of NK cells from a small fraction (~5%) of peripheral blood mononuclear cells (PBMCs) to therapeutically effective amounts are not widely accessible. Strategies that promote in vivo expansion rely on high-dose IL-2 in combination with lymphodepletion and suffer from severe treatment related toxicities as well as limited expansion. On the other hand, in vitro methods are available for robust NK cell expansion, but rely on prolonged in vitro co-culture with feeder cells that is costly and complex and requires high dose IL-2 (25,000-35,000 U) for persistence after injection into mice. An optimal NK cell expansion method would rely on off the shelf reagents, would not require long-term complex cultures, and would induce a rapid, sustained in vivo expansion of NK cells from unsorted PBMCs under low concentration of IL-2. We have developed a method to prepare particles from K562 cells engineered to express specific cytokines, and such particles stimulate NK cell expansion. Particles prepared using K562 cells expressing IL-21 and 41BBL (PM21 particles) cause highly specific expansion of cytotoxic NK cells from unsorted PBMCs that achieves 95% NK cells in ~14 days and ~10,000 fold expansion of NK in about 21 days. NK cell expansion is consistent using different preparations of PM-particles or leukocyte sources, and the PM-particles retain expansion efficacy with storage. The PM-mb21-41BBL particles were also used to stimulate in vivo NK cell expansion in NSG mice under ultralow IL-2 (1,000 units per injection, 3 injections per week, per mouse). Unsorted human PBMCs, either shortly pre-activated in culture for two days or not pre-activated, were injected (2 x 106 PBMCs per mice by i.p.) and human CD56+CD3- NK cells and other relevant hCD45+ lymphocytes were monitored in the peripheral blood over time and in tissues and fluids at the time of euthanization. In vivo NK cell expansion was observed in mice injected with PBMCs that were pre-activated with PM21 particles. The extent of NK cell expansion observed in the peripheral blood was in levels that would be relevant for clinical cancer treatment (>50,000 NK cells/mL of mouse blood 14 days post injection). In vivo NK cell expansion was further confirmed with analogous experiments using PBMCs that were stained with CellTrace Violet to monitor proliferation. Notably, NK cells were also found in spleen, bone marrow, lung, liver and brain. These results taken together provide proof of principle that PM-particle technology is effective for direct in vivo NK cell expansion from unsorted PBMCs under low dose IL-2.

Disclosures

No relevant conflicts of interest to declare.
Utilization Trend and in-Hospital Complications of Autologous Hematopoietic Stem Cell Transplantation in Multiple Myeloma in the United States: 13 Years Perspective
Authors: Savani Bipin N. , Mukherjee Akash , Savani Ghanshyambhai T , Ankit Chothani
Published: October 2019

Background: We aimed to explore utilization, complication and in-hospital mortality related to Autologous Hematopoietic Stem Cell Transplant (ASCT) in Multiple myeloma (MM) from 1998 to 2011 in United States.

Methods: We used Nationwide Inpatient Sample (NIS) database to isolate multiple myeloma cases undergoing ASCT. Study included adults age >18 years. Patient and hospital demographics were extracted from NIS. Peri-procedural complications were calculated using ICD-9-CM codes. Hierarchical multivariate regression models were formulated to assess predictors of in-hospital mortality and complication.

Results: Total of 6,957 (weighted-34,837) were available for analysis. Majority of patients were male (57%), white (57%) who presented to large bed size (78%),teaching (96%) hospital. Utilization of ASCT for MM increased from 5% in 1998 to 21.6% in 2011 (p<0.001). Despite temporal increase in mean age and comorbidites, mortality reduced (2.9 vs. 0.7, p<0.01) significantly from 1998 to 2011. In-hospital complications were common after ASCT with stomatitis (44.7%) being the most common followed by anemia requiring transfusion (28.5%), febrile neutropenia (16.5%) and bacteremia (15.0%). Mechanical ventilation and acute respiratory failure are the strongest predictors of in hospital mortality post ASCT followed by acute kidney injury,total Body irradiation (TBI) and bacteremia. Higher Charlson comorbidity index, female sex and TBI were associated with higher complication rates post ASCT.

Conclusions: Utilization of ASCT for MM patients has increased significantly since 1998 and even though complications are very common in patients undergoing ASCT (77.4%), in-hospital mortality is rare (<2%) and has decreased significantly over the years secondary to decreased use of pre transplant TBI and better transplant care.

Figure 1 Figure 1.

Figure 2 Figure 2.

Figure 3 Figure 3.

