bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of Recombinant Peptide as Substrate in FRET Based Protease Assays.
Authors: Zhang Lin , Lawson Heather , Harish Vallathucherry , Huff Jason D. , Knovich Mary Ann , Owen John
Published: October 2019

Proteases are enzymes that catalyze the breakdown of peptide bonds. They are essential to all physiologic and pathological processes. Some protease deficiencies are closely associated with certain disease conditions, such as alpha-1-Antitrypsin deficiency in COPD, Antithrombin III deficiency in clotting disorders, and ADAMTS13 deficiency in thrombotic thrombocytopenic purpura (TTP). Measurement of protease activity is of great interest in research, drug discovery and development, and for use as diagnostic or prognostic markers in disease states.

Traditional protease assay methods involve multiple steps and are very time-consuming. Recently, fluorescence energy transfer (FRET) based protease assays have become popular. These assays eliminate gel electrophoresis, radioactivity, and the need for special skills or complicated instruments. They require only one step incubation and allow high throughput screening of a large number of samples.

Generation of pure, reliable, high quality substrate is essential to the development and improvement of such assays. Most FRET based assays use synthesized peptide substrates, the size of which is restricted by the limitations of chemical synthesis. These substrates are usually labeled with a pair of two different fluor-quencher molecules. However, when two identical fluorescent molecules are in close proximity, their fluorescence emission can also be quenched. This phenomenon of autoquenching has been widely used in protein structural studies and even in single-molecule structural studies, but has never been used to produce FRET substrates. We hypothesized that this mechanism can be applied to the production of recombinant FRET substrates. We used the ADAMTS13 - von Willebrand factor (VWF) system to test this hypothesis. ADAMTS13 is a metalloproteinase that cleaves VWF between Y1605 and M1606. Kokame et al. have shown that the smallest effective substrate is a 73 amino acid (AA) peptide from the VWF A2 domain. A recombinant 73 AA peptide was generated using Qiagen pQE-100 His Tag vector and M15 E. coli expression system. Two native amino acids, Q1599 and P1611, which flank the ADAMTS13 cleavage site, were mutated to cysteines. These two cysteines were subsequently labeled with fluorescein molecules. The separation of these two fluoresceins by ADAMTS13 cleavage generated measurable fluorescence increase. There was a steady increase of fluorescence over time during the incubation of this recombinant peptide with pooled normal plasma (PNP) used as the source of ADAMTS13. There was only minimal change in fluorescence during incubation with heat inactivated PNP and plasma samples from known TTP patients with absent ADAMTS13 activity. The enzyme kinetic assay yielded an excellent linear relationship between the rates of fluorescence increase and ADAMTS13 enzyme concentration. Our study has provided proof of principle that fluorogenic recombinant peptides can be generated for FRET based protease assays.
Comparison of Anti-Angiogenic Activities of Thalidomide and Lenalidomide In Vitro.
Authors: Zhang Ling-Hua , Lu Ling , Wu Lei , Dredge Keith , Bartlett J. Blake , Schafer Peter , Muller George , Dalgleish Angus , Stirling David
Published: October 2019

Inhibition of angiogenesis is currently perceived as one of the promising strategies in the cancer therapy. Thalidomide (Thalomid®) and its Immunomodulatory Drug (IMiD®) analogs have entered into clinical trials for the treatment of various types of cancers. Lenalidomide (Revlimid®, CC-5013) is currently being assessed in oncology clinical trials worldwide. In this study we have compared the anti-angiogenic mechanisms of thalidomide and lenalidomide in a variety of experimental systems representing distinct events of the angiogenic process. Endothelial cell (EC) proliferation is stimulated by growth factors. Neither thalidomide nor lenalidomide have an appreciable inhibitory effect on growth factor-induced proliferation of human umbilical vein endothelial cells (HUVEC). In both the HUVEC and B16-F10 mouse melanoma tube formation assays, lenalidomide appears to be at least 10-fold more potent than thalidomide, the latter indicating that effects extend to vessels lined by tumor cells. However, in endothelial cell migration assays, thalidomide is more active than lenalidomide; in both the HUVEC and B16-F10 monolayer scratch migration assays, thalidomide is clearly more potent than lenalidomide in preventing the migration of cells into the wounded/scratched region. In assays of HUVEC migration towards specific angiogenic factors, such as VEGF, bFGF, and TNF-α, thalidomide is also more potent than lenalidomide. Interestingly, assays where the whole physiological process of angiogenesis can be studied show differential sensitivities. Lenalidomide is more potent in the rat aorta assay, whereas thalidomide is more potent in the human umbilical explant assay. Mechanistic studies on signal transduction events triggered by VEGF show that both thalidomide and lenalidomide partially inhibit Akt phosphorylation in VEGF-induced HUVEC. Furthermore, inhibitory effects on the phosphorylation of Gab1, a scaffolding protein upstream of Akt activation, have been observed. In summary, our data indicate that both thalidomide and lenalidomide interfere with key events in the angiogenic process and that they can be differentiated qualitatively depending on which component part is studied.
Repeat Borderline or Negative Platelet Factor-4 ELISA May Yield Positive Results in Patients with Clinical Suspicion of Heparin-Induced Thrombocytopenia.
Authors: Lakey Meredith A. , Arndt David W. , Marques Marisa B.
Published: October 2019

