bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Sample Preservation Method on Gene Expression and RNA Integrity in Acute Myelogenous Leukemia.
Authors: Ben-Ezra Jonathan , Ladd Amy C. , Dumur Catherine I. , Archer Kellie J. , Chesney Alden , Ferreira-Gonzalez Andrea , Wilkinson David S. , Garrett Carleton T.
Published: April 2020

Cryopreservation of patient bone marrow specimens allows for future purification of tumor cells for research or diagnostic purposes. It has not been determined whether this practice causes significant changes in gene expression. In order to evaluate this, we performed microarray analysis (Affymetrix U133A and U133A 2.0 gene chips) on mononuclear cells from 5 acute myelogenous leukemia (AML) patient samples (>70% blast count) that had been either a) cryopreserved, b) snap frozen, or c) resuspended in TRIzol and stored at −80°C. Samples remained frozen for a period of 1–4 weeks. While gene expression changes between groups were minor, there were some differences. Thirteen probe sets between TRIzol and cryopreserved samples, and 60 probe sets between TRIzol and snap frozen samples, varied significantly (>2-fold difference and p<0.01; t-test). While both non-TRIzol samples lost transcripts associated with mature, contaminating granulocytes, the snap frozen samples also lost transcripts associated with cell growth and metabolism. To determine whether the observed changes were enough to cause a misclassification of AML subtypes, we performed an unsupervised cluster analysis on the 15 original samples plus 15 new AML samples (13 unique cases), all of varying subtypes. RNA from all of these samples was undegraded. All samples passed strict quality control parameters, not limited to but including tumor content, % rRNA, cDNA and cRNA synthesis product size, and GAPDH 3′-5′ ratios. Differentially preserved samples originating from the same patient segregated together during the clustering process regardless of preservation method. Three main clusters emerged; cluster #1 (FAB-M0 and -M1, n=3), cluster #2 (FAB-M3, n=5), and cluster #3 (FAB-M2, -M4 and -M5b, n=22). In cluster #3, there were three sub-groupings. One group contained only M4 and M5b subtypes, another contained only the M2 subtype, and the third contained both M2 and M4 subtypes. RNA isolated from snap frozen samples was sometimes moderately to severely degraded. When we examined three snap frozen samples, not part of the above data set, exhibiting moderate RNA degradation, they all clustered incorrectly according to the above established groups. Given these results, we conclude that cryopreservation is an acceptable method of cell preservation for gene expression analysis, but snap frozen samples should be closely evaluated for RNA degradation before using in microarray analyses.
Characterization of Autoantibodies to PF4/Heparin in a Rat Model of Cardiopulmonary Bypass.
Authors: Arepally Gowthami M. , Homi H. Mayumi , De Lange Fellery , McCracken Emily K.E. , Goss Tina M. , Suvarna Shayela , Grocott Hilary P.
Published: April 2020

Patients undergoing cardiopulmonary bypass (CPB) represent a high-risk population for developing life-threatening complications of Heparin-Induced Thrombocytopenia and Thrombosis (HITT). Approximately 30-70% of patients undergoing CPB develop heparin dependent autoantibodies to PF4/heparin, which can provoke disease in susceptible individuals. To address the clinical relevance of bypass associated anti-PF4/heparin, we have created a rat model of autoimmune PF4/heparin in the setting of CPB. Sprague-Dawley rats were prepared for CPB using cannulae in the right external jugular vein for venous return to the CPB circuit and the ventral tail artery for arterial inflow. Rats were administered 0.3–0.4U/g heparin i.v. after cannulation, and were then connected to a miniaturized CPB system complete with a membrane oxygenator primed with 40 ml of blood from 2 syngeneic donors (each receiving 0.3U/g i.v. heparin). CPB was maintained for 75 minutes with a flow between 160–180 mls/kg/min (normal cardiac output in the rat) after which animals were decannulated and sacrificed for study over the ensuing 6 weeks. Control rats were subjected to a sham operation for which they received the same dose of heparin, as well as arterial and venous catheterization, but were not placed on CPB. Blood was collected from control animals or rats after CPB and tested for autoantibodies to rat PF4/heparin (rP+H) by ELISA. Rats were studied either after sham operation (n=6) or after CPB at the following time points: 15 minutes (n=15), 3 days (n=6), 16 days (n=25), and at 6 weeks (n=8). Rats undergoing CPB had markedly elevated anti-rP+H levels (as gauged by mean A450 nm ± SEM) 16 days after the procedure as compared to rats receiving sham operation (0.211 ± 04, 16 day CPB vs. 0.01 ± 0.01sham; P = 0<.0001 by ANOVA). High titer antibody responders from day 16 {Rat 77 (A450nm=0.84), Rat 80 (A450nm=0.81) and Rat 70 (A450nm=0.46)} were characterized for reactivity to rPF4 alone, rP+H or rP+H in the presence of excess heparin. Anti-rP+H from all three animals showed maximal reactivity to rP+H, but reduced binding to rPF4 alone or rat P+H in the presence of excess heparin. We additionally tested functional effects of anti-rP+H on rat platelets and human platelets, using a 14C-SRA. Rat platelets were not activated by anti-rP+H or a human HIT-like monoclonal antibody, KKO confirming the absence of activating FcγRII on rat platelets. Human platelets were activated by KKO (60% release), but not rat anti-P+H (<5% release). In conclusion, we demonstrate that autoantibodies occur in heparinized animals subjected to the platelet activating effects of CPB. This animal model will be critical for studying the many facets of bypass- related heparin sensitization, including: the immunobiology of HITT, non-FcR mediated thrombotic effects of anti-P+H, and for developing alternative therapies for HITT patients undergoing CPB.
Desulfated Heparin Blocks Platelet Activation Induced by HIT Antibodies / Heparin: A New Approach to Patient Management.
Authors: Prechel Margaret , Jeske Walter P. , McDonald Meredith K. , Drenth Amanda F. , Walenga Jeanine M.
Published: April 2020