Disclosures

No relevant conflicts of interest to declare.
Upfront Autologous Stem Cell Transplantation Overcome the poor Prognosis of Non-Germinal Center Subtype of diffuse Large B-Cell Lymphoma in Patients with Advanced Stage and Elevated Serum Lactate Dehydrogenase
Authors: Kim Yu Ri , Kim Soo-Jeong , Cheong June-Won , Kim Yundeok , Jang Ji Eun , Lee Jung Yeon , Yang Deok-Hwan , Lee Hyewon , Eom Hyeon-Seok , Min Yoo Hong , Kim Jin Seok
Published: October 2019

Introduction: The role of frontline autologous hematopoietic stem cell transplantation (ASCT) high-risk diffuse large B-cell lymphoma (DLBCL) is still controversial. We investigated the role of upfront ASCT as consolidation for high-risk DLBCL treated with rituximab containing chemotherapy according to molecular classification.

Methods: A total of 195 newly diagnosed DLBCL patients with advanced stage and elevated serum lactate dehydrogenase from three centers were retrospectively analyzed. All patients achieved more than partial response (PR) after completing conventional chemotherapy (cyclophosphamide, doxorubicin, vincristine, prednisone) with rituximab (R-CHOP). Molecular classification was performed according to Han's algorithm.

Results: One hundred fifty (76.9%) patients achieved complete response (CR) and 45 (23.1%) patients PR after frontline R-CHOP chemotherapy. Among these patients, sixty six (33.8%) patients were received ASCT. The 2-year overall survival (OS) was 82.9% and the 2-year progression-free survival (PFS) was 72.1%. Seven (10.6%) patients were relapsed after ASCT while 29 (22.5%) patients were relapsed in non-transplant patients. Patients who treated with ASCT showed superior OS and PFS (P=0.036, P=0.005). According to final response, ASCT showed superior OS and PFS in PR patients (P = 0.024, P = 0.009) while it did not in CR patients.

Among the 128 patients that underwent immunohistochemistry for molecular classification, 36 patients (28.1%) were classified to GCB type, 92 (72.9%) patients were non-GCB type. Twenty five (27.1%) non-GCB patients received ASCT showed significant survival benefit for OS and PFS (P=0.032, P=0.011) while GCB patients did not show the survival difference according to ASCT (Figure 1). In non-GCB DLBCL, ASCT was related with superior PFS both interim and final PR status (P = 0.006, P=0.028). There was no difference for OS and PFS between GCB and non-GCB type in ASCT patients while GCB patients showed superior OS and PFS in non-transplant patients (P = 0.048, P=0.009).

Conclusions: ASCT as consolidation improved OS and PFS in high risk DLBCL patients following R-CHOP chemotherapy. Especially, it could overcome the poor prognosis of non-GCB type DLBCL. Upfront ASCT could be considered effective treatment options for non-GCB type high risk DLBCL.

Figure 1. Overall survival and progression free survival according to autologous hematopoietic stem cell transplantation in non-GCB type DLBCL. Figure 1. Overall survival and progression free survival according to autologous hematopoietic stem cell transplantation in non-GCB type DLBCL. Figure 2 Figure 2.

Disclosures

No relevant conflicts of interest to declare.
Suppressing Hepcidin By Tocilizumab Effectively Improve Anemia in Inflammatory Diseases: Clinical Evidence and Basic Mechanisms
Authors: Yoshizaki Kazuyuki , Song Soken-Nakazawa J , Kawabata Hiroshi
Published: October 2019

Introduction

Dysregulated production of hepcidin is implicated in anemia of inflammation (AI) and interleukin-6 (IL-6) is a major inducer of hepcidin production. Increased imflammatory cytokines, especially IL-6 is response for pathogenesis of multicentric Castleman's disease (MCD) and rheumatoid arthritis (RA).

Patients and Method

In this study we study role of hepcidin and IL-6 in AI patients with MCD or RA, by investigating the effect of tocilizumab, an anti-IL-6 receptor antibody, on the serum hepcidin and relationship between hepcidin and iron-related parameters. The mechanism involving inflammatory anemia in MCD and RA was further analyzed in vitro by studied the transcriptional regulation of hepcidin in hepatoma-derived cell lines in the presence of cytokines, antibodies against cytokines, inhibitors of signal pathways and other hematopoietic factors.

Results

Our data showed that treatment with tocilizumab resulted in a rapid reduction of serum hepcidin-25 in MCD and RA patients. Although treatment with anti-TNF-a antibody also resulted in decrease of serum hepcidin, compared to tocilizumab therapy it decrease by a smaller margin. Furthermore, long-term reductions of hepcidin-25 were observed in ten MCD cases for one year after the start of tocilizumab treatment, which was accompanied by progressive normalization of iron-related parameters and improved disease activity. In in vitro experiments, IL-6, but not TNF-a or IL-1,-induced upregulation of hepcidin mRNA in hepatoma cell lines was completely inhibited with tocilizumab, and partially by EPO as well as TNF-a, but enhanced by BMP4 and MCD patient’s serum.

