bluuebunny/crossref_metadata_2025 huggingface dataset:
Pathogenic Role of Surface Platelet Factor 4 Complexes in Heparin-Induced Thrombocytopenia: Diagnostic and Therapeutic Implications.
Authors: Rauova Lubica , Zhai Li , Kowalska M. Anna , Arepally Gowthami M. , Cines Douglas B. , Poncz Mortimer
Published: April 2020

Heparin-induced thrombocytopenia (HIT) is caused by antibodies that recognize complexes between high molecular weight heparin and Platelet Factor 4 (PF4). Current therapy with direct thrombin inhibitors is not effective in all cases, likely because it acts downstream of antibody-induced platelet activation. More directed therapies to the underlying pathology in HIT may be more effective. Heparin and PF4 only bind HIT antibodies over a narrow molar ratio of reactants at which ultralarge soluble complexes are formed. We asked whether similar complexes form between PF4 and endogenous platelet glycosaminoglycans (GAG) and their pathogenic role in experimental HIT. Platelet surface GAG:PF4 complexes are indeed antigenic over a narrow molar range of reactants. Heparin is not required for either HIT-IgG or a HIT-like monoclonal antibody KKO to bind to PF4 on human or mouse platelet surfaces in vitro, but enhances antigenicity when very high levels of surface PF4 are present. Antigenicity is maximal at a PF4 concentration of 50 μg/mL (well within the range that can be achieved within a thrombus) and ~25 μg/mL heparin (~0.5 U/mL, which is within the therapeutic range) optimally enhances antigenicity when surface PF4 levels were increased 4-fold. Using transgenic mice lines each with platelets expressing a different level of hPF4, ranging from 0.5 – 6 X’s human platelet levels and all expressing FcRγIIA, were given KKO. The different lines developed thrombocytopenia proportional in severity and duration to hPF4 expression. A standard subcutaneous (sq) heparininzing dose (20 U/kg, sq daily) prolonged the duration of severe thrombocytopenia in high hPF4 expressing mice. We reasoned that altering the ratio of PF4 to GAG in either direction would alter antigenicity and could block the development of thrombocytopenia. In accordance with this concept, both high concentrations of anionic heparin (100 U/kg, sq daily) and cationic protamine sulfate (2 mg/kg, sq daily) decreased KKO binding in vitro and prevented KKO-induced thrombocytopenia in vivo as a demonstration of successful therapeutic intervention. These studies affirm a central role of surface GAG:PF4 complexes in the development of HIT, suggest ways to identify patients at high risk to develop HIT even prior to heparin exposure, and offers a new and rationale therapeutic paradigm based on disrupting surface antigen formation.
High-Dose Melphalan Chemotherapy with Autologous Stem Cell Transplantation in Patients with AL Amyloidosis: No Increased Mortality Using Induction and Mobilization Chemotherapy.
Authors: Schonland Stefan O. , Hegenbart Ute , Dengler Jolanta B. , Benner Axel , Hundemer Michael , Bochtler Tilmann , Ho Anthony D. , Goldschmidt Hartmut
Published: April 2020

High-dose chemotherapy (HDC) with autologous hematopoietic stem cell transplantation (ASCT) is the treatment of choice for patients (pts) with AL amyloidosis (AL) eligible for this therapy. Because of a high treatment-related mortality (TRM), pts admitted for HDC have to be selected by risk analysis. It is unclear whether pts should receive HDC directly after diagnosis or after an intensive pre-treatment with induction and/or mobilization chemotherapy. We have retrospectively analyzed all pts with AL admitted to our centre. The first aim of the study was to analyze the influence of prior chemotherapy on survival. Secondly, pts at high risk not to proceed to HDC should be identified by clinical parameters at first contact.

186 pts have been admitted to our centre since 1998 with the diagnosis of AL. 172 pts were ≤ 70 years of age and were tested regarding eligibility for HDC and ASCT. Major eligibility criteria were cardiac disease < NYHA stage IV and performance status (PS) < 4.

Eighty-three pts (median age 56 years, range 34–69; AL n=71, multiple myeloma >stage I with AL, n=10, NHL with AL, n=2) fulfilled the major eligibility criteria and started either (re-) induction (n=57) or mobilization chemotherapy (n=20) or went directly to HDC after G-CSF stimulated stem cell harvest (n=6). Induction treatment consisted of VAD/AD in 34 pts, pulsed dexamethasone (D) in 24 pts or other regimens in 2 pts. Mobilization chemotherapy included cyclophosphamide (C) in 2 pts, CAD (C/adriamycin/D) in 44 pts, ifosfamide in 13 pts and other regimens in 2 pts. Overall, in 72 pts stem cell mobilization has been performed. Sixty-five pts have been treated with HDC.

The estimated overall survival for the whole group is 60% (last event at 54 months, median follow-up 22 months). Fifty-six out of 65 transplanted pts are alive. Three pts died of TRM (5%), 6 pts died of AL progression. Eighteen pts (22%) have not been transplanted for several reasons which is comparable to the 16% reported by Oran et al. (BMT 2005). These included organ progression after induction chemotherapy in 13 pts and chemotherapy-induced mortality in 5 pts resulting in an overall TRM prior to HDM of 6%. Currently, only 2 out of these 18 pts are still alive, 11 pts died of organ progression after the decision against HDC. Our analysis shows that induction chemotherapy does not result in increased mortality prior to (6%) and after (5%) HDC compared to published data using G-CSF alone for mobilization (4% and 6%, Oran, BMT 2005). Significant factors for prediction not to proceed to HDC were number of organs involved (p=0.04) and NYHA stage (p=0.006) at first contact (multivariate analysis). Whether this intensive approach will finally lead to improved survival might be shown with a longer observation after HDC and ASCT.
High Dose Chemotherapy with Autologous Stem Cell Support Is Effective in Resistant and Refractory Hodgkin Lymphoma. A Single Center Analysis.
Authors: Cocorocchio Emilia , Peccatori Fedro , Bassi Simona , Gigli Federica , Radice Davide , Pruneri Giancarlo , Rabascio Cristina , Calabrese Liliana , Martinelli Giovanni
Published: April 2020

