bluuebunny/crossref_metadata_2025_split huggingface dataset:
Exosite Interactions Contribute to the Activated Protein C-Catalyzed Inactivation of Factor VIIIa.
Authors: Varfaj Fatbardha , Neuberg Julie , Wakabayashi Hironao , Fay Philip J. 
Published: October 2019

Abstract
Activated Protein C (APC) is an anticoagulant serine protease that proteolytically inactivates cofactors Va and VIIIa. Cleavage of human factor VIIIa occurs at Arg336 and Arg562 located within the A1 and A2 subunits, respectively. While cleavages are not ordered, the former site appears to represent a preferred cleavage site. Efficient catalysis requires binding of APC to a phospholipid surface and to the A3-C1-C2 subunit of factor VIIIa. The latter observation suggests that APC likely binds substrate via an exosite(s) thereby contributing to substrate specificity. A study was undertaken to evaluate contributions of substrate docking at the active site and exosite tethering to the APC catalytic mechanism. Recombinant, human factor VIII mutants where P1 Arg residues at 336 and 562 were substituted with Ala or Gln were constructed and stably expressed. Purified factor VIII was converted to factor VIIIa by thrombin and used as substrate to elucidate the mechanism of cleavages. Proteins mutated at Arg336 were also mutated at Lys338 because the latter residue may serve as an alternative APC cleavage site when residue 336 is mutated. Rates of inactivation of wild type and mutant factor VIIIa molecules and rates of cleavage at Arg336 and Arg562 by APC were monitored in the presence and absence of protein S. The R336A/K338A mutant was inactivated and cleaved at the 336 site approximately 20-fold slower than the wild type, whereas the R336Q/K338Q mutant was completely resistant to cleavage at the 336 site. These results indicate that residues other than Arg may be tolerated at the P1 site, whereas Gln yields a cleavage-resistant substrate. Indeed, the R336Q/K338Q/R562Q (triple Q) mutant was resistant to cleavage at both P1 sites. Furthermore, mutations retarding cleavage at residue 336 showed a dramatic decrease in rates of inactivation suggesting that cleavage at this site correlated with the inactivation of factor VIIIa. The importance of exosite interactions was explored by inhibition experiments examining the inactivation of wild type factor VIIIa in the presence of triple Q mutant factor VIIIa. Wild type factor VIIIa inactivation rates decreased as the proportion of triple Q factor VIIIa increased, indicating that the P1 mutant factor VIIIa effectively sequestered APC from the native substrate. Evaluation of inactivation rates suggested that APC possessed an ~8-fold greater affinity for the triple Q FVIIIa than the wild type factor VIIIa. Consistent with that observation, the Ki for triple Q factor VIIIa (29.5 ± 3.6 nM) was ~5-fold less than the Km for wild type factor VIIIa (133 ± 27 nM). Taken together, these results indicate that mutations in the P1 site that prevent cleavage may also retard dissociation of the enzyme-substrate complex. Overall, results from this study suggest that exosite interactions make a primary contribution to substrate affinity during APC-catalyzed inactivation of factor VIIIa.
Fibromodulin as a Novel Tumor-Associated Antigen (TAA) in Chronic Lymphocytic Leukemia (CLL) Which Allows Expansion of Specific CD8+ Autologous T Lymphocytes.
Authors: Mayr Christine , Bund Dagmar , Schlee Martin , Moosmann Andreas , Hallek Michael , Wendtner Clemens 
Published: October 2019

Abstract
BACKGROUND: Fibromodulin (FMOD), a collagen binding protein, was shown to be highly overexpressed in CLL cells compared to normal B lymphocytes by gene expression profiling. Therefore FMOD might serve as potential tumor associated antigen (TAA) in CLL, enabling expansion of FMOD-specific T cells. FMOD is physiologically expressed in articular cartilage, tendon and ligament. Furthermore, interactions of FMOD with the growth factor TGF-b were described and it may be a biologically relevant modulator of TGF-b activity.
Methods: Unpulsed native CLL cells and CD40 ligand (CD40L)-stimulated CLL cells as antigen presenting cells (APC) were used to expand autologous T cells from 13 patients.