Discussion

Our results suggest that, although multiple factors affect serum hepcidin levels, IL-6 plays an essential role in the induction of hepcidin. This accounts for the long-term ameliorative effect of IL-6 blockage with tocilizumab on anemia by inhibiting hepcidin production in MCD and RA patients.

Disclosures

No relevant conflicts of interest to declare.
The Prognostic Role of Diabetes Mellitus for Cardiac Complications in a Large Cohort of Well Treated Thalassemia Major Patients
Authors: Meloni Antonella , Gamberini Maria Rita , Rossi Giuseppe , Sorrentino Francesco , D'Ascola Domenico Giuseppe , Spasiano Anna , Filosa Aldo , Cuccia Liana , Dello Iacono Nicola , Forni Gianluca , Caruso Vincenzo , Maggio Aurelio , Pitrolo Lorella , Peluso Angelo , Midiri Massimo , Missere Massimiliano , Neri Maria Giovanna , Pepe Alessia
Published: October 2019

Epidemiologic as well as clinical studies confirm a close link between diabetes mellitus (DM) and heart failure (HF) in the general population. In a retrospective historical thalassemia major (TM) cohort, DM was demonstrated to lead to an higher frequency of cardiac complications also independently of cardiac iron status, but not prospective data are available. So, we determined prospectively the predictive value of DM for HF, arrhythmias and cardiac complications (HF, arrhythmias and pulmonary hypertension) in TM.

We followed prospectively 537 patients enrolled in the Myocardial Iron Overload in Thalassemia (MIOT) network.

Fifty-six patients were excluded because a cardiac complication was present at the time of the first Cardiovascular Magnetic Resonance (CMR), representing our starting point.

All the considered 481 TM patients were white (29.48±8.93 years, 263 females). Mean follow-up was 57.91±18.23 months. DM was present in the 9.8% of patients.

Cardiac events were recorded in 36 patients (7.5%). There were 18 episodes of HF, 16 arrhythmias and 2 pulmonary hyperthension.

DM was a significant predictive factor for HF (hazard ratio-HR=5.62, 95%CI=2.08-15.22; P<0.0001) as well as for arrhythmias (HR=3.70, 95%CI=1.18-11.65; P=0.025) and for cardiac complications globally considered (HR=4.26, 95%CI=2.04-8.92; P<0.0001).

DM remained a significant prognosticator for HF and cardiac complications also in a multivariate model including cardiac iron.

In conclusion, DM was found to be a strong predictor for HF, arrhythmias and cardiac complications. Our findings are relevant for the prevention of glucose disorder metabolism and they stress the need to intensify the chelation therapy in patients in whom excess pancreatic iron is found by MR, where available, or when patients develop glucose metabolism disorders.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Use of Nano-Formulated Hydroxyurea Enhances Drug Delivery to Pulmonary Artery Endothelial Cells
Authors: Mohtar Omar , Dragovic Doroteja , Tagne Jean-Bosco , Klings Elizabeth S
Published: October 2019

The vasculopathy of sickle cell disease (SCD), characterized by a chronic hemolytic anemia, endothelial dysfunction and recurrent vasoocclusive events, underlies most of the clinical complications. Recent work has suggested that hydroxyurea (HU) enhances endothelial nitric oxide (NO) production and reduces adhesion molecule expression which may contribute to its associated observed reduction in vasoocclusive events and improved survival in SCD. However, clinically HU is under-utilized, at least in part related to medication related toxicity, stressing a need for enhanced drug delivery. The use of nano-emulsions, a class of stable emulsions formed by a monolayer of phospholipids and/or biodegradable material such as chitosan, is a mechanism by which to enhance targeted cellular delivery of therapeutics. We hypothesized that a nano-formulation of HU would enhance drug delivery to the endothelium and may be a direct way of targeting endothelial dysfunction in SCD. Nano-formulated HU (nano-HU) was developed using a phospholipid approach and this compound was found to be biologically stable for > 6 months. Human pulmonary arterial endothelial cells (HPAECs) were co-incubated with 25µM, 125µM and 250µM HU, nano-HU, or the empty nano-emulsification preparation for 48 hours. HPAEC were isolated, and stained with propidium iodide (PI) for cell cycle analysis by FACS. HPAEC treated with 25µM nano-HU demonstrated the highest level of cell viability (55%) and similar levels of S phase inhibition as HPAEC treated with 125µM HU (where 10-20% cell viability was observed). RNA isolation of treated HPAECs was performed and qPCR for VCAM1 and SLX4, a regulator of DNA damage which promotes S phase inhibition was performed. 25µM nano-HU significantly decreased VCAM1 and increased SLX4 expression compared with 25µM HU suggesting increased efficacy at lower levels of toxicity. The impact of nano-HU on endothelial gene expression is currently being evaluated by RNA-seq. Nano-formulation of HU produces a molecule which is highly stable, and able to achieve higher intracellular concentrations with lower rates of toxicity in HPAECs .which provides the opportunity to gain greater understanding of the impact of this medication on the vascular endothelium and the potential for a more targeted and better tolerated delivery of this therapeutic to patients with sickle cell disease.

