bluuebunny/crossref_metadata_2025 huggingface dataset:
Fatal Bleeding Caused by Congenital Factor XIII Deficiency and Development of An Inhibitor in a Hispanic Male
Authors: Lorenzo Filipe R. , Haslem Derrick , Prchal Josef T. , Greenberg Charles
Published: April 2020

We report a case of fatal bleeding in a patient with mild congenital Factor XIII deficiency who also developed an inhibitor that interfered with fibrin stabilization. Plasma Factor XIII circulates as a tetramer composed of two A-chain and two B-chains and is bound to fibrinogen. After thrombin cleaves the A-chain at Arg 37, the B-chain dissociate producing Factor XIIIa. Factor XIIIa catalyzes covalent linkages between the fibrin molecules aligned in the fibrin clot. Factor XIIIa makes the fibrin resistant to disruption by urea and plasmin. Factor XIIIa levels greater than 1 % are needed to covalently stabilize a clot. We studied a 39 year old Hispanic male that presented with altered mental status, headache and a large left frontal parietal hemorrhage. Ultimately, he became non-responsive and was intubated. He had no major bleeding history but did have frequent nosebleeds. Routine blood counts and coagulation laboratory tests were normal. However, a qualitative Factor XIII assay was abnormal and the clot dissolved in 5M Urea. Furthermore, a 1:1 mixing study did not correct the defect suggesting the presence of an inhibitor. A small dose of cryoprecipitate was administered to the patient and this corrected the clot stability defect. Four days later, the defect recurred and 150 ml of cryoprecipitate was administered and despite correction of the fibrin stabilizing abnormality the patient died. Two of his sisters had a history of repeated hemorrhagic miscarriages in Mexico a finding consistent with congenital Factor XIII deficiency. DNA sequencing of the factor XIII a-chain gene was performed from the propositus’ mononuclear cells. A reverse transcription was done using an oligo(dT) 12–18 primer followed by nested PCR amplification and a heterozygous missense mutation was observed at codon 35 (G226T; Val35Leu). This mutation was than confirmed in propositus’ platelet cDNA. The Val35Leu substitution is close to the thrombin cleavage site, the G226T substitution is in the catalytic core domain. This case demonstrates that in some cases of Factor XIII deficiency there is residual Factor XIIIa activity in the mutant molecule that can prevent serious bleeding. However, patients may develop autoantibodies that further interfere with Factor XIII function and may suffer serious bleeding complications. Additional cases of Factor XIII deficiency may exist in patients that have mild bleeding problems.
Rituximab in the Treatment of Acquired Hemophilia a (AHA): A Novel Eradicating Therapy?
Authors: Mota Karla O. , Bastos Silvia Nakanishi
Published: April 2020

Background: Acquired inhibitors against factor VIII (FVIII), also termed Acquired Hemophilia A (AHA), occur rarely in the nonhemophilic population. Its incidence is approximately 1 to 4 per million/year. The incidence of AHA increases with age and a major peak is seeing in patients aged 68 to 80 years. These autoantibodies are associated with high rate of morbidity (90% of severe bleeding in affected patients) and mortality (8–22% of the cases). The diagnosis of AHA is based on the demonstration of an isolated prolongation of the activated partial thromboplastin time (APTT), not corrected by incubating the patient’s plasma with equal volumes of normal plasma (mixing study), associated with FVIII reduced levels and formal evidence of a FVIII inhibitor in a patient with no previous personal or family history of bleeding. In approximately half of affected patients there is no identification of relevant concomitant diseases. The bleeding pattern in AHA is characterized by hemorrhages into the skin, muscles or soft tissues, and mucous membranes. Rituximab (Mabthera®; Roche; Switzerland) is a chimeric monoclonal antibody against the pan B-cell antigen CD20 that induces a rapid in vivo depletion of normal B lymphocytes. Primarily developed to treat B-cell non Hodgkin Lymphomas, more recently, Rituximab has demonstrated effectiveness in a number of autoantibody-mediated diseases including AHA. Case Report: We treated a 69-year-old diabetic and hypertensive patient with AHA. In March 2007, the diagnosis was made by a prolonged APTT (ratio 2.74 sec), not corrected by the mixing study, and FVIII inhibitor 320 Bethesda units. The patient had no evidence of malignancy, autoimmune disease or use of drugs. His bleeding manifestations were large ecchymosis of upper and lower extremities for almost a year, and at admission to the hospital, a hematoma of left calf and gengival bleeding. First line therapy consisted of steroids and cyclophosphamide (CFM). The steroids had to be withdraw after one week of use, because of very difficult glycemic control. The CFM were use in the dose of 50mg/day adding an total accumulative dose of 3g. This protocol were used considering the incapacity of the patient to tolerate higher dose of CFM due to leukopenia. After treatment, the patient was evaluated and neither normalization of APTT nor improvement of clinical findings happened. Therefore, considering all the above, it was decided to use Rituximab 375mg/m2, weekly, for four weeks. In June 2007, the patient received and tolereted very well all the infusions. In August 2007, he had no bleeding symptoms and his APTT was normal. At his laterest visit to the clinic in July 2008, an year after finishing his treatment, he was still very well and his bleeding tests were normal.