We report retrospective data concerning 49 relapsed or refractory Hodgkin lymphoma pts, treated from 1995 in our institution with high dose chemotherapy (HDCT) with autologous peripheral blood stem cell (aPBSC) support. 41/49 had resistant disease, because presenting an early relapse (14/41), a late relapse (12/41) after CR induced by standard chemotherapy (CT) or persistence of disease (15/41) after initial CT. 8/49 had refractory disease, presenting stable or progressive disease within three months after the end of first line CT. Median age at transplantation time of 32 yrs (range 18–58 yrs). Before high dose chemotherapy, all pts received at least a standard chemotherapy (hybrid regimen for 4–6 cycles ± RT, according to the extension of disease). At the time of transplantation, 13 pts were in complete remission, 31 were in partial or very good partial remission and 5 were in stable /progressive disease. We used as myeloablative regimen mainly HDBEAM. Radiotherapy was performed in 9 pts after transplantation. In the subgroup of the 26 relapsed pts, 21 achieved a CR, 2 a PR and 3 a PD/SD; with a median follow up of 23 months from transplant (range 2 – 112 months), expected DFS and OS at 10 years are 90.5% and 76.9%, respectively. Analyzing separately early versus late relapsed patients, DFS expected at 10 years are 81.8% and 100%, respectively, and the OS expected are 71.4 and 83%, respectively. 11/15 pts with persistence of disease after initial CT achieved a CR, 2 PR, 2 presented SD/PD; with a median follow-up of 32 months from transplant (range 2 – 88 months), expected DFS and OS are 90.9% and 75%, respectively. Finally, 5/8 refractory pts achieved a CR (two of them progressed after transplant), 2 pts a PR (both progressed and died after transplant), 1 patient obtained a SD (progressed and died after transplant). No toxic deaths or second cancer or other severe late toxicity were recorded. Autologous transplantation is a safe procedure, considering the manageable early and late toxic effect; the clinical results achieved indicate that HDCT with aPBSC support is to be considered the first therapeutic option for those pts who present an early or late relapse after standard chemotherapy. The low number of refractory pts doesn’t allow any definitive conclusion about its role in this subgroup, but the relative high number of events observed seems to suggest that further analysis of biological features are warranted in order to better define the best therapeutic option.
Mitoxantrone and Melphalan Conditioning Regimen for Autologous Peripheral Blood Stem Cell Transplantation in Adults with Acute Myelogenous Leukemia.
Authors: Sprague Kellie , Padgaonkar Vaishalee , Klein Andreas K. , Chan Geoffrey W. , Miller Kenneth B. , Klingemann Hans
Published: April 2020

High dose mitoxantrone and melphalan (Mito/Mel) has been reported as an effective and well tolerated conditioning regimen for autologous peripheral blood stem cell transplantation (APBSCT) in patients with lymphoma. We review our experience with Mito/Mel as a conditioning regimen in 18 adults with intermediate or high risk acute myelogenous leukemia (AML) who underwent APBSCT. Median age at time of APBSCT was 51 years (range 19–70). Sixteen patients were transplanted in first complete remission (CR); two in second CR. All patients received mitoxantrone 60mg/m2 IV on day −4 and melphalan 180mg/m2 IV on day −1 followed by infusion of autologous peripheral blood stem cells on day 0. The 100 day survival was 94%. One patient died in CR of suspected pulmonary embolism 3 months post transplant. With a median follow-up of 24 months, 10 patients are alive. The median disease free and overall survival is 40 months and 47 months, respectively. All 18 patients received anthracycline based induction therapy prior to APBSCT. Cardiac toxicity was monitored by echocardiogram. A significant decline in left ventricular function developed in only one patient who had underlying diabetes and hypertension. We conclude that high dose mitoxantrone and melphalan is an easily administered conditioning regimen with a low risk of significant cardiac toxicity despite prior treatment with anthracycline based chemotherapy. This regimen when used for APBSCT in adults with AML results in favorable disease free and overall survival.
RICE/ICE Is an Effective PBSC Mobilising Regime in Patients with Aggressive Lymphoma.
Authors: Wilson Edward , Pawson Rachel , Beston Kathleen , Smyth John , Hatton Chris
Published: April 2020