Disclosures

Klings: Pfizer: Consultancy.
Vitamin D-Dependent Induction of Cathelicidin in Human Macrophages Results in Cytotoxicity Against High Grade B-Cell Lymphoma
Authors: Bruns Heiko , Büttner Maike , Fabri Mario , Mougiakakos Dimitrios , Bittenbring Joerg Thomas , Pasemann Shirin , Neumann Frank , Maurberger Anna , Kempkes Bettina , Mackensen Andreas , Gerbitz Armin
Published: October 2019

Infiltration by macrophages represents a characteristic morphological hallmark in high grade lymphatic malignancies such as Burkitt’s lymphoma (BL). Although macrophages can in principle target neoplastic cells and mediate antibody-dependent cytotoxicity (ADCC), tumor associated macrophages (TAMs) regularly fail to exert direct cytotoxic functions. The underlying mechanisms responsible for this observation remain unclear. Here we demonstrate that inflammatory M1 macrophages kill proliferating high grade B-cell lymphoma cells by releasing the antimicrobial peptide cathelicidin in a vitamin D-dependent fashion. Moreover, we show that cathelicidin directly induces cell death by targeting mitochondria of BL-cells. In contrast, anti-inflammatory M2 macrophages and M2-like TAMs in BL exhibit an altered vitamin D metabolism resulting in a reduced production of cathelicidin and consequently in inability to lyse BL cells. However, treatment of M2 macrophages with the bioactive form of vitamin D, 1,25D, or a vitamin D receptor agonist effectively induces cathelicidin production and triggers tumoricidal activity against BL cells. Furthermore, rituximab-mediated cytotoxicity of M2 macrophages is cathelicidin dependent and vitamin D treatment of 25D deficient volunteers improves rituximab-mediated ADCC against BL cells. These data indicate that activation of the vitamin D signaling pathway may activate antitumor activity of TAMs and improve the efficacy of ADCC.

Disclosures

No relevant conflicts of interest to declare.
ADAMTS13 Regulates Neutrophil Recruitment in a Mouse Model of Invasive Pulmonary Aspergillosis
Authors: Radsak Markus , Prüfer Steve , Ebner Katharina , Weber Michael , Reuter Sebastian , Stein Pamela , Scharrer Inge , Stassen Michael , Schild Hansjörg , Jurk Kerstin , Bosmann Markus
Published: October 2019

Von Willebrand factor (VWF) is secreted as an acute phase protein during inflammation. The main mechanism regulating the size and prothrombotic activity of VWF is the specific proteolytic activity of ADAMTS-13. To determine the relevance of this regulatory pathway for the innate inflammatory response by polymorphonuclear neutrophils (PMN), we employed a mouse model of invasive pulmonary aspergillosis (IPA) where PMN functionality is crucial for fungal clearance and survival. IPA was induced by intratracheal application of Aspergillus fumigatus conidia in wild-type (129/Sv/Pas) or Adamts13 deficient (Adamts13-/-) mice. After PMN depletion using a anti-Gr-1 specific antibody, all mice infected with Aspergillus fumigatus conidia developed neutropenia and succumbed due to lethal IPA. In contrast, all undepleted wild-type mice survived the infection. Interestingly, Aspergillus fumigatus infection in Adamts13-/- mice was lethal in 20% of the animals displaying a more severe course of IPA, as indicated by an increased fungal burden in lung homogenates along with increased levels of albumin and the inflammatory mediators IL-1β, IL-6, TNF-α, KC and MCP-1 in the bronchio-alveolar lavage fluid (BALF) compared to wild-type controls. Beyond this, we observed a decreased number of PMN in BALF of infected Adamts13-/- mice compared to wild-type mice. Lung histology sections demonstrated a more pronounced perivascular leukocyte infiltration in further support of a dysregulated inflammatory response in Adamts13-/- mice. Importantly, we observed no general defect in the activation of neutrophil effector functions as demonstrated by the normal induction of the oxidative burst, phagocytosis, degranulation, L-selectin shedding and apoptosis in response to formyl-peptide receptor agonists or exposure to Aspergillus fumigatus conidia or hyphae in vitro. Therefore, we conclude that the proteolytic regulation of VWF by ADAMTS-13 in an important mechanism to control PMN recruitment in the regulation of the innate inflammatory response in invasive fungal infections.