Conclusion: Rituximab should be considered a treatment option for AHA.
Seroprevalence of PF4/Heparin Antibodies among Blood Bank Donors
Authors: Pai Poulomi J. , McCracken Julianna E , Arepally Gowthami M
Published: April 2020

Heparin-Induced Thrombocytopenia (HIT) is classically viewed as a drug-induced immune disorder triggered by exposure to the anticoagulant drug, heparin. However, HIT may also be considered a dysregulated autoimmune response to two naturally occurring substances, Platelet Factor 4 (PF4), a platelet protein, and heparin, a tissuederived glycosaminoglycan. Until recently, studies of HIT and PF4/heparin antibody seroconversion have been exclusively focused on patients exposed to heparin or heparinlike drugs, including low-molecular weight heparin, the heparinoids and the synthetic agent, fondaparinux. Recent reports, however, indicate that PF4/heparin antibodies may arise spontaneously in some individuals in the absence of heparin (Warkentin Am J Med 2008; Hursting J Thromb Thrombolysis, 2008). We undertook this study to document the seroprevalance of “naturally-occurring” PF4/heparin antibodies in healthy subjects without prior heparin exposure. To study “healthy” subjects, we used blood bank donors as a surrogate population. Samples from blood bank units were obtained through Duke Transfusion services/American Red Cross (ARC). Donors met eligibility criteria for blood donation to the ARC and generally, represent a healthy population (http://www.redcross.org/services/biomed/0,1082,0_557_,00.html#hem). A Duke IRB waiver was issued due to use of anonymous samples. Plasma from donor units/segments were assayed in duplicate for the presence of PF4/heparin antibodies using a commercially available PF4/heparin ELISA (PF4 enhanced IgG,A,M kits, Genetics Technology Institute, Waukesha, WY). Positivity was defined by the manufacturer as A405nm ≥0.4. Positive samples were stratified into low, (A405nm=0.4–0.6), intermediate (A405nm=0.61–0.99) or high-positive values (A405nm≥1.0). High and intermediate positive samples were further isotyped using IgG and IgM specific antibodies (Sigma, St. Louis, MO). Of the 970 blood bank donors tested to date, 31 samples (3.2%) were positive using the manufacturer’s cut-off value. Repeat testing confirmed positivity in all 31 samples. Of those testing positive, 26 were low-positive (2.3% of overall cohort; 84% of all positive samples), 1 sample was intermediate positive (0.1% of overall cohort; 3.2% of positive samples) and 5 samples were high-positive (0.5% overall cohort; 16% of positive samples). Isotyping of 6 high/intermediate positive samples revealed IgG in 3 patients, IgM in the remainder. There was no correlation between antibody positivity and ABO blood type. In summary, our data reveals a high prevalence of PF4/heparin antibodies, the majority of which are low-positive values (~84%). Many of these low-positive samples likely reflect background “noise” of the assay and could be eliminated using a higher cut-off for positivity. We also found a seroprevalance of 0.5% of high positive antibodies, of IgG and IgM isotype, supporting the notion that some individuals may have “naturally-occuring”
Burden of Thrombocytopenia in Cancer Patients Undergoing Chemotherapy: a Retrospective Database Analysis
Authors: Aravind Suresh , Wu Ying , Ranganathan Gayatri , Nalysnyk Luba
Published: April 2020

Background. Along with anemia and neutropenia, thrombocytopenia is a common hematological adverse effect of chemotherapy. Prolonged and severe thrombocytopenia increases the risk of bleeding episodes. The goal of this study was to evaluate the epidemiology of thrombocytopenia among cancer patients on chemotherapy.

Methods. Pooled data over the period 2000–07 from a large electronic database of 15 community and 3 hospital-based outpatient oncology clinics in the US was analyzed. For this analysis, adult patients with a cancer diagnosis were grouped as: lung, breast, ovarian, H&N, colorectal, other solid cancers, hematologic and mixed cancers. Chemotherapies used were grouped into platinum, anthracycline, gemcitabine, taxane-based and other regimens. Incidence of Thrombocytopenia (defined as <150,000/μl) was examined by tumor and regimen.

Results. 47,159 patients [mean age 61, male 42%] who met inclusion criteria and 75,243 chemotherapy regimens were analyzed. Breast cancer patients constituted 19.5%, followed by NSCLC (14.9%), colorectal (11.9%), ovarian (3.1%), and H&N (2.5%). Platinum-based regimens accounted for 27.8% of all regimens. The incidence of post-chemo thrombocytopenia was highest among gemcitabine treated patients (64.2%), followed by platinum (55.5%). The lowest incidence was with taxane-based regimens (21.9%). Grade 3–4 (<50×109/L) thrombocytopenia was seen in 11% of patients on platinum or gemcitabine regimens, 2% of taxane-treated patients, and 5% of anthracycline-treated patients. Patients with NSCLC and ovarian cancer experienced highest incidence of thrombocytopenia. Thirty percent of patients (14,103) developed both anemia and thrombocytopenia during their chemotherapy. More than 90% of patients with thrombocytopenia grade 1–3 also had concomitant anemia grade 1 or 2. Twenty percent of patients with grade 4 thrombocytopenia also presented with anemia grade 3–4. Of these, 16.7% had NSCLC, 16.2% were breast cancer patients, and 9.1% were colorectal cancer patients. However, platelet transfusions were relatively rare (<5%) in these chemotherapy-treated patients.

Conclusions. Thrombocytopenia was relatively common in this large cohort of cancer patients undergoing chemotherapy. A substantial proportion of patients with thrombocytopenia also had anemia.
TPO Mimetics: Can More Than One Agent Be Used in a Patient?
Authors: Cromwell Caroline , Aledort Louis M.
Published: April 2020

Recently, advances in TPO mimetics have expanded therapeutic options for adult patients with chronic ITP. Seven second generation TPO agonists have been reported in the literature. Evidence suggests that non-peptide versus peptide TPO mimetics bind differently to the TPO receptor. Romiplostim, a subcutaneous peptide TPO mimetic has recently been FDA approved. Eltrombopag, Ligand L4665-08, and Aka-Rx 501 are oral non-peptide TPO mimetics undergoing clinical study.

The question arises if a patient fails or succeeds one TPO mimetic can the patient be treated with another agent and have a response. We present two cases of patients who were treated with two agents.