RESULTS: In CLL samples derived from 16 patients, high expression of FMOD by real-time RT-PCR was detectable in contrast to normal B lymphocytes. The number of T cells during four weeks of in vitro culture increased 2-fold with native CLL cells as APC and 3.5-fold with CD40L-stimulated CLL cells as APC. The amount of T cells recognizing HLA-A0201 binding FMOD-derived peptides detected by HLA-A2-dimer/peptide staining increased 10-fold during in vitro culture. The T cells expanded were also able to secrete IFN-g upon recognition of the antigen demonstrated by IFN-g-ELISPOT assays. T cells not only recognized HLA-A0201 binding FMOD peptides presented by TAP-deficient T2 cells, but also FMOD overexpressing autologous CLL cells in an HLA-A0201 restricted manner. Neither HLA-A0201 negative CLL cells nor non-malignant cells, i.e. PBMC from healthy donors or tonsilar B cells, were specifically recognized by T cells. When CD40L-stimulated CLL cells were used as APC, which were pulsed with FMOD peptides prior to coincubation with the T cells, significant higher amounts of T cells specifically recognized autologous CLL cells in IFN-g-ELISPOT assays P< 0.018).
CONCLUSION: FMOD was shown for the first time to be naturally processed and ( presented as TAA in primary CLL cells. This enables the expansion of autologous tumor-specific T cells which might be applicable in clinical vaccination trials or as a tool for a CLL-specific immune monitoring in the context of vaccination approaches including CD40L-gene modified autologous leukemic cells.
Lentiviral CCR5 Intrabody Gene Delivery Provides Protection and Enrichment during CCR5-Tropic Infection.
Authors: Swan Christina H. , Buhler Bernd , Tschan Mario P. , Barbas Carlos F. , Torbett Bruce E. 
Published: October 2019

Abstract
The molecular mechanism of human immunodeficiency virus type 1 (HIV-1) entry into target cells is a multistep mechanism. The viral envelope glycoproteins (env) binds first to CD4 and subsequently interacts with the V3 loop with a chemokine receptor, CCR5 or CXCR4, triggering the fusion event. Several findings suggest that viruses using CCR5 for entry (R5-tropic HIV-1) is the predominant species transmitted among patients. Importantly, CCR5 expression levels determine disease progression. CCR5 does not seem to be necessary for normal cell function, since individuals with a homozygous mutation (Δ32) do not appear to have clinical, immune alterations. Furthermore, these individuals are highly protected against transmission of R5-tropic HIV-1. Therefore, intervention strategies aimed at altering or blocking CCR5 expression may be beneficial for cellular protection and provide a clinical benefit against HIV-1 infection. We have constructed an HIV-1 derived vector, CAD-R5, expressing an intracellular single-chain antibody (intrabody) specific for CCR5 coupled to a KDEL endoplasmic reticulum retention signal. Intrabody expressing primary T-cells efficiently disrupted CCR5 cell surface expression with a 4.3-fold reduction in CCR5 mean fluorescence intensity (MFI) as compared to the control vector without the intrabody gene (7.9 and 33.6 MFI, respectfully). CAD-R5 transduced primary CD4 T-cells expressing the intrabody gene were resistant to R5-tropic HIV infection as shown by a 50 to 60-fold reduction in HIV-1 p24 concentration. Moreover, intrabody gene expressing cells demonstrated a selective advantage and enriched by 6.4-fold in a population of infected cells as compared to uninfected or infected cells containing vector without the intrabody gene. The SCID-human mouse model, produced by conjoining fetal human thymus and liver under the renal capsule, has become a staple for in vivo testing of T cell anti-retroviral therapy. When CAD-R5 transduced, fetal liver derived CD34+ stem cells were used for reconstitution of the human implants in SCID-hu mice (n = 2, 2 groups), 6-weeks later the human thymi displayed an average of 25% reporter gene expressing T cells. Thymocyte development was not altered by vector integration or loss of CCR5 cell surface expression as determined by the CD4+/CD8+ staining profile. Importantly, CCR5 intrabody expressing thymocytes were also highly resistant to ex vivo R5-tropic HIV-1 challenge with a 3-fold reduction in viral load. These results validate the efficacy of lentiviral delivered CCR5 intrabody mediated protection from R5-tropic HIV-1. The findings also underscore the potential advantage of intrabody gene delivery to CD34+ stem cells, which allows differentiation of protected T cell progeny.
CD34+ Cell Derived Macrophages Are Protected from HIV-1 Challenge by Intrabody-Mediated Reduction of CCR5.
Authors: Fischer Kimberlee M. , Just Margaret L. , Barbas Carlos F. , Torbett Bruce E. 