Disclosures

Radsak: Celgene: Research Funding.
Splenic Mastocytosis
Authors: ENDE NORMAN , CHERNISS EDWARD I.
Published: October 2019

1. A patient with splenic mastocytosis who had hypersplenism, periodic flushing and clotting defects is presented.

2. A correlation is shown between mastocytosis and elevated histamine and heparin levels in the spleen.

3. The patient is well nine months after splenectomy.

4. Wright’s stain or polychrome methylene blue stain of splenic tissue imprints is recommended as a method of demonstrating mast cells in the spleen.

5. Mast cell counts of normal spleen showed 0 to 0.25% of nucleated cells on imprint.

6. Splenic aspiration stained with Wright’s stain is satisfactory for demonstrating mastocytosis, though not dependable for true mast cell counts.

7. Cases of primary hypersplenism should routinely be reviewed for splenic mastocytosis.

8. Splenic mastocytosis is a variant of systemic mast cell disease in which involvement is predominately confined to the spleen.
A New Diagnostic and Therapeutic Approach Fibrinolysis and Hemorrhage
Authors: HEUSON J. C. , PEERS W. , TAGNON H. J.
Published: October 2019

1. The case is presented of a patient who underwent a cesarean section during which cardiac arrest occurred. Cardiac massage was successful in reestablishing normal heart action.

2. A severe hemorrhagic diathesis associated with fibrinolysis appeared during the period of shock associated with cardiac arrest.

3. The plasma of the patient obtained during the period of fibrinolysis was able to digest its own fibrin as well as a substrate of casein marked with radioactive iodine.

4. A new method of measurement of fibrinolysis based on the use of tagged casein is presented: the main advantage of this method is that the substrate is not contaminated with plasminogen or plasmin. In addition the products of the reaction are measured by their radioactivity. Therefore, the blank and readings in this method depend solely upon the substrate and are not contributed to by the other reagents used.

5. The proteolytic activity of the plasma from a patient with fibrinolysis was shown to be inhibited by the trypsin inhibitor from soy bean.

6. The intravenous injection of trypsin inhibitor in this patient was followed by the disappearance of fibrinolysis in her blood.
Autoimmune Hemolytic Anemia in NZB/Bl Mice Treated with the Corticosteroid Drug Betamethasone
Authors: CASEY T. P. , HOWIE J. B.
Published: October 2019

A controlled therapeutic trial of the corticosteroid drug betamethasone phosphate in the autoimmune hemolytic anemia of the NZB/Bl inbred strain of mice is described. In the treated group, in contrast to the controls, anemia was improved, positive Coombs tests became weaker or negative and there was a fall in titer of circulating antibody. The similarity of this disease in the mouse and in man is discussed. It is concluded that the NZB/Bl mice will prove useful in investigating the effectiveness and mode of action of therapeutic agents in autoimmune hemolytic anemia.
A Possible Immune Reaction Producing Spontaneous Remission in Leukemia
Authors: BURKETT LUTHER L. , FIELDS MURRAY L. , DIGGS LEMUEL W.
Published: October 2019

A patient is described in whom four phases of illness were recognized: (1) erythroleukemia, (2) brisk bone marrow plasmocytosis with Bence Jones protein in the urine suggesting multiple myeloma, (3) normality physically and of blood and bone marrow, with the exception of slight splenomegaly, (4) myeloblastic leukemia with death from Pseudomonas septicemia.

Local and reticuloendothelial plasmocytic reactions have been observed in experimental animals and in man with malignant disease, and this occurrence has been related to tumor inhibition and improved prognosis. Paraproteinemia and paraproteinuria have been noted in malignancies other than those arising from plasma cells or lymphocytes.

It is suggested that the plasmocytosis and abnormal protein in the case reported, as well as in other similar cases previously reported, may have represented an immune reaction induced by the malignancy and responsible for its remission.
Comparative Studies of Platelet Survival by Different Methods in the Rabbit
Authors: EBBE SHIRLEY , BALDINI MARIO , DONOVAN JANET
Published: October 2019

Four methods for measuring the survival of homologous platelets in rabbits were studied: (1) transfusion of nonradioactive platelet concentrates to thrombocytopenic recipients, (2) transfusion of concentrates of platelets labeled in vitro with Cr51-sodium chromate, (3) transfusion of concentrates of platelets labeled in vivo with P32-orthophosphate and (4) transfusion of whole blood labeled in vivo with P32-orthophosphate. The survival time of platelets in normal rabbits was 3-4 days.