RICE/ICE is an effective re-induction chemotherapy regime for patients with relapsed or refractory aggressive lymphoma prior to autologous stem cell transplantation. There is however a paucity of data on its effectiveness as a peripheral blood stem cell mobilising regime. We present data on its use in 23 patients, age 50 ± 15 (mean ± SD), with relapsed (n=16) or primary refactory (n=7) lymphoma (DLBCL (n=15), mantle cell (n=1), high grade transformation of follicular lymphoma (n=5), Hodgkins lymphoma (n=1), Anaplastic large cell lymphoma (n=1)). RICE (Rituximab 375mg/m2 (day 1), Etoposide 100mg/m2 (days 1–3), Carboplatin 5x(CrCl+25)mg/m2 (day 2) and Ifosphamide 5000mg/m2 (day 2)) was administered as re-induction chemotherapy (4 patients did not receive Rituximab) along with Filgrastim 10mcg/kg/day from day 5 until collection. Apheresis was attempted on day 15 in those patients (n=18) achieving a peripheral blood CD34+ve cell count greater than 10x103/ml. Peripheral blood stem cells were collected on a Gambro Cobe Spectra cell seperator processing a median of 2.5 blood volumes by standard protocols. Eight patients achieved a successful collection in the first apheresis session, six patients required a single further collection on day 16 and four patients required further collections on days 16 and 17. The median CD34+ve cell dose collected was 5.01x106/kg (range 0.5x106/kg − 17.4x106/kg) with only 2 patients achieving inadequate collections (0.5x106/kg and 1.37x106/kg). In total 70% (16 of 23) of patients successfully achieved PBSC collections of greater than 2.0x106/kg. The group of patients that failed to mobilise was not predictable in terms of known adverse markers such as marrow involvement, underlying low grade lymphoma or previous chemo/radiotherapy. In conclusion successful PBSC collection was achieved in the majority of patients treated with RICE/ICE although the number of patients is small and larger numbers will be required to confirm these results.
The Migratory Activity of Anti-CD19 cTCR-Redirected CIK Cells as a Useful Property for In Vivo Targeting of B-ALL Cells.
Authors: Marin Virna , Dander Erica , Biagi Ettore , Biondi Andrea , D’Amico Giovanna
Published: April 2020

Cytokine induced killer (CIK) cells are a population of ex-vivo expanded cells enriched in CD3+CD56+ natural killer T (NKT) cells with MHC-unrestricted cytotoxicity against several leukemia targets, except B-lineage acute lymphoblastic leukemia (B-ALL) cells. The trafficking machinery of NKT cells reflects their preferential localization in bone marrow, liver and spleen, which are also sites of B-ALL infiltration. In the present study we determined whether CIK cells maintain the original trafficking properties of NKT cells, by analyzing their pattern of expression of adhesion molecules, chemokine receptors and chemotactic activity. Moreover, we investigated the possibility to redirect the cytotoxic activity of CIK cells towards our selected target (B-ALL cells) by transduction with a retroviral vector encoding a chimeric T-cell receptor (cTCR) specific for the CD19 antigen. Similarly to freshly-isolated NKT cells, CIK cells expressed CXCR4, CCR6 and CCR7 (average expression 72%, 60% and 32% respectively), presenting chemotactic activity towards their respective ligands. The adhesion molecules CD49d and CD11a were highly expressed on >99% of CIK cells, potentially reflecting their capacity to adhere to the endothelium in bone marrow and non-lymphoid organs. To induce cytotoxicity against B-ALL cells, we transduced CIK cells with a retroviral vector containing the anti-CD19-ζ chimeric receptor and the GFP as marker gene, and analyzed their phenotypic characteristics (CD3/CD56 and chimeric receptor expression) and cytotoxic activity against REH B-ALL cell line and B-ALL cells in a 51Cr-release assay. Percentage of CD3+/CD56+ was not altered by transduction. CIK cells were efficiently transduced with the retroviral vector (average expression of GFP/chimeric receptor = 15%; n=8). Furthermore, CIK cells expressing the anti-CD19-ζ receptor showed a strong cytotoxic activity against the REH B-ALL cell line (average lysis, 59.9% at an effector:target ratio=30:1, range, 45.7%–69.5%; n=3) and against primary B-ALL blasts (average lysis, 60.2% at an effector:target ratio=30:1, range=39.7%–72.8%; n=3), that was not observed in CIK cells expressing the truncated-nonsignaling form of the receptor. In addition, the migratory capacities of the transduced CIK cells remain unchanged when compared to unmanipulated CIK cells. These findings suggest that CIK cells show a natural capacity to reach sites of B-ALL infiltration and proliferation, and can be redirected with a specific anti-CD19 cTCR, to becoming able to kill leukemic cells without affecting their migratory properties. Based on these preliminary findings we suggest that anti-CD19-modified CIK cells may be an attractive strategy for B-ALL immunotherapy.
A Sensitive Enzyme-Linked Assay of ADAMTS13 Antibodies for Diagnosis of TTP.
Authors: Raoufi Mojgan , Zhou Wenhua , Guinto Enriqueta , Grafos Nick , Ranzurmal Safi , Greenfield Robert S. , Rand Jacob , Tsai Han-Mou
Published: April 2020

Thrombotic thrombocytopenic purpura (TTP), a serious disorder characterized by the development of VWF-platelet rich thrombi in the arterioles and capillaries, requires reliable diagnostic assays for optimal management of the patients. Simple, reliable assays of ADAMTS13 inhibitors or antibodies are not yet widely available. To develop an ELISA for antibodies of ADAMTS13, we analyze the levels of patient IgG bound to immobilized recombinant ADAMTS13 in a microtiter plate format. The mean (±SD) IgG binding value is 5.1 ± 1.5 arbitrary units (AU)/mL among a group of 36 normal subjects and is 5.3 ± 2.7 AU/mL among 31 random patients (P > 0.1). Two (5%) of the normal group and 2 (6%) of the random group are outliers. When applied to patients with thrombotic microangiopathy, this assay yields IgG binding values ranging from 2.0 – 58.3 (median = 5.4) AU/mL in a group of 40 subjects with other thrombotic microangiopathy and 10.1 - > 60.0 (median 49.0) AU/mL in a group of 56 patients with acute TTP. Excluding 2 (5%) outlying samples, the mean (±SD) for the “other TM” group is 5.9 ±2.5 AU/mL, which is not different from the values of the normal and random subjects. In contrast, the IgG binding values are > 10.0 AU/mL in each of the acute TTP patients. Furthermore, heating the plasma samples at 56oC, presumably by dissociating ADAMTS13 from immune complexes, increases IgG binding of 4 weakly positive TTP samples from 10.1 – 12.7 to 28.3 – 154.3 AU/mL. Addition of native ADAMTS13 lacking the tag sequences decreases the binding of TTP IgG to 40% ± 20% of control, but does not affect the binding of 6 outlying non-TTP IgG (120% ± 20% vs. control, P < 0.001), suggesting that the IgG of non-TTP samples do not recognize ADAMTS13. In summary, the ELISA for ADAMTS13 antibodies is highly sensitive for detection of ADAMTS13 antibodies. A subset of the general population contains IgG molecules that react with the recombinant ADAMTS13 fusion protein but not with native ADAMTS13. Blocking with native ADAMTS13 enhances the specificity of the assay for TTP.