Published: October 2019

Abstract
The three main coreceptors that HIV-1 uses for viral entry are CD4 and the chemokine receptors CXCR4 and/or CCR5. Viral tropism dictates which chemokine receptors are used for viral entry. Naturally occurring CCR5 gene polymorphisms resulting in gene deletion (delta 32) results in loss of receptor expression and provides protection from CCR5 using viruses. There are no known deleterious effects of CCR5 loss in mice or in humans. The focus of our studies is to limit infection of myeloid cells by CCR5 using HIV-1s by decreasing CCR5 cell surface expression. We have successfully accomplished this by using HIV-1 vectors to deliver an intrabody directed against CCR5. The CCR5-specific intrabody gene, containing the endoplasmic KDEL sequence, produces a single chain antibody that sequesters CCR5 in the endoplasmic reticulum. We show that >= 93% of THP-1s, a myelomonocytic cell line, transduced with CCR5 intrabody genes stably expressed intrabodies for 6 months. Of cells expressing intrabody, CCR5 was decreased at least 80% on the cell surface. In macrophages derived from CD34+ cells, CCR5 gene expression was maintained at initial transduction levels throughout the 3-week culture period. Colony forming assays (CFU) were performed to assess the effect of intrabody expression on hematopoietic development. No differences were seen in numbers of CFU and colony types when comparing parental, control vector, and intrabody expressing cells (n = 3). To determine the level of protection afforded by CCR5 intrabody expression, macrophages were exposed to JR-FL, a CCR5 using HIV-1, at 0.3 and 0.51 multiplicity of infection. Cultures were maintained for 15 – 23 days and evaluated for p24, a measure of viral replication, at 3 – 8 day intervals. At the end of culture it was found that macrophages expressing CCR5 intrabody generated 42-, 57-, 52-fold less p24 as compared to parental and control vector containing macrophages. Thus, CCR5 intrabody expression drastically decreased viral load. Therefore, reduction of CCR5 through vector delivery of intrabody genes to CD34+ cells should reduce the likelihood of infection of myeloid cells, their function as viral reservoirs, and has promising therapeutic potential. Currently, studies are underway to evaluate this modality of treatment in humanized NOD/SCID mice.
Development of a Unique siRNA and Intrabody Combinatorial HIV-1 Vector to Knockdown CXCR4 and Protect Cells from HIV-1 Challenge.
Authors: Swan Christina H. , Tschan Mario P. , Barbas Carlos F. , Torbett Bruce E. 
Published: October 2019

Abstract
We overcome the problem of knocking down highly expressed gene function by combining intracellular single chain antibodies (intrabodies) and short interfering RNA (siRNA) to the same gene product. The chemokine receptor CXCR4 is expressed at high levels on T cells and is the co-receptor for pathogenic HIV-1s (termed X4-tropic). Furthermore, it has been shown that HIV-1 requires low amounts of CXCR4 for X4-tropic viral entry (Konopka and Düzgüne AIDS Res Hum Retro, 2002). In contrast to CCR5, due to the high levels of CXCR4, it has been more difficult to provide protection from HIV-1 challenge using conventional siRNA strategies. To address this issue a CXCR4 siRNA and intrabody gene combination HIV-1 vector has been generated. The CXCR4 intrabody is specific for the C-terminal domain of CXCR4 and is coupled to the KDEL endoplasmic retention signal and CXCR4 siRNA targets mRNA nucleotides 331–349 (Martinez, et al. AIDS, 2002). Using the myelomonocytic THP-1 cell line, a line that highly expresses cell surface CXCR4, it was found that CXCR4 intrabody or siRNA vectors mediated a 50% reduction in cells expressing CXCR4 as assessed by flow cytometry. In contrast, the level of CXCR4 cell knockdown increased to 82% using the CXCR4 siRNA and intrabody combinatorial vector. Moreover, the CXCR4 mean fluorescent intensity in cells containing the combination vector resembled the mean fluorescent intensity of cells analyzed with isotype control antibodies, i.e., little detectable CXCR4 expression as judged by flow cytometry. Similar CXCR4 knockdown trends were seen in primary CD4+ T-cells expressing the combinatorial vector as compared to vectors containing the intrabody gene or siRNA alone. HIV-1 X4-tropic challenge studies using a multiplicity of infection of 1 have shown that THP-1 cells expressing the combinatorial vector are resistant to infection (HIV-1 p24 production < 3 ng/ml), whereas THP-1 cells expressing the siRNA or intrabody gene vector were susceptible to infection (HIV-1 p24 > 15–35 ng/ml). These findings demonstrate that targeting both the message and protein product from the same gene is vastly superior to the targeting of either message or protein. The combinatorial vector is currently being tested in humanized mice for efficacy. Lastly, targeting a highly expressed cellular product at the message and protein level may prove beneficial for efficient gene knockdown outside the HIV-1 research and therapy field, such as for metastatic cancers associated with CXCR4/SDF-1 upregulation.