From comparison of the 3 methods using platelet concentrates, the following conclusions were drawn. (1) All the platelets in a platelet concentrate were capable of recirculating after transfusion. (2) Labeling with P32 or Cr51 did not damage platelets. (3) About one-third of the Cr51 was immediately eluted from viable platelets after they were transfused. (4) Further exchange of the label in vivo did not occur to a significant degree with either Cr51 or P32. (5) Cr51 did not elute from platelets during storage of the platelets. (6) Studies of rabbit platelets had applicability in predicting the behavior of human platelets.
Role of sialic acid in erythrocyte survival
Authors: Durocher JR , Payne RC , Conrad ME
Published: October 2019

The role of membrane sialic acid in erythrocyte survival is unclear, although there is evidence for a reduction in sialic acid and surface charge in older erythrocytes. We reduced the surface charge of human, rat, and rabbit erythrocytes by removing sialic acid with neuraminidase.Reduction in sialic acid correlated with decreases in electrophoretic mobility and loss of PAS staining of membrane glycoproteins on polyacrylamide gels. No changes in ATP levels or deformability were found. 51Cr erythrocyte survivals in rats and rabbits showed rapid clearance of desialylated erythrocytes with sequestration by the liver.These results suggest that reduction in erythrocyte sialic acid is a mechanism of erythrocyte destruction and may be important in erythrocyte senescence.
Intrinsic material properties of the erythrocyte membrane indicated by mechanical analysis of deformation
Authors: Evans EA , La Celle PL
Published: October 2019

Deformation of the erythrocyte membrane by the micropipette technique permits analysis of intrinsic material characteristics of the membrane and provides a means to differentiate purely membrane factors from such extrinsic factors as surface area-to-volume ratio. Using small micropipettes (less than 0.5 microns radius) to deform cells, it is evident that the red cell membrane behaves like a solid for periods of time up to 5–10 min of sustained deformation; for long periods of strain, permanent deformations occur, indicative of the semi-solid structural character. In the time range in which the membrane behaves like a solid, the material is linearly elastic up to strains of 400%, implying a loose network structure in the membrane plane, and evaluation of the elastic parameter mu (mu for normal discocytes equals 7 x 10(-3) dynes/cm) suggests that the elements comprising the network may have a molecular weight of approximately that of the water-soluble membrane protein spectrin. Whether the network system is cross-linked or simply a polymer solution remains unanswered. Experimental data indicate that plastic flow of the membrane under conditions of protracted strain may lead to permanent deformation of the membrane, whereas uniform dilation of the membrane, requiring over 1000 times more energy than for plastic flow, results in membrane failure and lysis. Analyses of the data from larger micropipettes of limiting mean cylindrical diameter show their utility in evaluating extrinsic factors, e.g., surface area-to-volume relationships, which are related to the capability of the whole cell to form a new configuration with implicit resistance to total surface area change, as the cell enters narrow channels of the microcirculation. Thus, micropipettes with diameters in the 2.7–3.0-microns range can provide sensitive comparisons of cellular deformability of erythrocytes.
Long-term remission from acute myelogenous leukemia after bone marrow transplantation and recovery from acute graft-versus-host reaction and prolonged immunoincompetence
Authors: Bleyer WA , Blaese RM , Bujak JS , Herzig GP , Graw RG Jr
Published: October 2019

A 19-yr-old boy has been in continuous complete remission from acute myelogenous leukemia for 3 yr after allogeneic bone marrow transplantation prepared with combination chemotherapy. During the first year post-transplant, however, the patient developed near-fatal graft-versus-host reaction followed by 11 severe viral and bacterial infections. Immune evaluation during this period revealed multiple defects which were not present prior to transplantation, nor present in the transplant donor: diminution of lymphoid tissue, decline of all immunoglobulin subtypes, deletion of secretory immunoglobulin, disappearance of isohemagglutinins, loss of antibody to diptheria and tetanus toxoids, cessation of cutaneous hypersensitivity to mumps antigen, and inhibition of serum opsonizing activity. The patient was also unable to develop normal humoral or cellular reactivity to brucella antigen, keyhole limpet hemocyanin, or dinitrochlorobenzene. This patient's course illustrates the severity and chronicity of immunoincompetence associated with allogeneic marrow grafting, the importance of early detection and rigorous treatment of infectious disease in these patients, and the need for improved immunologic reconstitution in human marrow transplantation. It also indicates that complete recovery from the immune defects is possible, and that long- term remission from acute myelogenous leukemia can be achieved with allogeneic marrow transplantation.
Arsenic intoxication as a cause of megaloblastic anemia
Authors: Westhoff DD , Samaha RJ , Barnes A Jr
Published: October 2019