Heparin-induced thrombocytopenia (HIT) is mediated by antibodies to heparin-platelet factor 4 (PF4) complexes which lead to platelet activation and aggregation with or without thrombosis. HIT diagnosis relies on clinical and serologic grounds. Although the detection of PF4 antibodies alone is not diagnostic of HIT without thrombocytopenia (relative or absolute) and/or thrombosis, a negative result does not exclude the diagnosis. Thus, it is expected that initially negative ELISAs may turn positive on repeat testing. We reviewed all PF4 ELISA (GTI Diagnostics, Waukesha, WI) assay reports generated at our institution from January 2003 to May 2005 in search of patients who had more than one test performed in this time-period. Of 1,128 tests performed, 181 (16%) were positive, 95 (8%) were borderline, and 852 (76%) were negative. Borderline results were considered those with an OD405 between 0.3 and 0.4 (cutoff value for positive) or two times that of the negative control of each run. No patients were evaluated using the serotonin-release assay. Nine of 70 patients (13%) with initially negative or borderline ELISAs had a positive result upon repeat testing from 2 to 63 days later (Table). All were inpatients, and 6 of 9 were from surgical specialties, mostly cardiac (5 of 6). Five of 8 patients (62.5%) with an initial positive PF4 ELISA result had either a negative or a borderline subsequent test from 7 to 225 days later (Table). The mean platelet count of the patients that had a nondiagnostic ELISA followed by a positive result was 98 x 109/L (N = 9) at the time of the initial test compared with 137 x 109/L at subsequent testing (p = 0.43 by paired Student’s t test). Of note, 2 patients had a normal platelet count when the ELISA result was positive. None had evidence of thrombosis. Borderline test results tended to be repeated sooner (mean of 16 days) than negative ones (mean of 21 days). We suspect that this difference reflects the clinicians’ high index of suspicion for HIT. Our findings suggest that in patients with a high pre-test probability for HIT (status post cardiac surgery with thrombocytopenia), there should be a low threshold for repeating an initial negative or borderline ELISA assay, thereby increasing detection and minimizing complications from this potentially fatal, yet common clinical entity.

Test Results of the 78 Patients With Repeat HIT ELISAs. Initial Test Result Repeat Test Result Number of Patients (% of Total) Mean Number of Days Between Tests (Range) NA: Not applicable Negative Positive 7 (13) 17 (2–63) (N=56) Negative 45 (80) 26 (1–216) Borderline 4 (7) 22 (8–36) Borderline Positive 2 (14) 21.5 (10–33) (N=14) Negative 6 (43) 17.5 (3–42) Borderline 6 (43) 9 (1–23) Positive Positive 3 (37.5) 34 (4–88) (N=8) Negative 4 (50) 26 (7–80) Borderline 1 (12.5) 225 (NA) Total 78
Case Report of Temozolamide Associated Steroid-Refractory ITP and Successful Treatment with Rituximab.
Authors: Reddy Sandeep , Janis Mark , Curti Mario , Minow Robert
Published: October 2019

We report the first case of temozolamide associated ITP and successful treatment with Rituximab. A 59 year-old male with grade III anaplastic astrocytoma is treated with resection followed by adjuvant temozolamide and gamma knife radiation therapy (XRT). He had been taking ramipril for hypertension, and atorvastatin for hyperlipidemia for greater than 1 year. He began taking phenytoin for seizure prophylaxis at the time of his original diagnosis in December of 2004. His CBC is normal at baseline. He recieves XRT from 2/1/05 until 2/15/05 with a normal platelet count of 142 on 4/21/05. He restarts temozolamide from 4/22 to 4/26 and presents with petechiae and platelet count 0. He receives platelet transfusion but platelet count remians at 3. A bone marrow biopsy confirms adequate megakaryoctes. He develops subcutaneous hemorrhage in the neck and is hospitalized for airway precautions. Prednisone at 2mg/kg and IVIG are given with no response. Rituximab is given weekly x 4 weeks at 375 mg/m2 with response by day 22 to platelet count of 123. The response is durable. See table 1.