Patient #1 was initiated on Aka-Rx 501 as she could not tolerate steroids and refused splenectomy. She was able to maintain a platelet count >50,000 for a period of six months without any bleeding. As the study did not have an extension, following a washout period she was enrolled in Ligand L4665-083 with a platelet count of 1,000. Within a week her platelet count rose to 97,000. Her platelet count, on a stable dose of Ligand progressively fell over two weeks and dropped to 2,000 with bleeding. She was then initiated on a higher dose of study medication.

The second patient had an eight year history of ITP refractory to IVIG and steroids. She was initially enrolled in AMG 531, and was able to maintain a platelet count greater than 50,000 for over six months. Her platelet count began to progressively decline to zero. Antibody tests were negative and a bone marrow biopsy was normal.

After a washout period, the patient was enrolled in AkaRx 501 Cl-003. One week later her platelet count rose to 64,000. She sustained a platelet count of greater than 30,000 and was initiated on the rollover study after four weeks. At week twelve, her platelet count as in the prior study fell to 0 with a concomitant CNS bleed.

These two cases demonstrate the use of two different TPO mimetics in a given patient. The first patient was treated with two oral non-peptide TPO mimetics, with a sustained response to one agent and a quick response followed by a rapid loss of response to the second agent. The second patient was treated initially with a peptide TPO mimetic followed by a non-peptide TPO mimetic, both times demonstrating an initial brisk response, long with one and short-lived with the second, which was eventually lost. These data suggest that more than one agent can be used in a given patient.
Use of the Platelet Indices for Differential Diagnosis of Pediatric Immune Thrombocytopenic Purpura (ITP)
Authors: Nakadate Hisaya , Kaida Miho , Furukawa Shinsuke , Ishii Masahiro , Higashihara Masaaki
Published: April 2020

Introduction: The prognosis of pediatric immune thrombocytopenic purpura ( ITP ) has a favorable prognosis, about 80 % of ITP children will be cured in 6 months with or without therapy, the rest of patients develop to chronic ITP. However, it is difficult to predict the course of this disease at the time of diagnosis. Recent advances of automated blood cell analyzer enable to measure various blood cell parameters. Among these parameters, platelet indices such as mean platelet volume (MPV), platelet size deviation width (PDW) and platelet-large cell ratio (P-LCR) has brought about some information for thrombocytopenia. We studied the significance of such platelet indices in the differential diagnosis of immune thrombocytopenic purpura ( ITP ) and their ability to discriminate the clinical courses of ITP.

Methods: Ninety-three patients were enrolled this study at our institution from 2004 to 2006. Their ages ranged from 1 to 17 years ( media 4.3 years ). Of 93, patients were divided into two goups for analysis, according to the diagnosis of ITP. 34 were diagnosed as ITP and 59 were diagnosed various blood diseases other than ITP. In these 59 non-ITP patients, five were with thrombocytopenia ( platelet count < 100.0 × 109/L ), the other without thrombocytopenia. Of 34 ITP patients, 29 were acute subtype and 5 were chronic. The Sysmex-XE2100 automated blood cell analyzer (Sysmex, Kobe, Japan) was used to measure the blood parameters, including the platelet indices.

Results: According to MPV, PDW and P-LCR, there were no significant differences between ITP patients and non-ITP. In children with ITP, there were significant inverse correlations between the platelet count and evaluated parameters, MPV (R2=0.376), PDW (R2=0.26) and P-LCR(R2=0.338). But no significant correlations were found between the platelet count and these evaluated platelet indices in non-ITP. Also, we have found all platelet indices were significantly higher in chronic ITP than in acute ITP. In particular, PDW (9.910 vs 13.080) and P-LCR (21.071 vs 33.220) showed marked differences between the two groups.

Conclusions: ITP is considered as a result of hyper-destruction of platelets. Increase of platelet count is regarded as decrease of destruction of platelets. Our data suggested that platelet indices, MPV, PDW and P-LCR showed the useful information for the degree of platelet destruction and the ability to predict the clinical courses of ITP.
Thyroid Disorder-Related Immune Thrombocytopenia: Clinical and Laboratory Characteristics
Authors: Cheung Eric M. , Naik Rahul , Keng Michael , Liebman Howard A.
Published: April 2020

Thyroid disorders have been associated with immune thrombocytopenia. However, there is little published data regarding the clinical and laboratory manifestations of patients with hyper- and hypothyroidism-related thrombocytopenia (Th-ITP) compared with those with euthyroid chronic immune thrombocytopenia (E-ITP). We reviewed the medical records for 135 patients who fulfilled American Society of Hematology criteria for chronic ITP.

Results: Of the 135 patients, 71 (52.6%) were screened for thyroid disorders. Of these 71 patients, 17 (23.9%) were diagnosed with thyroid disorders, including 5 (29.4%) with overt hyperthyroidism, 5 (29.4%) with subclinical hyperthyroidism, 4 (23.5%) with overt hypothyroidism, and 3 (17.6%) with subclinical hypothyroidism. Four patients were diagnosed with thyroid disorders prior to their diagnosis of immune thrombocytopenia while 13 were diagnosed after. The duration of ITP follow-up was similar for the E-ITP (46.1 mo; range: 1– 333 mo) and Th-ITP patients (50.5 mo; range: 1– 173 mo). Age (49.9 yrs for Th-ITP vs. 45.9 for E-ITP), gender (82.3% females for Th-ITP vs. 70.3% for E-ITP), initial platelet counts (17.27 × 109/L for Th-ITP vs. 18.87 × 109/L for E-ITP), and initial bleeding manifestations (64.7% for Th-ITP vs. 66% for E-ITP) were not significantly different between the two groups. Significantly fewer Th-ITP than E-ITP patients had platelet counts <50k at diagnosis (76.5% vs. 85.6%, p = 0.021). There was a trend for Th-ITP to have platelet counts <50k at the end of follow-up (23.5% vs. 8.5%, p = 0.064), despite having a similar duration of follow-up and receiving a similar numbers of individual therapies (2.82 vs. 3.13). However, fewer Th-ITP patients underwent splenectomy (17.65% vs. 38.98%, p = 0.087). Lupus anticoagulant and anti-cardiolipin antibodies were more common in Th-ITP than E-ITP patients, although this difference was not statistically significant (40% vs. 26.2%; p=0.086).