We have described a case of chronic arsenic intoxication associated with pancytopenia and megaloblastic erythropoiesis. The patient had the typical laboratory manifestations of effective erythorpoiesis due to a megaloblastic process, including macroovalocytes, mild pancytopenia, low reticulocyte index, increased marrow cellularity with erythroid hyperplasia, and morphologic evidence of megaloblastic maturation in the marrow. The patient's serum folate and vitamin B12 were normal, and the anemia regressed without therapy. Our case suggests that the combination of megaloblastosis with normoblastic or megaloblastic karyorrhexis,should raise the suspicion of arsenic intoxication in the mind of the observer. In addition, arsenic should be added to the list of agents causing a reversible megaloblastic anemia.
Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome
Authors: Lozzio CB , Lozzio BB
Published: October 2019

A cell-line derived from a patient with chronic myelogenous leukemia (CML) is described. The new cell-line, which has over 175 serial passanges in a 3 1/2-yr period, has the following characteristics: (1) CML cells started to proliferate actively since they were first incubated in culture media. A threefold increase in the total number of cells was observed during the first seven passages; the cell population increased by a factor of 10 to 20 every 7 days from passage 8 through 85; from 20 to 40 times from passage 86 through 150, and more than 40 times after 150 passages. (2) The majority of the nononucleated cells are undifferentiated blasts. (3) The karyotype of all the cells examined show the Philadelphia (Ph1) chromosome and a long acrocentric marker plus aneuploidy. The Giemsa-banding studies identified the Ph1 chromosome as a terminal deletion of the long arm of chromosome 22:del(22)(q12) and the long acrocentric marker as an unbalanced reciprocal translocation of one chromosome 17 and the long arm of one chromosome 15. (4) The CML cells do not produce immunoglobulins, are free of mycoplasma, Epstein-Barr virus, and herpes-like virus particles. (5) CML cells have no alkaline phosphatase and myeloperoxidase activities and did not engulf inert particles. (6) Cultured CML cells provide a constant source of a specific antigen. This CML cell-line represents a unique source of CML cells with meaningful indicators of malignancy for clinical and experimental studies.
Selection of patients for bone marrow transplantation in severe aplastic anemia
Authors: Camitta BM , Rappeport JM , Parkman R , Nathan DG
Published: October 2019

Despite androgens and intensive supportive care, satisfactory survival in severe aplastic anemia remains at 20% or less. Histocompatible bone marrow transplantation can restore normal hematopoiesis in approximately 40% of similarly severe individuals. Delay of transplantation for 3 wk after diagnosis allows time for proper evaluation and for many spontaneous recoveries. Further delay increases risks of fatal complications and decreases chances for successful transplantation while the incidence of spontaneous remission declines. When available, early histocompatible bone marrow transplantation may be the treatment of choice for severe aplastic anemia.
Abnormalities of factor VIII and platelet aggregation--use of ristocetin in diagnosing the von Willebrand syndrome
Authors: Weiss HJ
Published: October 2019

Ristocetin was used to study platelet aggregation in platelet-rich plasma and to assay the von Willebrand factor activity of factor VIII (VIII-VWF). Ristocetin-induced platelet aggregation (RIPA) was decreased in 13 of 18 patients with von Willebrand's disease (VWD) who had decreased plasma levels of VIII-VWF. The five patients with normal RIPA appeared to have mild VWD but did not constitute a separate subclass. RIPA was also abnormal in some patients with intrinsic platelet defects, but in no case was the defect corrected by normal plasma. The latter type of correction appears to be specific for VWD. Aspirin ingestion inhibited the second phase of RIPA (at low concentrations of ristocetin only) but did not affect the initial phase of aggregation or the level of VIII-VWF. We also studied a group of patients who had both abnormalities of the factor VIII complex and intrinsic platelet defects, such as impaired collagen-induced aggregation, as well. The findings in these patients and in those with typical von Willebrand's disease appear to comprise a spectrum of disorders (the von Willebrand syndrome) in which some abnormality of the factor VIII complex is associated with impaired platelet function. At present, ristocetin would appear to be a useful reagent for evaluating patients with bleeding disorders and for studying patients with the von Willebrand syndrome.
Morphologic and functional characteristics of bone marrow macrophages from imferon-treated mice
Authors: Fedorko ME
Published: October 2019