Figure Left: Binding of IgG to immobilized rADAMTS13-FLAG/His in four groups of study subjects. Right: Suppression of IgG binding by untagged ADAMTS13 in TTP but not in non-TTP samples. Figure. Left: Binding of IgG to immobilized rADAMTS13-FLAG/His in four groups of study subjects. . / Right: Suppression of IgG binding by untagged ADAMTS13 in TTP but not in non-TTP samples.
A Cyclophilin A-Independent Capsid Motif Renders HIV-1 Derived Vectors More Efficient at Transducing Human Cells.
Authors: Swan Christina H. , Chatterji Udayan , Gallay Philippe , Torbett Bruce E.
Published: April 2020

We recently identified naturally occurring HIV-1 capsid (CA) motifs, which evade both cyclophilin A (CypA)-dependence in human cells and TRIM-5α-CypA (TRIM-Cyp) restriction in owl monkey kidney (OMK) cells (Chatterji, et al. JBC, In press). These HIV-1 capsid motifs contained either one (G89V) or four (V86P/H87Q/I91V/M96I) amino acid substitutions within the CypA binding site, but only the four amino acid mutant was able to bind CypA, yet does not require CypA incorporation to infect human cells. OMK cells are naturally resistant to HIV-1 infection due to TRIM-Cyp, a negative host factor, which efficiently targets incoming wild type HIV-1 capsid and thereby prevents the proper uncoating of the CA core. However, addition of cyclosporine A, which blocks the interaction of CypA and CA, or mutations within the CypA-CA binding site (G89V) yields OMK cells permissive to HIV-1. Interestingly, HIV-1 infection with the V86P/H87Q/I91V/M96I capsid mutant was able to achieve similar levels of infection in OMK cells as does wild type HIV-1 with the addition of cyclosporine A, emphasizing that this motif allows infection despite TRIM-Cyp-CA interactions. Alternatively, the interaction between HIV-1 CA and CypA, is considered as a positive host factor in human cells. It has been proposed that CypA either “protects” the virus from endogenous restriction factors or allows proper CA core uncoating. Human cells lacking CypA or treated with cyclosporine A are highly resistant to infection by wild type HIV-1. Virus is blocked during the early stage of infection, at the pre-reverse transcription step. An important goal of human gene therapy is to efficiently deliver genes to non-dividing or quiescent cells such as hematopoietic stem cells and T-cells, respectively. Improvements on vector design such as self-inactivating LTRs, VSV-G envelope pseudotyping, various viral capsids, and enhancer elements have improved both the safety and efficiency of gene delivery. To identify capsid motifs that enhance transduction, we assessed whether the G89V and V86P/H87Q/I91V/M96I capsid motifs increased transduction efficacy in primary human cells as compared to vectors packaged with wild type HIV-1 gag/pol. Preliminary studies, at low levels of transduction (2000 pg p24 input ~1 MOI) in 293T cells, showed a 3-fold increase in GFP expression using the V86P/H87Q/I91V/M96I-gag/pol packaged vectors as compared to wild type or G89V-gag/pol vectors. Studies with primary PBMC and CD34+ hematopoietic stem cells are underway and will address whether the V86P/H87Q/I91V/M96I capsid motif will allow for greater transduction efficiency for non-cycling, quiescent and/or partially activated cells. Providing a means to enhance uniform vector transduction efficacy should allow for lower levels of vector input during transduction, thereby reducing the probability for insertional mutagenesis.
Can Health Care Plans Afford Hemophilia Costs? Yes.
Authors: Aledort Louis M. , Coates James
Published: April 2020

Hemophilia care has high annual recurring costs. 80–90% of these costs are to cover replacement therapy. Managed care programs have, in general, not recruited specialists to help determine the guidelines of care, the use of hemophilia treatment centers (HTC) by their patients or the multiple sources and charges for acquiring these expensive biologics. Aetna has 700–750 hemophilia patients in a typical year whose medical costs are upward of $60 million. These patients represent only.005% of the total membership, and 0.198% of the health care dollars spent. A hemophilia specialist was asked to evaluate these patients vis-á-vis site of care, optimal treatment plan, and potential savings for covered members, payers and employers.

Methods: working with the Aetna nurse case managers, individual patients were evaluated for the above issues.

Results: From June 2004 to June 2005, 190 patient files were reviewed. Of these, 18% had lifetime caps, and 30% were not treated in an HTC. 16% of these patients decided to transfer to an HTC for either their care or product procurement. The remainder decided not to pursue this path.