Conclusions: Thyroid disorders are commonly observed in patients with immune thrombocytopenia, but do not appear to significantly affect the response to therapy or long-term outcome in ITP patients.
The Investigation of the Clinical Values of Platelets Hemostatic Activity on Predicting Prophylactic Platelet Transfusion
Authors: Ding Jia-hua , Qian Hou-ming , Huang Cheng-yin
Published: April 2020

Objective To investigate the clinical values of platelets hemostatic activity on predicting the bleeding risk due to thrombocytopenia and threshold of prophylactic platelet transfusion.

Methods Activated plasma clotting time (APCT) and thrombelastography (TEG) were used to evaluate the platelets hemostatic activity of 127 patients with platelet count (Plt)<50×109/L.37/127 patients with bleeding symptoms and 90/127 patients without were involved in the test.4.5ml venous blood was collected in the tubes with 0.5ml 3.2% sodium citrate and detected the Plt,APCT and TEG.

Results Plt and APCT in the bleeding group and the non-bleeding group were(7.9±3.6)×109/L,104.0±6.1s and(23.0±10.1)×109/ L,68.4±9.4s, respectively. There were statistically significant differences between the two groups, P<0.01. The APCT was negatively correlated with Plt,r=−0.7613. The TEG maximum amplitude (TEG-MA)were 20.5±5.3mm in the bleeding group(n=15)and 43.4±6.3mm in non-bleeding group(n=20),p<0.01. Using APCT≥90s,TEG-MA≤30mm as the cut-off point to predict the bleeding due to thrombocytopenia, the sensibility and specificity were 100%,98.9% and 100%,93.3%, respectively. Using Plt ≤20×109/L, the sensibility was 100% and the specificity was 56.7%.

Conclusion The specificity of APCT or TEG-MA for predicting bleeding due to thrombocytopenia was better than that of Plt. APCT≥90s or TEG-MA≤30mm as trigger for prophylactic platelet transfusion would effectively prevent bleeding due to thrombocytopenia.
Platelet, CD40/CD40L, MPS-Cells: Partners in Crime
Authors: Corinaldesi Giorgio , Corinaldesi Christian
Published: April 2020

Several suggestions showed a strong correlation of platelet and CD40/40L; CD40/40L is a member of TNF superfamily, the genes is located on chromosome X, and it is expressed on endothelial cells, SMC, fibroblast and activated platelet and plays a pivotal role in inflammatory and expression of adhesion molecules, and it stimulates the release of cytokines. It helps identifing patients with atherosclerosis and an increased risk of thrombosis, peripheral and cardiovascular disease (CAD and CHF). CD40/40L was measured by flow cytometry, and s-CD40L by ELISA. In platelet activation CD40L is translocated to the platelet surface and it is accompanied by the release of sCD40L and RONS (reactive ossigen and nitrogen species) and this contributes to atherothrombotic disease and inflammatory process by forming (CD40-40L/GPIIb-IIIa, CD40-40L/Pselectin- PSGL) complex strictly correlated with levels of ICAM-1 and VCAM-1, induced TF-expression and release of MMPs. Activated platelets release chemokines, enhance adhesion and aggregation cytochine like-factors and coagulation factors. Patients with thrombotic events had more platelet-monocyte aggregate (36.8 +/− 12.4 %) than controls (14.2 +/− 2.1 %) and sCD40L above 4.5 ng/l, ICAM-1 levels 248 +/− 10.8 ng/ml vs 168 +/− 5.4 ng/ml, VCAM-1 764 +/− 18.6 ng/ml vs 532 +/− 8.6 ng/ml. The interaction of platelet and MPS-cells were enhanced by activation of P-Selectin 38.6 +/− 12.4 ng/ml vs 20.4 +/− 4.4 ng/ml, and MCP-1 286 +/−10.4 pg/ ml vs 132 +/− 5.8 pg/ml which are one of the major promoter of inflammation. This finding demonstrated that the development and progression of atherosclerosis is critically dependent on these molecules; this may propose new therapeutic target for future therapies.
The Effect of Omega-3 as a Dietary Supplement in a Mouse Model of Stem Cell Transplantation (SCT)
Authors: Al-Hashmi Sulaiman Ali , Sadeghi Behnam , Hassan Zuzana , Lindskog Magnus , Hassan Moustapha
Published: April 2020

Introduction: Several studies reported the importance of nutrition on SCT outcome. Many food components have been shown to affect the immune system and to interact with a number of drugs used in conditioning and in prophylactic treatment during SCT. Omega-3 fatty acids have immuno-modulatory effects via improving endothelial function, protection against oxidative DNA damage and by suppressing CD4+ T-cell function and proliferation. In the present study we examined the effects of omega-3 on the immune system in mice undergoing SCT.

Method: Female BALB/c mice, 8 – 12 weeks, were randomised to receive either diet enriched with menhaden fish oil (omega-3) or, corn oil (rich in omega-6). Both groups were fed for two weeks prior to conditioning (Day 0 was set to be the day of transplantation). Both groups injected intra-peritoneally with 80mg/kg busulfan (Bu) for four days (day –7 to day –4) followed by 200mg/kg cyclophosphamide (Cy) for two days (days –3 & -2). At day 0, half of the mice of both groups were killed and bone marrow, spleen and thymus were harvested and analysed with FACS to evaluate the lymphoid and myeloid cell phenotype. Serum was collected for analysis of cytokines. The remaining mice in both groups were transplanted with allogeneic bone marrow from 8 – 12 weeks male C57BL/6 mice (2.0 × 107 bone marrow cells and 3.0 × 107 spleen cells).