Heparin-induced thrombocytopenia (HIT) represents a disease spectrum triggered by an immune response to heparin. The most dramatic clinical expression of HIT is HIT antibody-driven thrombosis. Direct thrombin inhibitors (DTIs) are a promising new class of drugs for treatment of the acute phase of HIT; however, they have a narrow safety/efficacy window (high bleeding risk) and morbidity/mortality have not been eliminated. In addition, the high probability of developing thrombosis in HIT combined with extreme mortality, has led to a bias for prophylactic treatment. Thus, there remains a clinical need to identify optimal treatment options for patients with HIT. A new concept is to use an agent that can effectively compete with heparin in a manner that prevents the HIT antibody from inducing platelet activation, i.e., amelioration. We evaluated a 2-O, 3-O desulfated heparin (ParinGenix, Inc.; Tucson, AZ) to determine its ability to ameliorate HIT antibody/heparin induced platelet activation. The test agent was added to a mixture of known-reactive platelets and pre-formed immune complexes (heparin, PF4, HIT antibodies), and SRA and flow cytometry were performed. Due to the inherent biological variability of HIT antibodies, sera from four different patients (clinically diagnosed as HIT; SRA positive) were used. Two concentrations of heparin which reflect typical prevention (0.1 U/ml) and treatment (0.5 U/ml) clinical doses were used. The 2-O, 3-O desulfated heparin produced an amelioration of HIT antibody/heparin induced platelet activation as demonstrated in the SRA by inhibition of 14C serotonin release from activated platelets, and in flow cytometric analysis by inhibition of platelet microparticle formation and platelet cell surface P-selectin expression. Significant amelioration activity was initiated at 6.25 μg/ml 2-O, 3-O desulfated heparin and complete inhibition of the induced platelet activation (equal to the 100 U/ml heparin HIT assay ‘no response’) was achieved with 50 μg/ml of the agent. Since this new treatment approach blocks platelet activation caused by HIT antibody/heparin (not a characteristic of DTIs), we propose that a non-anticoagulant glycosaminoglycan (GAG) may be useful in improving the clinical management of patients with HIT. The concept of amelioration differs from all previous options for the clinical management of patients with HIT including the use of danaparoid, fondaparinux, DTIs and other drugs that target thrombin/thrombin generation inhibition. Although not directed at platelet activation inhibition, this type of GAG effects an inhibition of HIT antibody mediated platelet activation, which is the source of the pathophysiology of HIT. The data of this study suggest that this 2-O, 3-O desulfated heparin may be effective as either an adjunct or sole treatment of an ongoing HIT pathology, or as a preventive measure in patients who will be exposed to heparin.
A Novel Frameshift Mutation in a Case of Familial May-Hegglin Anomaly.
Authors: Cutler Jacky A. , Mitchell Mike J. , Savidge Geoffrey F.
Published: April 2020

The May-Hegglin anomaly is a rare, autosomal dominant condition that is characterised by thrombocytopaenia, anisocytosis, and Dohlë-like inclusion bodies in leukocytes. The disorder generally results in a mild bleeding tendency. May-Hegglin anomaly results from defects in the MYH9 gene, which encodes the heavy chain of nonmuscle myosin IIA. The gene maps to chromosome 22q12.3-q13.2, comprises 40 exons, and produces a 5.8kb mRNA transcript.

We present a case of familial May-Hegglin anomaly. This kindred was first identified, and published, in 1964; but no further analyses were carried out until recently.

The proband, now aged 80 years, has a life long history of easy bruising and epistaxis, although the symptoms have improved with increasing age. Her sister (deceased) and daughter reported similar symptoms: the daughter requiring splenectomy for thrombocytopaenia at age 9 years. The proband has also suffered from deafness for in excess of 60 years.

Recent investigations of the proband revealed a mild thrombocytopaenia, and the characteristic blood film of May -Hegglin anomaly. Platelet and neutrophil morphology were also consistent with the disorder. Karyotype analysis revealed good quality G banding throughout chromosome 22, and no gross alteration in the region 22q11.2., 22q13. Direct sequencing of the MYH9 gene identified a heterozygous deletion of a single base in a run of Gs at nucleotide positions 5818–5821 (codons 1940–1941). This frame shift mutation leads to the creation of a premature termination sequence at codon 1947, and the loss of the last fourteen amino acids. The deletion introduces a restriction site, and endonuclease digestion of a panel of normals failed to detect this change. Together with the fact that this is a conserved region across species (human, rat, rabbit, chicken), it is probable that this deletion is the causative mutation in this kindred.

This novel mutation represents the second reported defect affecting the tail-piece domain in May-Hegglin anomaly. The protein’s two heavy chains homodimerise in this region to form a tail, and multiple polypeptides associate through this tail domain to form bipolar filaments. Therefore, a mutation in this domain may exert its effect by the inhibition of myosin filament assembly.

To date, approximately 20 different mutations have been described in the MYH9 gene giving rise to not only May-Hegglin anomaly, but also the similar disorders of Fechtner syndrome, Sebastian syndrome and Epstein syndrome. In fact some mutations have been identified in patients with different diagnoses. The proband presented here has a long standing diagnosis of May-Hegglin anomaly, but does exhibit one symptom (deafness) more usually associated with Fechtner syndrome. The genetic, phenotypic and clinical overlap in these disorders suggest that these actually represent allelic variations of a single genetic disorder, which should more correctly be termed ‘MYHIIA syndrome’.
Hepatocyte Growth Factor Ameliorates Chronic Graft-Versus-Host Disease.
Authors: Imado Takehito , Iwasaki Tsuyoshi , Kuroiwa Takanori , Okamoto Takahiro , Hara Hiroshi , Sano Hajime
Published: April 2020