Response to Rituxan DATE PLATELET COUNT 6/1/05 3 6/16/05 9 6/23/05 104 6/30/05 123 7/21/05 118
Helixate®Nexgen in the Treatment of Patients with Hemophilia A: A Long-Term Pharmacovigilance Surveillance.
Authors: Haertel Sabine , Zacharias Claudia , Brackmann Hans-Herrmann , Roland Zimmermann , Harald Lenk
Published: October 2019

Comprehensive large-scale pharmacovigilance surveillances are very effective tools to collect data on products in the post authorization period. The evaluation presented here was set up to assess the long-term efficacy, safety and tolerability of the recombinant FVIII concentrate, Helixate®NexGen (in the US: Helixate®FS). Sixty-five hemophilia centers in Germany, Austria, Italy, France, and Sweden are participating in this ongoing international project. Previously untreated (PUP) and previously treated patients (PTP) at any age suffering from hemophilia A treated with Helixate®NexGen were eligible for the surveillance. Based on the standard schedule preferred at the centers, patients are routinely screened every 3 to 12 months. At these visits, the following parameters, as used routinely for these patients, were documented (non-interventional design): overall clinical response, pharmacokinetics, occurrence of bleedings, adverse drug reactions including the incidence of inhibitors, other laboratory safety parameters, coagulation parameters, relevant concomitant diseases, and medication. To determine the level of exposure, treatment modalities with Helixate®NexGen, including average factor consumption per month and exposure days are recorded. One hundred and ten patients have been included into the investigation up to now; data from 104 patients were available for this interim analysis including 2 PUPs. The median age was 25 years (range: 8 months – 68 years). One patient suffered from mild, eleven from moderate, and 89 from severe hemophilia A; in three patients the information on severity was missing. Most of the patients were treated prophylactically (92%, at least one infusion per week). Exposure to Helixate®NexGen during the pharmacovigilance ranged between 5 and 703 days. A total of 112 bleedings were documented during the observation period, with a range of 0 to 20 bleedings per patient per year. Efficacy of Helixate®NexGen was assessed as good or excellent in 97% of all cases. There were no cases of inhibitor development during the 2-year observation period, especially no cases of inhibitors when patients were switched from products produced in Chinese hamster ovary (CHO) cells to Helixate®NexGen, which is produced in baby hamster kidney (BHK) cells. The good tolerability of Helixate®NexGen was further supported by the fact that there were no other relevant side effects documented. In summary this pharmacovigilance supports the good tolerability and good/excellent efficacy data of Helixate®NexGen.
Comparison of aPTT (Platelin LS®) and Dilute Russell’s Viper Venom (Viperquik LA Test®) for the Screening of Lupus Anticoagulant in Patients with Venous Thromboembolism.
Authors: Roprigues Celso A. , Martinez Aline A. , Morelli Vania M. , Silveira Raniely C. , Noguti Maria Aparecida E. , Lourenco Dayse M.
Published: October 2019

INTRODUCTION: To rule out the presence of LA antibodies, two or more assays that are sensitive to these antibodies must be negative. The dilute Russell’s Viper Venom Time (dRVVT) and the activated partial thromboplastin time (aPTT) using sensitive reagents have been employed to detect LA. The aim of this study was to assess the concordance level of aPTT using Platelin LS and dRVVT as screening tests to identify LA in patients with venous thromboembolism (VTE).

METHODS: 94 patients (58 women, 62%) with VTE were evaluated. The detection of circulating inhibitor with Platelin LS and dRVVT was based on a prolonged clotting time and a prolonged 1:1 mixture of sample plasma and normal pooled plasma. The confirmation of the phospholipid dependency was performed only with dRVVT.