Results: Mice fed an omega 3-rich diet showed significant decrease in bone marrow and spleen cellularity compared to that observed in the corn oil group. In particular, the splenic B-cell population was severely affected, showing a 50% decrease in the omega-3 group compared to that seen in corn oil group. The T-cell populations of omega-3 fed mice were also affected, with a significant decrease by 10 to 20 % in the number of CD4+ cells. In contrast, no difference was observed for CD8+ cells, compared to corn oil-fed mice. In the thymus, the number of activated T-cells was decreased by 50% in the omega-3 compared to that observed in corn oil group. NK cells were decreased by 50% in the spleens of omega-3 fed mice. In bone marrow, the myeloid cells (CD11b+) were significantly lower in omega-3 fed mice compared to corn oil-fed controls. All transplanted mice that had received an omega-3 enriched diet died within the first week. These data can be compared to our previous results where more than 60% of mice receiving standard diet survive during the first week and of which at least 20% reach day+21.

Conclusion: Omega-3 administration as a dietary supplement for two weeks prior to conditioning regimen showed significant effects on the immune system in terms of a pronounced alteration in the balance of different cells lineages in both bone marrow and spleen. Rapid death of omega-3 fed animals after allogeneic transplantation was observed. Further studies are warranted to investigate the role of omega-3 on the outcome of stem cell transplantation.
Anti-CD3 Preconditioning Separates GVL from GVHD Via Modulating Host Dendritic and Donor T Cell Migration in TBI-Conditioned Recipients
Authors: Li Nainong , Chen Ying , He Wei , Yi Tangsheng , Zhao Dongchang , Zhang Chunyan , Lin Chia-Lei , Forman Stephen , Zeng Defu
Published: April 2020

Host dendritic cells (DCs) play a critical role in initiating graft versus host disease (GVHD) and graft versus leukemia (GVL), and separation of GVL from GVHD remains a major challenge in the treatment of hematological malignancies by allogeneic hematopoietic cell transplantation (HCT). Here, we show that preconditioning with anti- CD3 mAb before conditioning with total body irradiation prevents GVHD but retains GVL in a HCT model of MHC-mismatched C57BL/6 donor to BALB/c host. Prevention of GVHD is associated with inhibition of donor T expression of homing and chemokine receptors and inhibition of GVHD target tissue expression of chemokines. Furthermore, inhibition of donor T expression of gut homing a4b7 and chemokine receptor CCR9 by anti-CD3 preconditioning results from reduction of CD103+ DCs in draining mesenteric lymph nodes, which is associated with downregulation of DC expression of CCR7, a receptor required for tissue DC migration to draining LN. These results indicate that anti-CD3 preconditioning reduces not only tissue release of chemokines but also prevents tissue DC migration to draining LN and subsequently reduces draining LN DC’s capacity in imprinting donor T tissue tropism. Therefore, modulation of host DCs by anti-CD3 preconditioning before HCT represents a new approach for separating GVL from GVHD. (Li and Chen contribute equally.
Association Between Acute Graft Versus Host Disease and Lung Injury after Allogenic Hematopoietic Stem Cell Transplantation
Authors: Liu Qifa , Luo Xiaodan , Ning Juan , Xu Dan , Fan Zhiping , Sun Jing , Zhang Yu , Xu Bing , Wei Yongqiang
Published: April 2020

To investigate the characteristics of chest computed tomography (CT) and pathogenesis of acute graft versus host disease(GVHD)-induced lung injury after allogenic hematopoietic stem cell transplantation. In 47 patients with acute GVHD of grade II–IV, CT showed abnormalities in 20 patients in which 17 cases were suspected of acute GVHD-induced lung injury. Of 17 patients, CT revealed diffused interstitial infiltrate in 5 cases, diffused interstitial and alveolar infiltrate in 7 cases,diffused interstitial and segmental lobar alveolar infiltrate in 5 cases. The levels of serum IFNγ and TNFα in patients were higher than those of the healthy group. The histopathology of lung showed disorganization, epithelial cell damage, interstitial fibroplasias and interstitial T cell or macrophage infiltrate. In 24 patients who survived more than 6 months, 3 cases each in cases with and without lung injury developed late-onset noninfectious lung injury; 11 patients had abnormal pulmonary function including 7 patients in 9 patients with lung injury and 4 patients in 15 patients without lung injury. We concluded that T cell, macrophage, IFNγ and TNFα might play a role in the pathogenesis of acute GVHD-induced lung injury. Acute GVHD-induced lung injury might progress to late-onset noninfectious lung injury.
Construction and Pre-Clinical Evaluation of An Anti-CD19 Chimeric Antigen Receptor
Authors: Kochenderfer James , Feldman Steven , Zhao Yangbing , Xu Hui , Black Mary , Morgan Richard , Dudley Mark , Wilson Wyndham , Rosenberg Steven
Published: April 2020