Since chronic graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation (HSCT), a new strategy to prevent chronic GVHD is necessary. We previously showed that hepatocyte growth factor (HGF) protected against lethal effects of acute GVHD and promoted T cell reconstitution in murine models (J Clin Invest107;1365:2001, Blood 2004 in press). We have now investigated whether HGF can protect against chronic GVHD. (C57BL/6 x DBA/2) F1 (BDF1) recipient mice were lethally irradiated (9 Gy) and co-transplanted with 5 x 106 bone marrow cells and 1.5 x 107 spleen cells from C57BL/6 donor mice. At the same time, mice were injected intramuscularly with 8 microgram of the human HGF gene, or with a control mock vector, every week using liposomes containing the hemagglutinating virus of Japan. Untreated GVHD recipients showed significant atrophy of the spleen and the thymus on day 56 after transplantation, while HGF treatment effectively prevented this atrophy. Splenic T cells from untreated GVHD recipients proliferated and produced significant amounts of IL-4 after being exposed to irradiated BDF1 spleen cells in vitro, while those from HGF-treated GVHD recipients did not. These results suggest that host-reactive type 2 T helper (Th2) cells were eliminated in the HGF-treated GVHD recipients by protection against thymic damage caused by acute GVHD, thereby preventing the progression to chronic GVHD. We next used DBA/2 into BDF1 models to determine the effect of HGF on another type of chronic GVHD. Chronic GVHD was induced by injection of DBA/2 spleen cells (9 x 107) into non-irradiated BDF1 mice. HGF treatment effectively prevented proteinuria and histological changes associated with glomerulonephritis. Liver sections of untreated GVHD recipients revealed prominent primary biliary cirrhosis (PBC)-like changes, as indicated by lymphocyte infiltration into the periportal area of the liver. HGF treatment effectively prevented these histological changes. HGF treatment greatly reduced the number of splenic B cells and the serum IgG and anti-DNA antibody levels. Furthermore, HGF treated GVHD recipients showed down-regulation of IL-4 mRNA expression in the spleen, liver and kidneys. IL-4 induction from DBA/2 CD4+ T cells stimulated by irradiated BDF1 CD11c+ dendritic cells (DCs) was significantly inhibited by addition of recombinant HGF (10 ng/ml) in vitro. Taken together, these results indicate that HGF can prevent chronic GVHD by eliminating host-reactive Th2 cells in the thymus, or by inhibition of Th2 cell induction by interaction with DCs.
Perforin and Fas Ligand Are Required for the Regulation of Alloreactive CD8+ T Cells.
Authors: Maeda Yoshinobu , Levy Robert B. , Reddy Pavan , Liu Chen , Teshima Takanori , Ferrara James L.M.
Published: April 2020

We evaluated the role of Fas ligand and perforin, the major T cell-mediated cytotoxic pathways that regulate T cell homeostasis, in a CD8+ T cell mediated model of graft-versus-host disease (GVHD) where donor and recipients differ at a single MHC class I antigen (B6 → bm1). Lethally irradiated (11Gy) bm1 mice were transplanted with T cell depleted BM and CD8+ T cells from either wild type (wt) or cytotoxic double deficient (cdd, deficient in both pathways) B6 donors. We hypothesized that cdd CD8+ T cells would be unable to mediate significant GVHD. Contrary to our hypothesis, recipients of cdd donor CD8+ T cells demonstrated significantly greater histopathologic damage from GVHD and increased serum levels of IFN-gamma and TNF-alpha compared to controls (Table 1). In order to understand this increase, we evaluated the in vivo expansion of donor T cells in these recipients as well as in BMT recipients of allogeneic CD8+ T cells from FasL deficient (gld) and perforin deficient (pfp−/−) donors. CD8+ wt T cells expanded until at day 10 after BMT, followed by a rapid decline. By contrast, cdd CD8+ T cells expanded continuously up to day 30 after BMT, peaking at almost one hundred times the number of wt T cells. gld T cells showed kinetics similar to wt T cells, whereas the pfp−/− T cells showed a significantly greater peak on day 10 but a similar contraction by day 30. Percentages of annexin+ cdd donor CD8+ T cells were significantly reduced compared to the other groups. Persistence of host antigen presenting cells did not account for the unrestrained expansion of cdd donor T cells because host dendritic cells were not detected in either the spleen, BM or gut of recipients of cdd CD8+ T cells on day 6 after BMT. In addition, alloantigen expression on epithelial target cells did not enhance GVHD because B6 donor cdd T cells induced equivalently lethal GVHD in [bm1 → B6] and [bm1 → bm1] chimeras (MST of 30 days and 27 days, respectively). We conclude that both perforin and Fas ligand pathways are required for alloreactive CD8+ T cell populations to contract after their initial expansion during a GVH reaction and that the absence of both these pathways results in donor CD8+ T cell unrestricted expansion and more severe GVHD.

Table 1

GVHD score (gut) IFN-g (pg/ml) TNF-a (pg/ml) CD8+T cell (x10e6) Annexin+CD8+(%) cdd vs. wt, *P<0.05 Wt 4.0±0.4 110±12 6.5±2.7 0.9±0.2 81±3.3 Cdd 5.7±0.3* 263±71* 64.6±3.2* 80.1±4.0* 62±3.6* Pfp−/− ND ND ND 2.6±0.7 73±5.1 Gld ND ND ND 1.7±0.4 80±0.6
Cyclophosphamide Plus Allogeneic CD4+ T Cell Infusion Induces Anti-Lymphoma Immunity Despite Lack of Graft-Versus-Host Disease (GVHD) or Sustained Engraftment.
Authors: Jalla Sanju , Wang Jie , Luznik Leo , Fuchs Ephraim J.
Published: April 2020