Table I Patients Issue 1) 4.5 % Altered regimen, product or product procurement 2) 3 % Contemplating altered regimen, product or product procurement 3) 17 % Physician contacted regarding optimal therapy 4) 9.5 % Prices of product negotiated downward 5) 12 % Product procurement switch to PHS pricing 6) 3 % Adhering to previous regimen Table II $ Savings (using above categories) Real Potential 1) 2,978,028 2) --- 353,972 3) 0 4) 1,157,504 5) 4,334,523 6) --- 1,904,360 Total 8,470,055 2,258,332

The potential savings represent those who are either contemplating change but, in the main, are physicians and/or patients who chose not to accept the recommendations made. Realized annualized savings of more than $8 million affect all aspects of the care system. It is particularly revelant to those with lifetime caps. If this model were expanded for other high cost disorders, medical expenses could diminish and premium inflation could eventually decrease. Hemophilia biologics have continued to increase in costs from cryoprecipitate in the 1960s to current recombinant products. Fostering access to HTCs substantially improves quality of care and reduces medical expenditures. In conclusion, we believe that by using this approach, hemophilia care and its costs can be appropriately managed.
Non-Hemolytic Antibody Induced Loss of RBC Antigen during Transfusion of Crossmatch Incompatible Blood.
Authors: Zimring James C. , Hair Gregory A. , Chadwick Traci E. , Deshpande Seema S. , Anderson Kimberly M. , Hillyer Christopher D. , Roback John D.
Published: April 2020

Background: Transfusion of red blood cells (RBC) into patients with anti-donor RBC antibodies (crossmatch incompatible transfusion) can result in antibody mediated hemolysis. Less well appreciated is the ability of anti-RBC antibodies to specifically remove their target antigen from donor RBCs without compromising cell survival. This phenomenon has now been reported for the major clinically significant blood group antigens, including Rh, Kell, Kidd and Duffy. Although this has been described multiple times in humans, no mechanistic elucidation has been accomplished. In an effort to investigate the mechanism of this process, we describe the first animal model of non-hemolytic antibody induced RBC antigen loss.

Methods: mHEL mice express the model antigen Hen Egg Lysozyme (HEL) as a cell surface protein on RBC. Since mHEL mice are on a C57BL/6 background, the mHEL antigen represents a single antigenic difference between donor RBC and recipient mice. Immunizing C57BL/6 mice with HEL/CFA results in the generation of high titer IgG anti-HEL responses rendering the mice crossmatch incompatible with mHEL RBC. This system was utilized to study the effects of transfusing mHEL RBC into crossmatch incompatible recipients.

Results: Similar to the antibody induced antigen loss observed in humans, transfusion of donor mHEL RBC into crossmatch incompatible mice results in selective loss of HEL antigen from donor RBC without affecting other blood group antigens or reducing the circulatory lifespan of the donor RBC. In addition, recovered RBC that have lost their antigen have normal morphology. This process is antigen specific and occurs in mice that have received passive injections of anti-HEL antisera. A spleen is not required for antigen loss to occur. However, antigen loss does not occur in animals with a targeted deletion of the FcγIII receptor. Although polyclonal anti-HEL antisera consistently causes antigen loss, and IgG1 and IgG2b are the predominant subclasses of anti-HEL IgG in the antisera, no antigen loss is observed in response to purified monoclonal anti-HEL antibodies of the IgG1 and IgG2b subclass.

Conclusion: These studies demonstrate that antibody induced antigen loss is a process that involves interaction of RBC, anti-RBC IgG and FcγIII receptors, thus providing mechanistic insight into the phenomenon of antigen loss during incompatible transfusion. The lack of antigen loss in response to monoclonal anti-HEL IgG1 or IgG2b suggests that antigen loss occurs in response to a minor IgG subtype in antisera, depends upon biological properties of the antibody (such as affinity), or that additional serum cofactors are involved.
Use of Complementary and Alternative Medical Practices in a Pediatric Sickle Cell Population.
Authors: Faust Douglas S. , Gardner Renee , Rayford Christopher
Published: April 2020

There is a substantive body of literature on the use of Complementary and Alternative Medicine (CAM) therapies among adult populations generally, and chronically ill populations specifically, however less is known about its utilization with sick children. The use of CAM approaches to the treatment of sickle cell (SC)disease and the associated pain crises has focused largely on the use of hypnosis, musculoskeletal manipulations, or relaxation techniques for acute pain relief. The more general use of CAM therapies in the treatment of sickle disease by families is less well surveyed. Numerous studies have found utilization of CAM therapies in up to 70% in both general and sick adult populations, with somewhat lower incidence in pediatric populations. Adults attending sickle cell clinics with their children were asked to participate in structured interviews detailing the use of vitamins and supplements, physical and somatic therapies, and spiritual/holistic therapies, working from a standard list of interventions used in other studies. 47% of the sample regard treatment with CAM therapies as harmless, however the majority of respondents indicated they were aware of the potential for a potential interaction between prescriptive medication and CAM practices. Most believed that CAM therapies were widely used by adults, and both healthy and physically ill children. In this sample, 80% of those sampled reported the use of CAM therapies in the home, but only 53% reported using CAM therapies with the chronically ill child. The most frequently endorsed CAM approaches included prayer (70%), relaxation (55%) and general vitamins (45). Removing prayer as an alternative failed to significantly reduce the incidence of CAM usage with either the family or the physically ill child. Of the various supplements considered in the treatment of SC disease and discussed in the literature, none of the families reported use of Niprisan, one family reported use of aged garlic, two families reported use of Noni Juice, and 17% of the families reported the use of iron supplements for their child with SC. Further research with other chronically ill pediatric populations is underway, with additional data to be gathered on the use of cultural or regional folk remedies, typically not sampled in other CAM surveys.
A Clinical Database Supports Better Patient Care, and Clinical Research in a Sickle Cell Center at a Large County Hospital.
Authors: Jonas William S. , Cooper Melanie , Platt Allan F. , Beasley JoAnn , Palmer Sheila , Khoury Hanna J. , Eckman James R.
Published: April 2020