The iron storage macrophage has been isolated from the marrow of Imferon-treated mice and studied in vitro by morphologic, histochemical, and functional tests and isotope labeling techniques. These macrophages on stained preparations are large, many times binucleate cells (up to 150 mu), and show Prussian blue reactivity. In Epon-embedded, stained thick sections they contain elongated narrow basophilic inclusions. These macrophages are actively phagocytic and pinocytic; histochemical studies show that these cells are rich in acid phosphatase, nonspecific esterase, and PAS diastase-resistant activity. Iron storage macrophages do not incorporate the 3H-thymidine. The electron microscopic appearance of this macrophage shows that the cell has ferritin free in the cytoplasm and several types of cytoplasmic granules: those with large quantities of electron-dense ferritin and/or hemosiderin (type A), elongated granules (type B) with moderately electron dense homogeneous matrix and some ferritin at the periphery, and granules with heterogeneous content (type C). The above findings demonstrate that the iron storage cell is a mature macrophage which contains hydrolases, ferritin, and a unique population of cytoplasmic granules which are lysosomal in nature. There is some evidence to suggest that the unusual lysosome (type B granule) occurs after macrophages have ingested erythrocytes.
Inhibition of intravascular fibrin deposition by dipyridamole in experimental animals
Authors: Gurewich V , Lipinski B , Wetmore R
Published: October 2019

Intravascular fibrin deposition was induced in rabbits by endotoxin, the infusion of fibrin monomer (FM), and by the infusion of thrombin and EACA. A previously developed radioisotope technique was used to measure the fibrin deposits in various organs. Dipyridamole treatment of rabbits caused significant inhibition of fibrin deposition in all three experimental models. The drug also inhibited platelet consumption and, in the thrombin- and EACA-infused animals, fibrinogen consumption as well. The results obtained with dipyridamole were compared with the effect of thorotrast. It was concluded from this comparison that the effect of dipyridamole could not be attributed to inhibition of the reticuloendothelial system. It is postulated that dipyridamole inhibits the final step at which soluble FM is precipitated as fibrin in vivo.
Fibrinogen ""New York"--an abnormal fibrinogen associated with thromboembolism: functional evaluation
Authors: Al-Mondhiry H , Bilezikian SB , Nossel HL
Published: October 2019

A 54-yr-old woman presented with a 23-yr history of repeated life- threatening thromboembolism. The presence of a qualitatively abnormal fibrinogen was suggested by the demonstration of delayed and incomplete coagulation of plasma or partially purified fibrinogen by thrombin or Reptilase. Two brothers showed a similar in vitro defect but were clinically not affected. The plasma fibrinogen concentration was 0.50- 1.64 mg/ml when estimated by heat turbidity, clottability, or immunologic techniques. The serum contained 80–820 mug/ml of unclottable fibrinogen-related materials even after 24 hr exposure to thrombin. The fibrinogen-related material in the serum showed faster anodal mobility an immunoelectrophoresis than that of normal plasma. Immunodiffusion studies with rabbit antihuman fibrinogen antiserum showed lines of identity between control plasma and the patient's plasma and serum. Studies of the kinetics of thrombin action on fibrinogen demonstrated impaired release of fibrinopeptide A and B and defective polymerization of preformed fibrin monomers. The maximum amount of fibrinopeptide A released by exhaustive treatment with thrombin was similar (per milligram protein) for both the patient's and control fibrinogen. This abnormal fibrinogen varient is tentatively designated fibrinogen “New York”; its possible identity with one of the previously described abnormal fibrinogens has not been excluded.
Some factors affecting fibrinogen precipitation by ristocetin: ultrastructure of precipitates
Authors: Ts'ao C , Green D , Rossi EC
Published: October 2019

Fibrinogen in aqueous solution is precipitated by the antibiotic ristocetin. This reaction is inhibited by albumin and facilitated by low temperature. Resolubilized fibrinogen clots in the presence of thrombin. Ristocetin-precipitated fibrinogen takes the form of fibrils or clumps, composed of irregularly spaced, structure-less particles. The addition of ristocetin to washed platelets suspended in fibrinogen- containing media produces fibrinogen clumps in both the media and in the surface cannalicular system of the platelets. The changes in light transmission (aggregation curves) are due to both platelet aggregation and fibrinogen clumping. The role of the latter is confirmed by the observation that the addition of ristocetin to inert latex particles suspended in fibrinogen solution produces typical aggregation curves. This phenomenon is prevented by the addition of albumin to the media. We conclude that (1) if fibrinogen is present in any artificial system, albumin should be included in the media to prevent fibrinogen precipitation; and (2) statements about aggregation of any particulated materials by ristocetin should not be based solely on light- transmission changes, but should also include a description of the morphologic appearance.