Can We Do Better? Identifying Orthopedic Surgery Patients at High Risk for Venous Thromboembolism Despite Thromboprophylaxis.
Authors: Kahn Susan R. , Schiff Renee L. , Shrier Ian , Hammouda Wahbi , Zukor David 
Published: October 2019

Abstract
Background
Routine administration of thromboprophylaxis is the standard of care for patients undergoing major orthopedic surgery due to the very high rate of postoperative venous thromboembolism (VTE). However, because prophylaxis is not 100% effective, a significant proportion of patients who receive thromboprophylaxis develop post-operative VTE (“breakthrough VTE”). Clinical and surgical characteristics that are associated with the development of VTE in orthopedic surgery patients who receive standard thromboprophylaxis have not been well characterized.
Objectives
To evaluate the incidence of and risk factors for breakthrough VTE in patients who underwent major orthopedic surgery at a tertiary care hospital in Montreal, Canada.
Methods
Charts from consecutive patients who underwent total hip arthroplasty (THA), total knee arthroplasty (TKA) or hip fracture surgery (HFS) (hip pinning or hemiarthroplasty) from August 1, 1999 to April 30, 2000 were abstracted using standardized case report forms. Data were collected on patient characteristics (including VTE risk factors and postoperative complications), surgical characteristics (including type of surgery, operative factors, type of anesthetic) and thromboprophylaxis regimen. The results of any tests performed for clinically suspected VTE were documented, and associations between potential risk factors and objectively confirmed VTE were examined.
Results
Over the 9 month study period, 310 patients underwent major orthopedic surgery (34% THA, 30% TKA, and 36% HFS) and received standard thromboprophylaxis with either dalteparin or enoxaparin (mean duration of prophylaxis: 7 days). Of 83 suspected VTE, 44 (7 proximal DVTs and 37 distal DVTs; 14% of study population; median onset 6 days post-op) were confirmed with objective testing (compression ultrasonography). Multivariate analyses showed that knee surgery (odds ratio [OR] 4.8, 95% confidence interval 2.3, 10.1) and type of low molecular weight heparin (enoxaparin: protective) (OR 0.39, 95% confidence interval 0.20, 0.80) independently predicted VTE. Patient characteristics such as previous VTE, malignancy, hormonal therapy or varicose veins were not associated with VTE.
Conclusion Despite receiving standard thromboprophylaxis, 14% of patients undergoing major orthopedic surgery developed symptomatic breakthrough VTE. Factors that independently predicted VTE in our population were TKR surgery and type of low molecular weight heparin. Our results suggest that TKR patients may warrant more aggressive post-operative physiotherapy and ambulation and adjunctive prophylactic measures such as pneumatic compression. In addition, due to the heterogeneity of different low molecular weight heparin compounds, direct comparison of the effectiveness of enoxaparin with dalteparin for orthopedic prophylaxis in prospective, randomized trials seems warranted.
Efficacy of Patient Self Testing (PST) for Management of the Hypercoagulable Patient.