Recent evidence suggests that tumor-bearing animals contain CD8+ T cells that can respond productively to a tumor vaccine, but that these T cells do not respond because of insufficient help from tumor-specific CD4+ T cells, which have either been inactivated or turned into anti-tumor suppressor T cells. We therefore devised a strategy to augment anti-tumor immunity by administering cyclophosphamide (Cy), to eliminate suppressor CD4+ T cells, followed by combining autologous tumor cell vaccination and infusion of partially MHC-mismatched, or haploidentical, CD4+ T cells as a source of T cell help for endogenous CD8+ T cells. Interestingly, the combination of Cy followed by haploidentical T cell infusion, with or without vaccine, induced potent systemic anti-tumor immunity resulting in cure of 40-50% of BALB/c mice harboring the A20 B cell leukemia/lymphoma. Depletion of CD8+ T cells from the infusate abrogated GVHD but did not compromise anti-tumor immunity. Allogeneic donor spleen cells that contained CD8+ T cells engrafted durably and caused lethal GVHD. In contrast, the combination of Cy plus CD8+ T cell-depleted spleen cell infusion induced only transient engraftment, peaking on day 7 and declining to undetectable levels by day 14. In the absence of Cy conditioning, allogeneic donor spleen cell infusions did not induce detectable chimerism beyond day 3. In summary, Cy plus allogeneic CD4+ T cell infusion induces potent anti-tumor immunity in a mouse model of B cell leukemia/lymphoma. Potential mechanisms of the therapeutic effect include direct tumor cytotoxicity by CD4+ T cells or allogeneic CD4+ T cell help for endogenous, tumor-specific CD8+ T cells.
Growth Arrest-Specific Gene 6 (Gas6) Deficiency in Mice Alleviates Graft-versus-Host Disease (GVHD) in the Liver.
Authors: Burnier Laurent , Herbert Jean-Marc , Savi Pierre , Collen Désiré , Conway Edward M. , Carmeliet Peter , Izui Shozo , Angelillo-Scherrer Anne
Published: April 2020

GVHD is the single most important complication of allogeneic bone marrow transplantation (BMT), mediated by donor-derived alloreactive T cells. Gas6, a vitamin K-dependent protein, interacts with phospholipid membranes via its γ-carboxyglutamic acid-containing tail and binds to the receptor tyrosine kinases Tyro3, Axl and Mer via its carboxy-terminal globular domain. Gas6 has been implicated in reversible growth arrest, survival, proliferation, cell adhesion, hemostasis and erythropoiesis. Preliminary experiments have shown that Gas6 deficient (Gas6-/-) mice have a leukocyte trafficking defect into inflamed tissues. We hypothesized that donor Gas6-/- T cells might induce less GVHD than wild-type (WT) T cells in MHC-mismatched BMT. We therefore compared the ability of Gas6-/- FVB (H-2q) bone marrow cells to produce GVHD in lethally irradiated MHC haplo-mismatched Gas6-/- and WT B6 mice (H-2b). Gas6-/- and WT B6 received 9 Gy total body irradiation and 107 cells from bone marrow of FVB mice (H-2q) of the same genotype. (Gas6-/- B6 mice received Gas6-/- FVB cells and WT B6 mice were injected with WT FVB cells.) On Day 28, recipients were sacrificed and chimerism was confirmed by expression of H-2q haplotype in recipient bone marrow. Neither group had clinical evidence of GVHD. However, histologic studies revealed lesions in the livers of both genotypes, which were notably less severe in Gas6-/- mice. The size and number of portal space leukocyte infiltrates were reduced in Gas6-/- mice (40 ± 6.6 % of portal spaces infiltrated), compared to WT mice (76.8 ± 6.5 % of portal spaces infiltrated, P < 0.01). Apoptosis of liver cells was also reduced in Gas6-/- mice. We hypothesized that diminished leukocyte trafficking in the Gas6-/- mice may be due to defects in cytokine-induced expression of adhesion molecules. We therefore isolated primary capillary endothelial cells from Gas6-/- and WT mice, and assessed their response to TNF-α. In contrast to WT endothelial cells, Gas6-/- endothelial cells responded without increases in VCAM-1, ECAM-1, E-selectin, IL-1 or IL-6. This resistance to TNF-α was reversed when the Gas6-/- endothelial cells were co-treated with recombinant Gas6, suggesting that the effect of Gas6 on vascular endothelial function is critical for normal leukocyte trafficking in GVHD. Overall, these results provide strong rationale for considering Gas6 and/or its receptors as a potential therapeutic target to minimize GVHD.
Transjugular Liver Biopsy in Hemophilia Is Safe with a Minimum of Two Doses of Factor.
Authors: Ragni Margaret V. , Jaworski Kristen , Boggio Lisa , Brettler Doreen B. , Hoots W. Keith , Kuriakose Philip , Rutherford Cynthia Jean , Zajko Albert B.
Published: April 2020