The Georgia Comprehensive Sickle Cell Center at Grady Health System was enabled in 1985 by an appropriation of the Georgia State Legislature. The Center developed as the World’s first dedicated 24 hour acute care center is committed to patients with sickle cell syndromes. The Acute Care Center is equipped with 12 observation beds and patients are treated for up to 8 hours before a decision about hospital admission is made. Continuity follow-up clinics are held in the adjacent clinic area with 6 exam rooms, a waiting room, reception area, staff offices, and a multimedia teaching center for patients. Care is provided by a multidisciplinary team of physicians, nurses, physician assistants, nurse practitioners, clinic assistants, secretarial support, social workers, psychologists, and a clinical nurse specialist in psychiatry. The state contract required 16 demographic characteristics on each patient seen by the center staff. To accomplish this, a relational database was developed to manage this large dataset. This database was initially established in R-Base format but subsequently transitioned to Windows based Access in 1995. The database was designed and implemented mainly to coordinate patient care containing demographics, problem lists, current medications, radiology results and laboratory data. The database also is utilized to manage the business aspects of the Center including making appointments, tracking visits, monitoring transfusions, ordering supplies, and tracking inventory. The database also provides support for research by identifying patients who qualify for research studies, tracking participation in clinical trials, correlating molecular genetic characterization with clinical findings and outcomes. The database presently has over 3500 registered patients. There are over 1608 active patients defined as being seen in the past two years. Phenotypes and ages are outlined in Table 1. The number of active patients in many age groups allows for cross-sectional, age specific analysis of complications in sickle cell disease. The database also facilitates identifying subjects for intervention trials by entry of study inclusion and exclusion criteria as search criteria for inquiries. The use of the database as a clinical management tool captures information during patient care activities minimizing the need for separate data entry. Data from 20 years of following patients in this data set also provides information on natural history, frequency of complications, and impact of interventions in patients with sickle syndromes

Table 1. Phenotypes and Ages of Active Patients Phenotype HbSS HbSC Sβ+ thal Sβo thal Other Unkn Total Number 711 214 112 18 237 316 1608 Percent 44% 13% 7% 1% 15% 20% 100% Age 0 – 9 10–19 20–29 30–39 40–49 50–59 ≥ 60 Number 321 339 414 283 172 58 21
Dietary Hypercholesterolemia Enhances Heparin-Induced Thrombocytopenia/Thrombosis: A Prothrombotic Risk Factor in a Transgenic Mouse Model.
Authors: Reilly Michael P. , Dessain Scott K. , Taylor Scott M. , Kowalska M. Anna , Poncz Mortimer , Sachais Bruce S. , Cines Douglas B. , Arepally Gowthami , Williams Kevin Jon , McKenzie Steven E.
Published: April 2020

Heparin-induced thrombocytopenia/thrombosis (HIT/T), the most frequent drug-induced immune thrombocytopenia, is a common cause of life- and limb-threatening thrombosis. Although heparin/platelet factor 4 (PF4) antibodies are detected in many patients treated with heparin, there is little understanding of why only a subset of patients develops thrombosis. We recently produced and characterized the first mouse model that recapitulates the salient features of the disease. A second generation model, designated IIA/hPF4-mPF4KO, expresses human FcγRIIA and PF4 but lacks endogenous mouse PF4. These models allow systematic investigations of factors that contribute to pathogenic consequences of HIT/T antibodies. In the current study, we hypothesize that hypercholesterolemia, a known stimulus for atherosclerosis, endothelial dysfunction, and platelet hyperreactivity, would augment thrombosis in our mouse model of HIT/T. Age and sex-matched IIA/hPF4-mPF4KO mice were fed an atherogenic diet (Paigen diet) (AD; 15% cocoa butter, 1% cholesterol, 0.5% cholate) (n=10) or maintained on standard diet (SD; 4.5% fat, no cholate) (n=10). Mice fed the AD for only 4 weeks had significantly increased cholesterol levels (173 ± 29 mg/dl vs. 50 ± 18 mg/dl for SD-fed; p < 0.0001). Mice were then injected with 30 U heparin and KKO, a mouse monoclonal heparin/PF4 antibody. The mean nadir platelet count in AD-fed mice was 34.6% ± 9.1 lower than that in the SD-fed mice (p< 0.0001). Thrombin-anti-thrombin III (TAT) levels, which reflect thrombin generation in vivo, in the AD-fed mice increased from 32 ± 5 μg/at baseline to 79 ± 16 μg/l (p < 0.0001) coincident with the platelet nadir. In contrast, SD-fed mice, with a less profound fall in platelets, showed no increase in TAT. Histological examination showed multiple platelet-fibrin thrombi in lungs and livers of AD-fed mice, whereas the SD-fed mice showed no histological evidence of thrombosis. Thus, in our mouse model, short-term diet-induced hyperlipidemia significantly increases the severity of heparin/PF4 antibody-mediated thrombocytopenia and thrombosis. Our studies provide evidence that a specific host factor can enhance the pathologic effects of heparin/PF4 antibodies in vivo and contribute to thrombotic risk in patients with HIT/T. An increased understanding of the contribution of prothrombotic factors not only will facilitate identification of patients with heparin/PF4 antibodies who are at increased risk of thrombosis, but also provide novel approaches to treatment of HIT/T.
Effects of HLA-C and HLA-DQB1 Mismatching on Outcome of Unrelated Donor Marrow Transplantation for Patients with Severe Aplastic Anemia.
Authors: Kojima Seiji , Yabe Hiromasa , Kato Koji , Kigasawa Hisato , Sakamaki Hisashi , Tsuchida Masahiro , Kato Shunichi , Morishima Yasuo , Kodera Yoshihisa
Published: April 2020