Adoptive T cell immunotherapy can cause regression of established malignancy. One promising approach is to transfer genes encoding chimeric antigen receptors (CARs) that specifically recognize tumor-associated antigens to T cells before the T cells are adoptively transferred to patients. We have constructed a CAR that consists of an anti-CD19 single chain variable region (scFv) that is coupled to a portion of the CD28 costimulatory molecule and the signaling component of the CD3-zeta chain. CD19 is a promising target for immunotherapy because most malignant B cell express CD19, but the only normal cells that express CD19 are B cells, B cell precursors, and perhaps follicular dendritic cells. We have demonstrated that gamma-retroviruses encoding the anti-CD19 CAR can be used to transduce human T cells and that these transduced T cells specifically recognize CD19+ targets. To transduce T cells, we stimulated T cells with the anti-CD3 monoclonal antibody OKT3 on day 0 then conducted sequential retroviral transductions on day 2 and on day 3. Transductions were performed by spin-loading retroviruses onto RetroNectin (Takara) coated culture plates followed by overnight incubation of the OKT3- stimulated T cells on the plates. Forty-five to sixty-seven percent of T cells expressed the anti-CD19 CAR as measured by flow cytometry 7–8 days after transduction (n=8). Anti-CD19-CAR-transduced CD8+ and CD4+ T cells produced IFNg and IL-2 specifically in response to stimulation with CD19+ target cells. The transduced T cells specifically killed primary chronic lymphocytic leukemia (CLL) cells. T cells from CLL patients that were either untreated or previously treated with fludarabine plus rituximab could be transduced and induced to proliferate sufficiently to provide enough cells for clinical adoptive T cell transfer. In addition, we adapted this protocol for use in CLL patients with very high peripheral blood leukemia cell counts by depleting CD19+ cells using magnetic bead sorting prior to OKT3 stimulation. In preparation for a clinical trial that will enroll patients with advanced B cell malignancies, we have generated a producer cell clone that produces GALV (Gibbon ape leukemia virus)-enveloped gamma-retroviruses encoding the anti-CD19 CAR, and we have produced sufficient retroviral supernatant for the proposed clinical trial under good manufacturing practice (GMP) conditions.
Murine Leukemias with Insertional Mutations at Lmo2 Are Highly Predicitive of Leukemias Induced Following Gene Therapy in SCID-X1 Patients
Authors: Dave Utpal P. , Akagi Keiko , Tripathi Rati , Cleveland Susan , Thompson Mary , Yi Ming , Stephens Robert , Downing James R. , Jenkins Nancy , Copeland Neal G.
Published: April 2020

Five SCID-X1 patients successfully treated with autologous bone marrow stem cells infected ex vivo with an IL2RG-containing retrovirus subsequently developed T-cell leukemia and four contained insertional mutations at LMO2, a known T cell oncogene. Indirect genetic evidence suggests a role for deregulated IL2RG expressed from the retroviral vector in tumor formation, although this remains controversial. Here we show that the genes and signaling pathways deregulated in murine leukemias with retroviral insertions at Lmo2 are strikingly similar to those in human leukemias with deregulated LMO2 and are highly predictive of the leukemias induced following gene therapy in SCID-X1 patients. We also provide genetic evidence suggesting that deregulated IL2RG and LMO2 expression cooperate to induce leukemia but are not sufficient and require additional cooperating mutations. The highly concordant nature of the genetic events giving rise to mouse and human leukemias with mutations at Lmo2 are an encouraging sign to those wanting to use mice to model human cancer and may help in designing safer methods for retroviral gene therapy.
Lipid Emulsion as Antisense Oligonucleotides Vector That Inhibits P-Glycoprotein Expression in a Sarcoma Cell Line through LDL Receptor
Authors: Levy Debora , Debes Adriana Aguiar , Celestino Andrea Turbuck , Schreier Shirley , Maranhão Raul , Bydlowski Sergio Paulo
Published: April 2020

It is well known that antisense oligonucleotides (OAS) are able to inhibit gene expression in a sequence-specific way. The potencial use of an artificial lipid nanoemulsion (LDE) as a vector to carry OAS has been described. LDE was shown to bind 3’-cholesteryl- OAS (C-OAS) and to be internalized into cells through LDL receptors. Here we further explore these findings by examining the capacity of this complex to inhibit MDR-1 gene expression in a sarcoma cell line (MES-DX), which express P-glycoprotein. LDE was prepared as described (Bydlowski et al. ,1995). The capacity of LDE to bind C-OAS was examined by fluorescence spectra analysis using a Hitachi F4500 fluorimeter. Human plasma was obtained from healthy blood donors and LDL, HDL and lipoprotein free serum (LPDS) were separated by sequencial ultracentrifugation. C-OAS/LDE complex was incubated with HDL (apoE donor) before cell experiments were performed. Binding of [3H] LDE/C-OAS complex to LDL receptors from MES-DX cells was studied by competition assay. Two different C-OASs, both complementary to regions flanking the AUG initiation codon were used. Inhibition of MDR-1 gene expression was evaluated by RT-PCR. The binding constant for C-OAS/LDE was 4,2 × 10−3M−1 indicating a high specific capacitiy of conjugation.The C-OAS/LDE complex was shown, by the competition assays and confocal microscopy, to bind to LDL receptors and then to be internalized into cells. Both C-OAS/ LDE complexes strongly inhibited (70% inhibition) the MDR-1 gene expression after 48 hours of cell incubation. This inhibition was not observed when LDE was used alone or complexed with scrambled OAS sequences. The results show that this nanoemulsion binds to cholesteryl-OASs. Moreover, this nanoparticle is an efficient carrier for OAS to target cells expressing LDL receptors. This complex is able to internalize oligonucleotides into cells specifically through the LDL receptor-mediated pathway. The internalized ODN was able to act on nucleic acid sequences as determined by the inhibition of MDR-1 gene expression. Therefore, LDE/C-OAS is promissing nanoparticle complex to be used in gene therapy studies.
Bone Loss Study in Type 1 Gaucher Disease by Two Different Technologies
Authors: Roca Mercedes , Latre Paz , Alfonso Pilar , Irun Pilar , Civeira Ignacio , Meriño Erardo , Pocovi Miguel , Giraldo Pilar
Published: April 2020

Background: Bone disease is the most important source of disability in type 1 Gaucher disease (GD1). High percentage of patients present generalized osteopenia and osteoporosis with reduced bone mass. It is important to know the risk of bone mass lost in order to avoid the bone resorption. The standard technique to evaluate bone disease is Dual-Energy X-ray Absorptiometry (DEXA) that measures bone density in the axial skeleton. Nevertheless other procedure to exam bone density using ultrasound (US) can measure the amount of mechanical energy transmitted by the bone, as well as their speed of transmission (BUA).