Over 80% of adults with hemophilia are infected with hepatitis C (HCV) infection through exposure to clotting factor concentrates 20 or more years ago. Although liver biopsy is considered the gold standard to assess the severity of HCV liver disease, the majority of those with hemophilia do not undergo biopsy, even by the less invasive transjugular route. Little is known about the actual or perceived bleeding risks which may affect patient decisions about biopsy. Further, little is known about the minimal hemostatic dose of factor replacement which may affect physician decisions about biopsy. Of 161 hemophilic men enrolled in the multicenter HIV and HCV in Hemophilia (HHH) study which evaluates extent of and risks for HCV disease progression, 112 (69.6%) have decided against biopsy. Among the reasons, given by 75 of the latter, were fear of the procedure, in 23 (30.7%); lack of insurance, in 11 (14.7%); past biopsy, in 11 (14.7%); lack of time, in 10 (13.3%); lack of perceived indication, in 6 (8.0%); presence of an inhibitor, in 5 (6.7%); fear of bleeding, in 5 (6.7%); and other illness or uncertainty, in 4 (5.3%). The 49 (30.4%) agreeing to liver biopsy were more likely than those declining biopsy to be HIV+ (53.1% vs. 34.8%, p < 0.05) and less likely to have an inhibitor (0% vs. 13.4%, p = 0.002), but were as likely to have hemophilia A, 81.6% vs. 83.0%; be over 35 years of age, 49.0% vs. 53.1%; Caucasian, 81.6% vs. 85.7%; and HCV genotype 1, 80.8% vs. 79.5%, all p > 0.05. At the time of biopsy, the median platelet count was 185,000/ul (38,000–366,000/ul), the median PT was 12.0 seconds (9.4–15.4 sec), and the median INR was 1.0 (0.9–1.2). All patients received two doses of factor, and 25 (71.4%) received a third dose; three (8.6%) received a fourth dose for unrelated procedures. The median pre- and post-biopsy F.VIII doses were 49.5 U/kg, 26.6 U/kg at 1–4 hours and 45.0 U/kg at 24–48 hours; and the median F.IX doses were 74.0 U/kg, 44.3 U/kg, and 45.0 U/kg, respectively. No biopsy-related bleeding occurred. Minor adverse events included fever in two, liver function abnormality in one, and duodenal ulcer bleeding in one. The median Knodell, Ishak, and Metavir scores did not differ significantly by HIV status (+ vs.), type hemophilia (A vs. B), age (>35 vs ≤ 35 yr), HCV genotype (1 vs. non-1), or race (Caucasian vs. non-Caucasian). Although preliminary, these findings suggest that fear of the procedure is the most common deterrent to liver biopsy in patients with hemophilia. Yet, when performed by the transjugular route, liver biopsy appears to be safe, with excellent hemostasis achieved with a minimum of two doses of factor, including a 100% dose pre- and a 50% dose post-biopsy. These findings will require confirmation in larger numbers of subjects.
Incidence and Outcome of Discontinuation of Prophylactic Treatment among Young Adults with Severe Hemophilia.
Authors: van Dijk Karin , Fischer Kathelijn , van der Bom Johanna G. , Scheibel Elma , Ingerslev Jørgen , van den Berg H. Marijke
Published: April 2020

Introduction

Most of the current discussion about prophylaxis for severe hemophilia patients is on the dose and when to start. However, as hemophilia is a chronic disease, it is important to evaluate the duration of prophylaxis. The aim of this study was to study and compare adherence to prophylaxis and outcome of severe hemophilia patients.

Methods

All patients with severe hemophilia A and B (factor VIII/IX<0.01 IU/ml), born between 1970 and 1980 and treated in Copenhagen, Århus (Denmark) or the Van Creveldkliniek, Utrecht (The Netherlands) were studied. Data on treatment were collected from the patients’ files from 1972 until 2003. In addition, a questionnaire on adherence to prophylaxis was used. For assessment of outcome the clinical score according to Gilbert and the radiological Pettersson score were used.

Patients were categorized according to adherence to prophylactic treatment: patients who never discontinued prophylaxis (never), patients who temporarily discontinued prophylaxis (temporarily) and patients had switched to on demand regimen (permanently).

Results

83 patients were studied. Median follow up was 19 years (range 6–29). Median age at start of prophylaxis was 5.9 years (interquartile range (IQR) 4.0–8.7). 34% of patients stopped taking prophylaxis temporarily and 35% stopped taking prophylaxis permanently at a median age of 21.5 years (IQR 18.4–24.4). Follow up since the last stop was 3.6 years (IQR 1.4–7.9), the annual number of joint bleeds on on demand treatment was 3.0 (IQR 1.4–8.7).

The median clinical score was 3.0 points (IQR 1.0–6.0) in patients who never or temporarily stopped and 4.0 (IQR 0.0–6.3) in patients who permanently stopped taking prophylaxis. Pettersson scores were available for the Dutch patients and the median Pettersson score was 13 points (IQR 5–23) for patients who never or temporarily stopped and 13 (IQR 1–24) for patients who stopped permanently. The proportion of patients who discontinued prophylaxis and outcome parameters were similar for the Dutch and Danish patients.

Conclusion

Two thirds of young adults with severe hemophilia on prophylaxis discontinue prophylaxis at least once. One third permanently stop taking prophylaxis, while maintaining a low joint bleed frequency. Four years after switching to on demand treatment, outcome in these patients is similar to outcome in patients who continue taking prophylaxis.
Factor VIII Half-Life and Clinical Characteristics of Severe Hemophilia A.
Authors: van Dijk Karin , van der Bom Johanna G. , Mauser-Bunschoten Eveline P. , Roosendaal Goris , Lenting Peter J. , de Groot Philip G. , van den Berg H. Marijke
Published: April 2020

Introduction

Patients with severe hemophilia A have considerably different factor VIII half-lives. Whether this is associated with clinical characteristics has not been reported. The aim of this study was to describe the effect of half-life on the clinical characteristics of patients with severe hemophilia.

Patients and Methods

Patients were selected from a single-centre cohort of 214 patients with severe hemophilia, born between 1944 and 1995. To improve efficiency we measured factor VIII half-life in the patients with the most severe and the mildest clinical phenotypes of severe hemophilia. Patients were selected according to age at first joint bleed, annual joint bleed frequency, clotting factor consumption and radiological Pettersson scores.

A first blood sample was taken after a period of 72 hours in which the patient did not use factor VIII. After infusion with 50 IU factor VIII/kg, blood was collected at 15, 30 minutes and 1, 3, 5, 24, 30, 48 and 60 hours. From 1972 onwards, data on joint bleed frequency, clotting factor use and age at first joint bleed were collected from the patients’ files. Pettersson scores were performed at five-year intervals. For calculations of annual clotting factor use (IU/kg/yr) and number of joint bleeds per year, the last 5 years of follow-up were used. Linear regression analysis was used to assess the relation between clinical characteristics and factor VIII half-life.