Prior studies assessing the effects of HLA mismatching on outcomes of unrelated donor marrow transplantation (UBMT) have yielded conflicting results. Our previous study revealed that mismatching of HLA-A and -B antigens, and not of HLA-DRB1 antigen, was the most crucial risk factor for survival for patients with SAA who received UBMT (Kojima S et al, Blood100: 799, 2002). Because only the data of HLA-A, -B, and DRB1 were available, the role of HLA-C and DQB1 mismatching was not clarified in that study. The current study aimed to evaluate the effects of HLA-C and DQB1 mismatching. From Feb 1993 to April 2003, 260 consecutive patients with acquired aplastic anemia (AA) received UBMT through the Japan Marrow Donor Program. We selected 79 recipient-donor pairs in which molecular analysis of HLA-A, -B, -C, -DRB1, and DQB1 were performed. Patient ages ranged from 3 to 46 years (median, 15 years). Various preconditioning regimens were used by individual centers. In 69 patients (87%), cyclosporine and methotrexate were used for the prophylaxis against GVHD. Of the 79 pairs, 26 (33%) were found to be matched at HLA-A, -B, -C, -DRB1, and DQB1; 18 (23%) were mismatched at a single HLA antigen (6 HLA-A or -B, 7 HLA-C, 5 HLA-DRB1 or HLA-DQB1); 31 (39%) were mismatched at two HLA antigens (5 HLA- and HLA-DQB1, 18 HLA-A or -B and HLA-C or -DRB1 or -DQB1, 8 HLA-C and HLA-DRB1 or -DQB1); and 9 (11%) were mismatched at 3 HLA antigens. Transplant-related toxicities such as graft failure, grade 3 to 4 acute GVHD, and chronic GVHD were found in 7/74, 14/70, and 16/59, respectively. In a univariate analysis, any single HLA mismatching including HLA-A, -B, or -C did not increase the incidence of acute GVHD. Of 79 patients, 44 survived. The 5-year survival rate did not differ between recipients transplanted from a full-matched donor (68.5%) and those from any single HLA mismatched donors (66.7 to 80.0%). In addition, the 5-year survival rate was not worse (62.5%) in patients transplanted from HLA-C and HLA-DRB1 or -DQB1 mismatched donors. However, the 5-year survival rate was significantly worse (38.9%) in recipients who transplanted from HLA-A or -B mismatched and -C or -DRB1 or -DQB1 mismatched donors and it was 0% in patients who were mismatched at 3 antigens. In a multivariate analysis, the relative risk for HLA-A or -B and -C or -DRB1 or -DQB1 mismatching was 4.24 (95% CI, 1.6–11.5) and for 3 antigen mismatching, it was 20.0 (95% CI, 5.6–71.6). The current JMDP study showed that mismatching of a single HLA antigen did not result in increased mortality. However, in patients with HLA-A or -B mismatching, additional mismatching at HLA-C, -DRB1, or -DQB1 had a significant adverse effect on survival. Matching of HLA-C and -DQB1 should be incorporated into algorithms for unrelated donor selection.
Identification of Novel Erythropoietic Genes and Alleles Using a Large-Scale Chemical Mutagenesis Screen in Mice.
Authors: Jane Stephen M. , Sutton Rosemary , Rank Gerhard , Auden Alana , Alexander Warren , Kile Benjamin , Hilton Douglas , Cunningham John M. , Curtis David
Published: April 2020

Genome-wide chemical mutagenesis screens have been successfully performed in zebrafish to identify genes involved in erythropoiesis. We have now executed a large-scale screen for genes involved in red cell production in mice by using N-ethyl-N-nitrosurea mutagenesis. Five thousand G1 mice were screened by automated haematological analysis at 7 weeks of age for abnormalities of their red cell indices that lay more than 3 standard deviations outside the normal population average. Mice with co-incident abnormalities in white cell or platelet counts were excluded. Six mutant lines were identified with microcytosis (RBC1-6) that was heritable with subsequent backcrossing. Three of these lines (RBC 1, 4, 5) were found by gene mapping and candidate gene sequence analysis to harbour different mutations in the transferrin receptor gene (TfR) that led to embryonic lethality in their homozygous state. These mutations were clustered in the sequence encoding the domain involved in receptor homodimerisation, providing, for the first time, in vivo functional correlations of the previous structural analyses. The RBC3 line was also mapped to the interval containing the TfR gene, although no mutations were present in the TfR coding region or splice donor/acceptor sites in this line, raising the possibility of a regulatory sequence mutation, which we are investigating. The RBC2 line was mapped to chromosome 8, and found to contain a splice acceptor mutation in the ankyrin gene. This mutation results in a 22 bp deletion with a resultant frame shift in the regulatory domain. Mice homozygous for this mutation are born jaundiced with massive splenomegaly and a blood film consistent with severe spherocytosis, and die within 6 weeks of progressive anemia. As such, this line represents a new mouse model of severe hereditary spherocytosis. The final line, RBC6 displays a profound hypochromic, microcytic anemia in the homozygous state at birth, with apparent normality of all other organ systems. The mutation has been mapped to a chromosomal interval containing only a few genes, none of which have been previously linked to red cell production or differentiation, and therefore represents a novel erythropoietic gene/allele.
ADAMTS13 Levels Support Hypothesis of Distinct Mechanistic Pathways for Early Versus Late-Onset of Thienopyridine-Associated Thrombotic Thrombocytopenic Purpura (TTP).
Authors: Kwaan H.C. , McKoy June M. , McCaffrey Kathryn R. , Yarold Paul R. , Kim Benjamin , Zakarija Anaadriana , Bandarenko Nicholas , Pandey Dilip K. , George James N. , Zheng X. Long , Carson Kenneth R. , Bennett Charles L.
Published: April 2020