Aims: In order to evaluate bone disease indicators in GD1, we have planned a comparative analysis in GD1 by both techniques to exam individual variability in bone mineral density and to study the response to therapy.

Patients and Methods: 24 adults GD1 patients diagnosed in last 5 years and 15 healthy controls stratified by age and gender were analyzed. Bone marrow changes have been evaluated by a semi quantitative score (S-MRI, Roca et al 2006 Eur J Radiol). To analyse the bone density we have used the Z-score quantification in L5 and femur head, calcaneus US was performed simultaneously determined BUA and bone density, in addition GBA genotype, clinical characteristics and subrogate biomarkers profile included chitotriosidase, CCL-18, MIP- 1b were analyzed. The advantages/disadvantages of each procedure and the correlation of results with the clinical characteristics, S-MRI, response to therapy, have been analyzed.

Results: Osteopenia in GD is present in 85% of cases, it progresses with age and has been correlated with the severity of the disease and response to therapy. BMD evaluation by DEXA in GD1 is well documented but the special physio-pathological characteristics of GD make the use of US feasible and BUA results provide more information about the risk of skeletal complications.

Comments: More experience is needed in order to precisely define the role of bone US exam in the management of GD patients. This work has been partially sponsored by FIS 07/90938 and CIBERER U-752
Dose Delay Amongst Cancer Patients Undergoing Chemotherapy
Authors: Wu Ying , Aravind Suresh , Nalysnyk Luba , Ranganathan Gayatri
Published: April 2020

Background. Cancer patients often have their chemotherapy dose delayed or reduced to mitigate chemotherapy-induced side effects such as thrombocytopenia, neutropenia, anemia, etc. In situations where the patient is responding to the chemotherapy, such dose delay and dose reduction may minimize the effectiveness of the chemotherapy and lead to poorer clinical outcomes. In this study, we analyzed the characteristics of dose delay among cancer patients on chemotherapy in the US.

Methods. Pooled data over the period 2000–07 from a large electronic database of 15 community and 3 hospital-based outpatient oncology clinics in the US were analyzed. Adult patients with a cancer diagnosis were grouped as: lung, breast, ovarian, H&N, colorectal, other solid cancers, hematologic and mixed cancers. Chemotherapies used were grouped into platinum, anthracycline, gemcitabine, taxane-based and other regimens. Among patients with complete chemotherapy plan information, each subsequent cycle of the chemotherapy regimen after the first cycle was examined. The planned cycle duration was compared to the actual duration between the start of one cycle and start of the next. Any delay of more than 7 days was defined as a cycle delay. Demographic and clinical characteristics of patients with delays were further analyzed.

Results. The dataset included 47,159 cancer patients (mean age 61, male 42%) and 75,243 chemotherapy regimens. A total of 26,317 patients (113,175 chemo cycles) had complete chemotherapy plan information in the database. Of these, 5,961 patients (22.7%) experienced chemo delays. A total of 9,293 chemotherapy cycles (8.2%) were delayed. The average chemo delay was 17 days (SD, 8 days; median, 14 days). 25% of the delays were more than 3 weeks. Tumor types of the 5,961 patients with cycle delays included, colorectal cancer 20.3%, NSCLC 18.6%, breast cancer 15.2%, ovarian 4.4%, and H&N 2.9%. The reasons for delaying chemo were not specified in the database. However, 60% of these patients experienced anemia (Hb<11g/dL) and 62% had thrombocytopenia (<150,000/μl) at some point during their chemotherapy. Baseline and clinical characteristics of patients with chemo delays were similar to the overall population in the database.

Conclusions. Chemotherapy cycle delays were relatively common in this large group of cancer patients. While the decision to delay therapy was multi-factorial, further research is needed to investigate the reasons, preventable measures, clinical and cost outcomes of such delays.
Measurements of Heterogeneity of Bone Marrow Cellularity
Authors: Goldenberg Alec , Haglof Karen , Kelley Priscilla , Davis Geri , Liu Cynthia , Ibrahim Sherif
Published: April 2020

Introduction: Quantifying cellularity is an integral component of bone marrow examinations. Estimates of marrow cellularity may influence the diagnostic interpretation of bone marrow samples. The accuracy of cellularity estimates may be influenced by the variable distribution of cellular elements within the marrow space. To better understand the degree of heterogeneity of bone marrow cellularity, we undertook a study to quantify the variable distribution of bone marrow cells in bone marrow core biopsies.

Method: 8 gauge bone marrow core biopsies of 20 patients were retrospectively reviewed by 2 hematopathologists (SI,CL). The specimens were recovered from the posterior superior iliac crest using standard technique with 8G Snarecoil biopsy needles by 3 operators (KH, PK, GD). The percent cellularity was determined in sequential 0.2 X 0.4 cm portions of the core biopsies by each of the hematopathologists. Cellularities were recorded in 10% increments.