Results

Factor VIII half-life was measured in 42 patients and ranged from 7.4–20.4 hours, with a median of 11.8 hours. One hour increase in factor VIII half life was associated with a decrease of 96 (SD 45) IU clotting factor use per kg per year (p<0.05). Joint bleed frequency was similar in patients with a shorter and a longer factor VIII half-life. Median number of joint bleeds was 2.9 per year (interquartile range (IQR) 1.1–4.4) in patients with a factor VIII half-life shorter than 12 hours and 2.6 per year (IQR 1.0–4.8) in patients with a factor VIII half-life longer than 12 hours (p=0.84). Patients with a factor VIII half-life shorter than 12 hours had a median Pettersson score of 52 points (IQR 12–61) and patients with a factor VIII half-life longer than 12 hours had a median Pettersson score of 29 points (IQR 16–52; p=0.90).

Conclusion:

Patients with a shorter factor VIII half-life need more clotting factor to prevent joint bleeds and subsequent arthropathy than patients with a longer factor VIII half-life.
A Non-Human Primate Model for Lentivirus-Mediated Anti-HIV RNAi Strategies.
Authors: Beagles Karen , Beard Brian , Rossi John , Yee Jiing-Kuan , Hu Shiu-lok , Trobridge Grant , Kiem Hans-Peter
Published: April 2020

AIDS remains a significant health problem worldwide despite the advent of highly active antiretroviral therapy (HAART). Although substantial efforts have been made to develop a vaccine there is still no cure and alternative strategies are needed to treat HIV infection and to control its spread. Our goal is to evaluate lentiviral vectors that inhibit HIV replication by RNA interference (RNAi) in a non-human primate SHIV model to develop a hematopoietic stem cell (HSC) gene therapy for AIDS. SHIV89.6 P is a chimeric virus comprised of an SIV genome that contains the tat, rev and env genes of HIV and infects both T lymphocytes and macrophages. Infection of non-human primates with SHIV89.6P results in significant decreases in CD4+ T cells as early as 4 weeks post infection, and is currently the best large animal model available to test gene therapy strategies for AIDS. We present here data showing efficient transduction of M. nemestrina CD34+ cells with an HIV-based lentiviral vector and RNAi-mediated inhibition of SHIV89.6 P replication in a hybrid T/B lymphocyte cell line (CEMx174). Although others reported a block to transduction of M. mulatta CD34+ cells with an HIV-based lentiviral vector, we observed efficient transduction rates (» 50%) of M. nemestrina CD34+ cells, comparable to transduction rates observed in human CD34+ cells (» 60%). To determine effectiveness of anti tat/rev shRNA to inhibit SHIV89.6P in vitro, a human T cell/B cell hybrid cell line (CEMx174) was transduced with a lentiviral vector expressing a short-hairpin RNA (shRNA) targeted to both HIV tat and rev sequences that also contained either a GFP reporter gene or a MGMT(G156A) resistance gene at MOIs of 1.3 and 3 respectively. Polyclonal populations of CEMx174 cells transduced with the GFP and MGMT(G156A) vectors were challenged with a 2.15x103 TCID50 dose of SHIV 89.6P. One week post challenge, expression of both tat and rev transcripts was reduced 88% and 97% respectively in these cultures as measured by real-time PCR. In summary, we have shown efficient HIV-based lentiviral transduction of M. nemestrina cells and efficient inhibition of SHIV infection by shRNA against HIV tat and rev thus providing a useful model to test lentiviral-mediated anti-HIV RNAi stem cell gene therapy in vivo.
Anemia and Cardiovascular Disease (CVD) Synergistically Interact To Increase the Risk of Frailty in Community-Dwelling Older Women.
Authors: Chaves Paulo H.M. , Woodman Richard C. , Fried Linda P.
Published: April 2020

Background: Moderate-to-severe anemia has been shown to adversely affect health outcomes in patients with cardiovascular disease (CVD). Less is known about the potential conjoint impact of mild anemia and CVD on frailty status in community-dwelling older adults. The objectives of this cross-sectional study were two-fold: (1) to assess the risk of frailty associated with mildly low Hb in community-dwelling older women with and without CVD, and (2) to test whether the association between Hb and frailty differed according to CVD status.

Methods: Data from women 70–80 years old who participated in the baseline assessments of the Women’s Health and Aging Studies (WHAS) I and II were analyzed. Analysis was restricted to those with Hb >/= 10g/dL, and known frailty and CVD status. CVD was defined as angina, myocardial infarction, and/or congestive heart failure. Frailty was defined according to previously validated criteria; i.e., 3 or more of the following: slowness (walking speed<20th percentile), unintentional weight loss >10%, weakness (grip strength <20th percentile), low energy expenditure (bottom 20th percentile of Kcal estimated using the Specific Activity Scale), and self-reported low energy. A fully-adjusted logistic regression model that controlled for demographics, diseases (including depression), renal function and cognition was used to determine the probability of being frail as a function of Hb and/or CVD.

Results: In the analytic sample of 670 community-dwelling older women, 14% were frail, 12.8% had WHO-defined anemia (Hb <12 g/dL), 23.6% had CVD, and 17.1% had both anemia and CVD. Those with CVD were more likely to have anemia, though statistical significance was only borderline (OR 1.6, 95%CI 1.0–2.5, p=.077). Both anemia and CVD were independently associated with frailty in the fully-adjusted model (respectively, OR 2.3, 95%CI 1.1–4.8; and OR 2.2, 95%CI 1.2–4.2). When Hb was treated as a continuous variable, even mildly low Hb such as 11.5 g/dL (OR 3.0; 95%CI 1.5–6.0, p=.002), and low-normal Hb such as 12.5 g/dL (OR 1.3; 95%CI 1.0–1.8, p=.044) were independently associated with frailty, as compared to mid-normal Hb such as 13.5 g/dL. The relationship between frailty status and Hb varied according to CVD status (p-value for multiplicative interaction <.035) (Figure below).