Authors: Zucker Marcia L. , Jacobs Helena , LaDuca Frank M. , Kessler Craig 
Published: October 2019

Abstract
The PREVENT trial showed strong evidence of the importance of long term low intensity oral anticoagulation therapy (OAT) in patients diagnosed with hyper-coagulable disorders following an initial venous thrombo-embolic event. The current study was designed to compare patient compliance, satisfaction, safety and time in range (TIR) with OAT monitored by either PST or standard laboratory methods. All patients enrolled had a history of at least one hyper-coagulable disorder and had experienced one or more thrombotic episodes prior to enrollment. Patients randomized to the PST arm were directed to test weekly using the ProTime Microcoagulation System (ITC, Edison, NJ); those in the control arm were directed to return to the clinic monthly for Prothrombin time/International Normalized Ratio (PT/INR) testing. Additional testing was encouraged for both groups in response to changes in diet, medication or clinical condition. Of the patients screened for the trial, eight patients randomized to the control arm completed the evaluation period and twenty-three patients randomized to the PST arm have completed at least 6 months of testing. Time in range was determined by the method described by Rosendaal where the INR results between two test dates are assumed to be evenly split between the two test results. The PST patients tested on average once every 9 days (range 5 – 16 days); demonstrating a mean TIR of 73% (range 45 – 90%). The control patients tested at an average frequency of once a month (range 0.3 – 3 months); demonstrating a mean TIR of 73% (range 24 – 96%). There were no statistical differences in the TIR or time above or below range between the two groups. Satisfaction surveys completed at the time of enrollment and after 3 and 6 months of PST showed strong endorsement of PST use. This pilot study demonstrates the utility of PST in the management of OAT in patients with a number of idiopathic coagulopathies and congenital hypercoagulable states (e.g., Protein C deficiency, hyperhomocysteinemia) as an alternative to routine clinical management without compromise to the patient’s well-being.
Sequential Therapy with Cytarabine and Bismuth-213 (213Bi)-Labeled-HuM195 (Anti-CD33) for Acute Myeloid Leukemia (AML).
Authors: Mulford Deborah A. , Pandit-Taskar Neeta , McDevitt Michael R. , Finn Ronald D. , Weiss Mark A. , Apostolidis Christos , Morgenstern Alfred , Divgi Chaitanya R. , Larson Steven M. , Scheinberg David A. , Jurcic Joseph G. 
Published: October 2019

Abstract
HuM195, a humanized anti-CD33 monoclonal antibody, targets myeloid leukemia cells and has activity against minimal residual disease. To enhance the potency of native HuM195 and avoid nonspecific cytotoxicity seen with β-emitting radioimmunoconjugates, the α-emitting radiometal 213Bi was conjugated to HuM195. The feasibility, safety, and antileukemic activity of therapy with 213Bi-HuM195 were shown in a phase I trial (Jurcic et al. Blood 2002). Because of the short-range (50–80 μm) and high linear energy transfer (8400 keV) of α particles, radioimmunotherapy with 213Bi is ideally suited for the treatment of residual disease. To determine the effects of 213Bi-HuM195 after partial cytoreduction with chemotherapy, we treated 25 patients (median age, 67 years; range, 49–80) with cytarabine 200 mg/m2/day for 5 days followed by 213Bi-HuM195 in a phase I/II trial. Fourteen patients had relapsed or primary refractory AML; 5 patients had previously untreated de novo AML, and 6 patients had untreated secondary AML. Sixteen patients had intermediate-risk cytogenetics, and 9 had poor-risk cytogenetics. During the phase I portion of the study, cohorts of 3–6 patients were treated with 0.5, 0.75, 1, and 1.25 mCi/kg. At the 1.25 mCi/kg dose level, 2 of the 4 patients had dose-limiting myelosuppression (grade 4 leukopenia lasting ≥ 35 days) and 1 patient died of progressive pneumonia. Therefore, the maximum tolerated dose was determined to be 1 mCi/kg. No responses were seen at the first two dose levels. Seven of the 19 patients (37%) who received 1 mCi/kg (n=15) or 1.25 mCi/kg (n=4) responded. There were 2 CRs lasting 9 and 12 months; 3 CRp (CR with incomplete platelet recovery) lasting 1, 2, and 6 months and 2 PRs lasting 3 and 8 months. The median time from initiation of chemotherapy to recovery of leukocyte counts was 34 days (range, 21–59 days). Delayed count recovery was attributed to persistent leukemia in 6 patients. Neutropenic fever occurred in all patients, and 19 patients had documented infections. At the phase II dose level, two of the 15 patients died of progressive infections, and one had grade 4 hyperbilirubinemia. The most common extramedullary toxicities were transient, low-grade elevations in liver function tests (n=19) and serum creatinine (n=11). Thirteen patients had infusion-related reactions following the first injection of 213Bi-HuM195, typically characterized by reversible grade 1 or 2 fever and/or chills. One patient had orthostatic hypotension and syncope after the first antibody infusion associated with concomitant bacteremia. Sequential administration of cytarabine and 213Bi-HuM195 is tolerable and can produce complete remissions in patients with AML.