Results: The mean age of the patients was 73.2 ± 1.8 years. There were 12 males and 8 females. 5 patients had monoclonal gammopathies. Anemia, multiple myeloma and thrombocytopenia were each diagnosed in three patients. 2 patients demonstrated myelodysplasia and 1 patient each had acute leukemia, leukocytosis, non-Hodgkin’s lymphoma and thrombocytosis. The mean white blood cell count, hemoglobin and platelet count were 8.7 (range 3.8–42.8), 12.2 (range 10.1–15.3), and 233 (range 91–226), respectively. The mean length of the core biopsies was 1.78 ± 0.09 cm (range 1.4–3.3) and the median number of 0.2 × 0.4 cm portions examined per core biopsy was 8 (range 5–12). In total, 165 portions were examined by each hematopathologist independently. The cellularity of 12 and 11 portions could not be determined by each of the hematopathologists, respectively, as a result of biopsy artifacts. No core biopsy showed a consistent cellularity within the examined portions, each core demonstrating a range of cellularities. Only 2/20 and 1/20 of the core biopsies, as examined by each hematopathologist, respectively, demonstrated 2 consistent cellularities. A median of 4 different cellularities were identified in each core. The mean range of cellularities of each core’s portions was 43 ± 4.6 %, and 46.5 ± 4.9 %, as determined by the 2 hematopathologists, respectively, which was statistically equivalent (paired t-test p=0.349).

Conclusions:

Bone marrow cellularity is heterogeneous. Cellularities of different portions of core biopsies vary significantly. Average differences between minimal and maximal portional cellularities may be as high as 46%. The accuracy of bone marrow cellularity measurements is related to the size of the biopsy specimen.
Pharicin a, a Novel Natural Compound That Induces Mitotic Arrest and Catastrophe of Cancer Cells by Perturbing Microtubule Dynamics and the Spindle Checkpoint
Authors: Chen Guo-Qiang , Dai Wei , Xu Han-Zhang , Li Dao
Published: April 2020

Microtubule poisons such as taxol represent a potent and effective class of anticancer compounds. In the past decades, great efforts have been directed to identify novel natural products with a mode of action similar to taxol but with a minimal side effect. In this study, we report the functional characterization of a new ent-kaurene diterpenoid termed pharicin A, which was originally isolated from Isodon pharicus leaves, a perennial shrub frequently used in Chinese folk medicine for tumor treatment. Pharicin A induces mitotic arrest in Jurkat, Raji and HeLa based on their morphology, DNA content, histone H3 serine-10 phosphorylation, and mitotic marker protein analyses. Pharicin A stabilizes the formation of mitotic spindles, which is coupled with a rapid accumulation of Cdc20 as well as an increased phosphorylation of Cdc27 and BubR1. Pharicin A treatment also results in an enhanced interaction between Cdc20 and spindle checkpoint components including Mad2 and BubR1. Moreover, pharicin A-induced mitotic arrest in HeLa cells is tightly associated with the presence of lagging/missegregated chromosomes at spindle pole regions, which are highly enriched in BubR1, CENP-E and Sgo1. Although pharicin A stabilizes microtubules both in vitro and in vivo, it induces mitotic arrest in taxol-resistant Jurkat cells. Combined, our study strongly suggests that pharicin A represents a novel class of small molecule compounds capable of perturbing microtubule dynamics and the spindle checkpoint in tumor cells, which merits further preclinical and clinical investigations for cancer drug development.
An in Vitro and in Vivo Evidence That Human Rhabdomyosarcomas (RMS) Express Functional CXCR7 Receptor: Overlapping and Distinct Role of CXCR4-SDF-1 and CXCR7-SDF-1/ITAC Axes in Regulating Metastatic Behavior of RMS Cells
Authors: Grymula Kasia , Tarnowski Maciek , Maksym Radoslaw , Kucia Magdalena , Ratajczak Janina , Ratajczak Mariusz Z
Published: April 2020

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of adolescents and children that frequently infiltrates the bone marrow (BM). We have demonstrated that the a-chemokine stromal-derived factor (SDF)-1-CXCR4 axis (Blood2002;100:2597, Cancer Res. 2003;63:7926, Cancer Res.2007;67:2131) plays an important role in RMS metastases. With the recent description of CXCR7, a new receptor for SDF-1 that also binds the interferon-inducible T cell-alpha chemoattractant (ITAC) chemokine, we become interested in the role of the CXCR7-SDF-1/ITAC axis in RMS metastasis. To address this issue, we evaluated 7 highly metastatic alveolar RMS (ARMS) and 3 less metastatic embryonal RMS (ERMS) cell lines. We found that all RMS cell lines express CXCR7 in contrast to CXCR4, which was expressed by 7 of 10 RMS cell lines only. CXCR4 was expressed at a much higher level by highly metastatic ARMS lines while CXCR7 was expressed at a high level on the less metastatic ERMS lines. More importantly, CXCR7 receptor on RMS cell lines was functional after stimulation with ITAC and SDF-1 as evidenced by MAPKp42/44-, AKT-, and STAT-3 phosphorylation, chemotaxis, cell motility, and adhesion assays. We also noticed that CXCR7 undergoes rapid internalization after stimulation with SDF-1 and ITAC, is a lipid raft-associated receptor, and, surprisingly, has a downregulated expression in response to hypoxia. We also noticed that similarly to CXCR4, signaling from activated CXCR7 was not associated with increased RMS proliferation or cell survival. Thus, we conclude that both CXCR4 and CXCR7 are not affecting proliferation of RMS cells; however they play an important role in both SDF-1- and ITAC-directed metastasis. For the first time, we also identify ITAC as a new regulatory chemokine that is actively involved in this process. In conclusion, since CXCR7 is more important in adhesion and becomes down-regulated during hypoxia, this changes the balance in SDF-1 signaling through both receptors (CXCR7/CXCR4). As a result, SDF-1 activates more robust CXCR4 receptor, which promotes chemotactic responses of RMS cells. As a biological consequence, this switch in SDF-1 receptor signaling increases the metastatic potential of RMS by mobilizing tumor cells to egress from the tumor into the peripheral blood and lymph and migrate to new tissue locations. We also conclude that targeting of the CXCR-SDF-1 axis alone without simultaneous blockage of CXCR7 will be an inefficient strategy to inhibit metastasis of RMS cells. This is currently being tested in our laboratories in immunodeficient mice inoculated with human RMS cells.