RESULTS: Among the 94 patients, an abnormal test was found in 24 patients (26%) with aPTT and in 61 patients (65%) using dRVVT. After mixing tests, aPTT ratio remained long in 25% of patients with abnormal aPTT, and in 9.8% with long dRVVT. Five patients had a prolonged mixing study identified by both tests, which resulted in a substantial agreement between the two tests (Kappa= 0.78). Confirmatory tests for LA were positive in 5 out of the 6 patients with long dRVVT mixture, resulting in a prevalence of LA detected by dRVVT of 5.3%. The 5 patients with LA detected by dRVVT also had prolonged mixture with aPTT.

CONCLUSION: Our results indicate that aPTT with Platelin LS is highly associated with the presence of LA detected by dRVVT and may be suitable as a screening test for LA in patients with VTE.
Isolated, Idiopathic Splenic Vein Thrombosis, Presenting with Massive Lower GI Bleeding.
Authors: Agheli Aref , Arora Alka , Khalil Maged , Naik Seema , Dumlao Theresa , Steier William , Kalavar Madhumati
Published: October 2019

Isolated, also called idiopathic, splenic vein thrombosis (SVT) is a very rare clinical condition, which usually results in left-sided portal hypertension and isolated fundal varices. This syndrome is a rare cause of mostly upper, gastrointestinal bleeding. There are only a few hundreds of cases reported in the literature. Colonic varices are even much rarer, 0.07% post mortem autopsies, and 0.2% in a prospective large endoscopic trials. Pancreatic disorders, including malignancies are the most common underlying causes for SVT. Congenital aneurysm of the splenic vein is one of the theoretical explanations of the Isolated, Idiopathic SVT.

Case report: A 53 year old Caucasian female with history of hypertension, and no history of smoking or alcohol abuse, presented with chronic lower gastrointestinal bleeding. Upper endoscopy and flexible colonoscopy revealed perigastric varices without any source of acute bleeding. A bleeding scan demonstrated marked splenomegaly and source of bleeding from left colon. Mesenteric angiogram during venous phase showed splenic vein thrombosis and extensive perigastric varices. In addition, a single large left colonic varix from the lower pole of the spleen was identified as the source of bleeding. The patient was treated with splenic artery embolization with coils, followed by splenectomy, without any major complication. Coagulation studies 8 weeks after the procedure did not show any hypercoagulable state.

Conclusion: The Isolated, Idiopathic SVT, itself is a very rare syndrome. Our center has reported four cases of SVT, secondary to medical conditions, such as; pancreatic malignancy, MRSA sepsis, and multi-organ failure. Upper gastrointestinal bleeding has been more frequently reported than lower bleeding. Interestingly, in our case report, a single colonic varix secondary to SVT was proved to be the cause of chronic lower gastrointestinal bleeding. SVT should be suspected in any patient with a triad of gastric varices, splenomegaly, and normal liver function tests, who presents with gastrointestinal bleeding secondary to left sided or so called “sinistral” portal hypertension. Mesenteric angiography with venous phase is the gold standard for the diagnosis of SVT, as endoscopic studies may not be diagnostic of this syndrome. Splenectomy is the only and definitive procedure of choice in the patients with isolated SVT, followed by post splenectomy vaccination.
Interaction between Paracetamol and Warfarin: A Double-Blind Placebo-Controlled Randomised Study.
Authors: Mahe Isabelle , Bertrand Nathalie , Drouet Ludovic , Simoneau Guy , Sollier Claire Bal dit , Mazoyer Elizabeth , Caulin Charles , Bergmann Jean-François
Published: October 2019

Background: Paracetamol is the gold standard antalgic and antipyretic therapy for patients receiving oral anticoagulants. Paracetamol and anticoagulant are therefore often concomitantly prescribed. The literature reports conflicting data about this association, concluding sometimes to the existence of an interaction and sometimes not. The primary objective of the study is to assess the effect of paracetamol on the anticoagulant effect of warfarin in usual conditions.

Methods: Patients on stable oral anticoagulation (warfarin with a target INR between 2 and 3) for at least 1 month have been included in a prospective randomised double blind study, cross-over controlled versus placebo. They randomsuccessively received for a 14 days-period paracetamol (at 1g four times daily) and placebo. Both periods were separated by a wash-out period lasting at least 2 weeks. INR, factors II, V, VII, X were measured during each period before the intake of the study-treatment and 12h after the intake at day 2, 4, 7, 9, 11 and 14.