Conclusion: In community-dwelling older women, mildly-low/low-normal Hb and CVD were independently associated with frailty status. More importantly, presence of both of these risk factors was associated with a synergistic increase in frailty risk above that expected on the basis of their independent effects. These results are particularly relevant for the development of novel screening and intervention approaches to prevent frailty in older adults.

Figure Figure
Developing National Estimates of the Out-of-Pocket Costs of Cancer: The Importance of Including Patients with Lymphoma.
Authors: McCaffrey Kathryn R. , Carson Kenneth R. , Kutikova Lucie , Fisher Matt , Pickard Simon , Schumock Glen , Bowman Lee , Bennett Charles L.
Published: April 2020

Background: While Congress has mandated that the NIH provide a national estimate of the cost of cancer, almost all cancer cost studies have focused exclusively on breast cancer. No study has reported comparison data for persons with Hodgkin’s disease (HD) or non-Hodgkin’s lymphoma (NHL). These illnesses have a high cure rate, and affected persons are likely to experience significant economic hardships. Many participate in the workforce during treatment and long after the treatment is complete. Herein, we report preliminary results from an ongoing study on the out-of-pocket direct medical and non-medical costs for a cohort of patients with lymphoma and provide contextual comparison with a cohort of breast cancer patients who received care at the same cancer center (Arozullah, Supportive Oncology, 2004).

Methods: 178 breast cancer and lymphoma patients provided information on out-of-pocket costs for the preceding 3-month period; 12% had a diagnosis of HD or NHL. In total, 120 lymphoma patients will be interviewed for this study. Direct medical costs are costs related to medical care such as medications, procedures, and doctor visits. Direct non-medical costs are costs related to cancer, but not medical care, such as costs for meals, transportation, parking, and phone calls.

Results: The majority of both lymphoma and breast cancer patients were < 65 years old, married, and employed. All patients had healthcare insurance coverage, with the majority insured with private plans. In comparison to women with breast cancer, persons with HD/NHL had similar mean monthly out-of-pocket cost expenditures, $635 versus $728. For lymphoma patients, factors associated with high direct medical costs included ≤ 12th grade education ($1,585/month) and HD ($1,133/month).

Conclusion: Mean monthly out-of-pocket expenditures are similar for HD/NHL and breast cancer. Direct medical out-of-pocket expenditures for lymphoma vary. Direct medical expenditures are greatest for HD ($1,130), intermediate for aggressive NHL and breast cancer ($512–$597), and lowest for indolent NHL ($180). Comprehensive economic analyses of cancer should include a range of malignancies.

Average Monthly Out-of-Pocket Costs for Lymphoma and Breast Cancer Patients.

Direct Medical Cost Direct Medical Cost Direct Non-Medical Cost Direct Non-Medical Cost Lymphoma Breast Lymphoma Breast Household Income < $60,000 $381 $664 $40 $111 ≥ $60,000 $599 $553 $159 $161 Education ≤ 12th grade $1,585 $610 $72 $118 > 12th grade $437 $653 $141 $122 Diagnosis < 6 Months $577 $487 $114 $135 ≥ 6 Months $333 $660 $128 $130 Total Lymphoma $516 .. $119 .. HD $1,133 .. $155 .. AggressiveNHL $512 .. $167 .. Indolent NHL $180 .. $166 .. Total Breast Cancer .. $597 .. $131
The Critical Role of the NKG2D Receptor in CD8+CTL and CD8+CD56+ NKT Cell Cytotoxicity.
Authors: Wu Jia-Yan , Hill John M. , Ernstoff Marc , Meehan Kenneth R.
Published: April 2020

We previously demonstrated the ability to grow and ex vivo expand mobilized peripheral blood mononuclear cells (PBMNC) from myeloma patients into aggressive cytotoxic effector cells (Blood102; 422b, 2003). These experiments were designed to test the function and mechanism of tumor cell killing of these cells. Peripheral blood stem cells (PBSC) were collected from myeloma pts after mobilizing with cyclophosphamide and rhG-CSF and cultured in Aim-V serum-free medium at 37 and 5% CO2. After 2 hrs, the non-adherent cells were removed and placed in culture with Aim-V, IL-2 (50 IU/ml) and OKT-3 (50 ng/ml) for 7 days. Cytotoxicity of the expanded cells was tested on Day 0 and Day 7 using a chromium release assay. To identify the cytotoxic potential of cell subsets, cell populations were depleted using the Auto MACS Magnetic Cell Sorter (Miltenyi Biotec Auburn, CA) and cytotoxicity assays were repeated. Since the CD8+ cell(s) were the most cytotoxic, the CD8+ cells were isolated and their mechanisms of tumor cell killing were evaluated by testing killing through MHC Class I, T cell receptor or the NKG2D receptor. The ex vivo expanded population was extremely cytotoxic and killed RPMI 8226 myeloma cells at 60% lysis (+/− 1.6%) (E:T of 100:1) when compared to 3.9 % on day 0 (+/− 0.8%). CD8+ or CD8+CD56+ cell subsets contributed to > 83.3 % (+/− 1.5%) of the killing. Blocking the TCR pathway (Redirected Cytotoxicity Assay) had no effect on killing and blocking the MHC Class I molecules decreased cytotoxicity by only 6%. When the NKG2D receptor was blocked, cytotoxicity by the CD8+ cells decreased by 48% (+/− 2%), demonstrating the critical role of the NKG2D in these CD8+ cell populations. The expanded cytotoxic effector cells aggressively lyse myeloma tumor cells in a MHC and non-MHC restricted fashion. These ex vivo expanded CD8+ cells likely acquire the NKG2D receptor and kill tumor cells in a non-MHC restricted manner. Since MHC expression is often low or absent on myeloma cells and the NKG2D ligands are fairly specific to tumor cells, the infusion of these ex vivo expanded cells following transplant may improve clinical outcomes.
Ceftazidime Versus Levofloxacin for Antibacterial Prophylaxis in Allogeneic Hematopoietic Cell Transplant (HCT) Recipients.
Authors: Frieze Deborah A. , Guthrie Katherine A. , Nichols William G.
Published: April 2020