Background: Ticlopidine and clopidogrel, thienopyridine derivatives used for cerebrovascular events and coronary artery syndromes, are the first and third most common causes of drug-related TTP reported to the FDA. Ticlopine-associated TTP has been characterized as having later onset, having higher relapse rates, and being more responsive to plasmapheresis—findings associated with ADAMTS13-deficiency. Based on a limited number of ADAMTS13 measurements, Moake (NEJM 2002) and Bennett (NEJM 2000) speculated that antibody-mediated ADAMTS13-deficiency appeared to be the cause of both ticlopidine- and clopidogrel-associated TTP.

Methods: Clinical and ADAMTS13 laboratory data for TTP associated with ticlopidine (n=116 and 12 cases, respectively) and clopidogrel (n= 46 and 7 cases, respectively) were reviewed.

Results: Clinical Evaluation: Mortality of ticlopidine-associated TTP was 34.5%. Mortality of clopidogrel-associated TTP was 22.2%. Overall moretlaity rate was 31%. Among 162 thienopyridine-associated TTP cases, late-onset TTP (89% of the cases) occurred after > 5 days of thienopyridine use and was 4-times more common with ticlopidine than clopidogrel. Early onset TTP (11% of the cases) occurred within 5-days of thienopyridine initiation and was 4-times less common with ticlopidine than clopidogrel. Laboratory Evaluation: Among 12-ticlopidine and 7-clopidogrel-associated TTP patients, laboratory assays prior to plasmapheresis identified severe ADAMTS13 deficiency 3.3-fold more often with ticlopidine (92% versus 29%, p < 0.01) and presence of inhibitory autoantibody almost 3-fold more common with ticlopidine (83% vs 30%) than clopidogrel. Among thienopyridine-associated TTP cases, patients with severe ADAMTS13-deficiency (<5% of normal) experienced higher relapses rates (15% versus 0%).

Conclusions: Contrary to previously proposed mechanistic hypotheses, thienopyridine-associated TTP appears to manifest as an ADAMTS13-deficiency mediated syndrome (primarily with > 5 days of ticlopidine) that has occasional relapses and an ADAMTS13-normal syndrome (primarily with < 5 days of clopidogrel) that is not associated with relapse. Although in the same drug class, the metabolites of ticlopidine and clopidogrel are different, which may account for the distinct mechanistic pathways. Immunologically mediated ADAMTS13 -deficient TTP, like ticlopidine-associated TTP, may result from immunogenic exposures that occurred 1 to 2 weeks prior to syndrome onset. In contrast, ADAMTS13-normal TTP cases, like clopidogrel-associated TTP, may result from recent exposure that occurred within 5 days prior to TTP onset leading to direct endothelial cell injury.
Immunogenetic Profiling of Follicular Lymphoma Reveals Universal N-Glycosylation Sites Introduced into the B Cell Receptor by Somatic Mutation and Suggests Relevance to Lymphoma Pathogenesis.
Authors: Ottensmeier Christian H. , McCann Katy J. , Johnson Peter , Stevenoson Freda K.
Published: April 2020

Immunogenetic analysis of B-cell malignancies can provide important information that relates to the cellular origin and clonal history of these lymphomas and give clues as to possible pathogenic mechanisms. In follicular lymphoma (FL), immunoglobulin variable region (V) genes are commonly somatically mutated and display intraclonal heterogeneity consistent with location in the germinal centre (GC). In this analysis of 44 cases of FL we find that, with minor exceptions, both the VH and VL gene usage reflects that of the normal B cell repertoire, indicative of a common antigenic drive and in support of a final transforming event in the GC. We have previously reported a high incidence of potential N-glycosylation sites in the VH genes of FL, which have been introduced by the process of somatic mutation. Here we have assessed both the VH and VL genes and find that sites are universally present and further demonstrate that they are available for functional glycosylation. The majority of sites are found in VH (81%) and are located predominantly within CDR2 and CDR3, with few sites present in the FRs. Sites are also evident in VL (45%) where they are focused mainly in CDR3 and CDR1. A minor subset (10%) has sites in VL only. In total, 26 different N-glycosylation motifs were observed, with NIS being the most commonly used. The natural motif in the V4–34 germline gene appears unimportant, and can be lost. Scrutiny of the somatic mutations giving rise to these motifs reveals that the acquisition of sites was predominantly (73%) achieved by a single amino acid (aa) replacement to Asn at position 1 of the motif, either with or without an additional, non-essential aa replacement at another position. Common ‘hotspots’ were observed within the CDR2 for the VH gene segments V3–23, V3–48, V3–07 and V3–15. It appears likely that the acquisition of N-glycosylation sites in the antigen-binding site during somatic mutation in the GC and the subsequent addition of oligosaccharides is important to the lifestyle of FL and may provide a critical second tumorigenic event. In turn, it may be possible to exploit this seemingly essential feature to develop novel therapeutic approaches.