Results: 20 patients were included. On paracetamol, the mean maximal INR increase observed was 1.01 ± 0.45 versus 0.27 ± 0.28 on placebo, p=0.003. The mean maximal INR was higher on paracetamol than on placebo (3.45 vs 2.66, p=0.01). A clinically significant increase of INR (above 0.5) was observed in 17 out of 19 patients on paracetamol as compared to 3 out of 19 patients on placebo. Factor II, VII, X levels were significantly decreased on paracetamol while there was no difference in factor V levels.

Discussion: Paracetamol at 4g/d induced a significant increase in INR in patients on a stable anticoagulation with warfarin. It results in an increase in the risk of bleeding of the warfarin. The mechanism of the interaction may be a metabolit of the paracetamol, inhibiting of enzymes involved in the synthesis of the vitamino-K dependent clotting factors.
Identification, Characterization of a Novel Zinc Finger Protein (HZF1) Gene and Its Roles in Erythroid Differentiation and Megakaryocyte Differentiation.
Authors: Zhang Jun-Wu , Peng Han , Du Zhan-Wen
Published: October 2019

A significant number of transcription factors contain evolutionarily conserved zinc finger motifs. The classical C2H2 zinc finger motif, which employs two cysteine and two histidine residues to coordinate a single zinc ion, is a maim type of the zinc finger proteins. Many of the identified C2H2 type zinc protein have been demonstrated to be transcription factors that play important roles in differentiation and development of cells and tissues of higher organisms. In this study, we obtained some novel expression sequence tags (ESTs) containing C2H2 type motifs by reverse transcription-polymerase chain reaction (RT-PCR) using RNAs derived from hemin-induced K562 cells. A cDNA encoding novel zinc finger protein (designed as HZF1) was obtained by screening the human bone marrow cDNA library using one of the ESTs as the probe. The cDNA sequences (2013 nucleotides) have been submitted to the GenBank databases under accession No. AF244088.1). Three transcripts of HZF1 gene were explored by PCR amplification of cDNAs derived from hemin-induced K562 cells. The cDNA sequences (2632 nucleotides) of the longest transcript have been submitted to the GenBank databases under accession No. DQ117529). These transcripts may result from different splicing of the pre-mRNA of HZF1 but the differences between them are only involved in 5′ non-translation region of HZF1 mRNA. BLASTN analysis revealed that HZF1 gene has four exons and three introns. The putative protein consists of 670 amino acid residues including continuous 15 C2H2 and 2 C2RH zinc finger motifs. This structure characterization and the nuclear location of the protein suggest that HZF1 may function as a transcription factor. HZF1 mRNA was detected in ubiquitous tissues and various hematopoietic cell lines. Increased HZF1 mRNA expression was observed following hemin-induction or phorbol myristate acetate (PMA)-induction of K562 cells. Both of the antisence method and RNA interference assay revealed that repression of the intrinsic expression of HZF1 blocked the hemin-induced erythroid differentiation and reduced the PMA-induced megakaryocytic differentiation of K562 cells, which suggested that HZF1 play an important part in erythroid differentiation and megakaryocytic differentiation.
NCoA4 Containing Transcriptional Complexes Modulates Response of Myelomonocytic Leukemic Cell Line to Retinoids and VD3.
Authors: Baudet Aurelie , Delva Laurent , Balaguer Patrick , Piquemal David , Marti Jacques , Commes Therese
Published: October 2019

Large scale analyses of transcriptome improve comprehension of complex processes such as differentiation or cell proliferation. SAGE libraries construction of the AML model U937 allowed the identification of new markers of myelomonocytic differentiation induced by retinoids and vitamin D3 (VD3) (Piquemal et al., 2002). Those molecules act through ligand-dependent transcription factors of the nuclear receptors family: RAR, RXR and VDR. Among differentially expressed members of transcriptional complexes, the most relevant was the co-regulator NCoA4 (Nuclear receptor Coactivator 4). This protein that modulates interactions between transcription factors, RNA polymerase II and chromatin remodeling factors, was initially described as a co-activator of the androgen receptor (AR). Its activity has been extended to receptors of estrogens (ER), peroxisome-proliferating activators (PPAR), retinoid X (RXR) and recently to VD3 receptor (VDR).