Antibacterial prophylaxis for patients with neutropenia after HCT may decrease morbidity and mortality. We sought to determine whether levofloxacin prophylaxis is as effective as ceftazidime (which has been utilized at our center for the past decade) for this indication. Consecutive cohorts of allogeneic HCT recipients received ceftazidime (2 g IV q8h, N=221) or levofloxacin (750 mg PO or IV, N=103) as antibacterial prophylaxis during neutropenia (absolute neutrophil count < 500 cells/μL). The two groups were comparable with regards to age, gender, underlying disease, conditioning regimen, and donor match. Compared to the levofloxacin cohort, significantly more patients in the ceftazidime cohort received bone marrow rather than peripheral blood stem cells (p=0.007). Overall, 71 (32%) of patients in the ceftazidime cohort had at least one bacterial infection as compared to 31 (30%) in the levofloxacin cohort; time to first infection was similar as well. Eleven percent of ceftazidime patients and 8% of levofloxacin patients experienced 2 or more infectious episodes. In total, the ceftazidime cohort had 104 infections (mean 0.47 infections per patient) and the levofloxacin cohort had 44 infections (mean 0.43 infections per patient). Breakthrough infections with skin-colonizing gram-positive cocci were modestly more prevalent in the levofloxacin cohort than in the ceftazidime cohort (38.6% of infections vs. 34.6%), as were enteric gram-negative rods (6.8% vs. 5.8%). Oral and gastrointestinal gram-positive cocci (22.7% vs. 30.8%), and environmental gram negative rods (11.4% vs. 12.5%), were more prevalent in the ceftazidime cohort. Of the seven viridans streptococci infections isolated in the ceftazidime cohort, only one was resistant to levofloxacin, yet all of the five isolates in the levofloxacin cohort were resistant. Patients in the levofloxacin cohort had a significantly higher probability of having a fever, as compared to patients in the ceftazidime cohort (p=0.004). Changes in antibiotic regimen were more likely to occur in the levofloxacin cohort (87 of 103 patients) as compared to the ceftazidime cohort (154 of 220 patients), p=0.0002. Relapse-free survival in the first 200 days after HCT, however, did not differ by prophylaxis regimen. Our data demonstrates that levofloxacin is an appropriate alternative to ceftazidime for use as antibacterial prophylaxis in allogeneic HCT recipients, and may be more cost effective given its oral bioavailability. Prospective, randomized trials are needed to elucidate the ideal prophylaxis regimen.
Herpes Zoster (HZ) Infection in the Post-Hematopoietic Stem Cell Transplant Pediatric Population May Be Preceded by Transaminitis.
Authors: Berman Jason N. , Berry Winter , Wang Molin , Neuberg Donna S. , Guinan Eva C.
Published: April 2020

HZ infection is a common complication following hematopoietic stem cell transplantation (HSCT). We reviewed the incidence, complications, and associated risk factors in a post-HSCT pediatric population at a single center and examined whether antecedent blood counts or transaminase levels (AST, ALT) might predict the onset of infection prior to the development of skin manifestations. Seventy-eight children between the ages of 2 months and 21 years developed HZ among the 309 patients, on whom data was available, who underwent HSCT from January 1995 to December 2002. Underlying diagnoses included: acute leukemia (n=108), chronic myelogenous leukemia (n=22), myelodysplastic syndrome (n=24) and other conditions (n=155). Two hundred patients underwent an allogeneic HSCT (98 sibling donor, 102 unrelated donor) and 109 underwent an autologous HSCT. The incidence of HZ was 25% by 10 months and 40% by 70 months post-HSCT. Post-herpetic neuralgia occurred in 21/73 (29%; 95% CI=19%, 41%) evaluable patients, disseminated skin involvement in 15/74 (20%; 95% CI= 12%, 31%) patients and visceral organ involvement in 6/75 (8%; 95% CI= 3%, 17%) patients. Acyclovir prophylaxis for cytomegalovirus (CMV) antigen positivity was given for 21 days post-BMT in 49/75 (65%) patients and had no impact on the incidence or onset of HZ, nor did immune compromise (defined as history of chemotherapy or underlying primary immune deficiency) prior to HSCT. Risk factors for development of HZ included: patient age-greater than 10 years (p<0.0001), allogeneic HSCT (p<0.001), and conditioning with total body irradiation (TBI) (p<0.059). Type of allogeneic donor did not add significantly to the model. Graft-versus-host-disease (GVHD) increased the interval to HZ infection (p<0.0018). There was insufficient evidence to attribute this surprising result to CMV prophylaxis. However, prior GVHD did increase the risk of disseminated skin involvement (p<0.016). There was a significant change in ALT level in the month preceding diagnosis of HZ (p<0.02), with 22/37 (59%) evaluable patients having a progressive increase. This rise in ALT level was not associated with GVHD (p=0.60). Changes in AST levels, white blood cell and platelet counts were not significant. Forty-four patients were admitted to hospital for HZ infection for a median of 6 days (range 2–39). Length of stay for allogeneic patients was significantly affected by skin dissemination and visceral involvement (p<0.023 and p<0.034, respectively) and not by donor type, prior immune status, GVHD or TBI. Inclusion of HZ infection post-HSCT in the differential diagnosis of patients with a rising ALT level, particularly if > 10 years of age after allogeneic HSCT with TBI conditioning may allow for earlier initiation of therapy with less morbidity and decreased need for hospital admission.