Using real-time semi-quantitative PCR, we found that NCoA4 is specifically expressed during the monocytic differentiation of U937 but not during the granulocytic differentiation of NB4 cell lines. These results were confirmed by analysis on normal and in vitro-differentiated leukemia primary cells. Moreover, its early induction, within 6 hours of retinoids and VD3 treatment on U937 cells, suggests that its expression may be controlled by one or several nuclear receptors. Because of cross-talks between retinoids and VD3 pathways, we used NB4-LR2 cells in which RAR is knock-down. In this cell line, NCoA4 is expressed in a VDR-dependent fashion reinforcing the hypothesis that the coregulator is specifically involved in the VD3-monocytic differentiation of leukemic cells.

Next, to explore the role of NCoA4, U937 cells were stably transfected to constitutively over-express the protein. The doubling time of this cell line (U-NCoA4) reaches to 48 hours against 24 hours in U937 cells. Concerning ligand-induced growth arrest, these cells are particularly sensitive to the RXR and VDR agonists while no significative difference was observed after treatment by the RAR agonist or by any combination. In addition, over-expression of NCoA4 induces a slow down of differentiation, as shown by expression of CD11b and CD14 myelomonocytic markers. Thus, in U-NCoA4, except for the RAR agonist, treatment for 72 hours corresponds to treatment for 48 hours levels of U937 cells.

To conclude, in term of growth arrest, NCoA4 over-expressing cells are particularly sensitive to RXR and VDR agonists. Thus, natural ligands present in the culture medium might reduce or delay proliferation, inducing the same effect on differentiation. In order to have a large view of networks, transcriptome of U-NCoA4 was analyzed by real time PCR on Low Density Array composed of a hundred messengers extracted from U937 SAGE libraries. Analysis is currently in progress.
Insulin-Like Growth Factor 1 Induced Expression of Vascular Endothelial Growth Factor, Which Is Dependent on MAP Kinase and Phosphatidylinositol 3-Kinase Signaling in Human Bone Marrow Stromal Cells.
Authors: Liu Gexiu , Tang Guangxiao
Published: October 2019

INTRODUCTION: Administration of bone marrow stromal cells after traumatic brain injury provides functional benefit. Its protective mechanisms include neurogenesis and angiogenesis. Different traumatic brain injuries have shown that vascular endothelial growth factor (VEGF) induced neuroprotection, neurogenesis, and angiogenesis. Insulin-like growth factor 1 (IGF-1) is a 7.5-kDa peptide with structural homology to proinsulin. In the adult organism, the liver is the major source of IGF-1. Although IGF-1 is very low in the normal adult rat brain, a number of studies have shown that IGF-1 is strongly induced in the CNS, and exerted its mitogenic and trophic effects on a variety of cell-types after different traumatic brain injuries, suggesting its repair roles after brain damage. We tested the hypothesis that IGF-1 induces expression of VEGF in human bone marrow stromal cells, and its signaling pathway.

METHODS: Human bone marrow adherent cells were cultured, and were passaged in DMEM/F12 containing 10% FBS. The fifth passage cells were identified as stromal cells. Subconfluent cells were used, and were washed twice in PBS, then cultured in serum-free DMEM/F12. After overnight incubation, cells were exposed to IGF-1 (100 ng/ml) in the presence of no kinase inhibitor or a 1 h pretreatment with 50 μM PD98059, or 200 nM wortmannin. Cells were harvested after 2 h for analysis of active phosphorylated kinases (Akt and MAPK) or after 24 h for analysis of VEGF protein and mRNA. VEGF mRNA was detected by semi-quantitative RT-PCR, and VEGF protein by ELISA, and kinases by Western blot.

RESULTS: 100 ng/ml IGF-1 increased significantly both VEGF mRNA and VEGF protein, which were very low level in control cells. 24 hours after treatment, ratio of RT-PCR product between VEGF and β-actin reach to (38.93±6.73)% from (18.61±4.25)% of control cells (P<0.01). VEGF protein increased to (123.45±20.86)pg/ml from (46.97±8.91)pg/ml (P<0.01). Moreover, IGF-1 induced active phosphorylated kinases (Akt and MAPK). And PD98059 or wortmannin inhibited effects of IGF-1.

CONCLUSION: IGF-1 enhanced expression of VEGF mRNA and VEGF protein in human bone marrow stromal cells, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling. These results suggest that VEGF be involved in therapeutic effects of bone marrow stromal cells in ischemic disorders, including stroke.
Molecular Diagnosis of Invasive Fungal Infections: Species Identification and Quantitative Analysis by Broad-Spectrum PCR Assays.
Authors: Baskova Lenka , Preuner Sandra , Lion Thomas
Published: October 2019

Invasive fungal infections (IFI) play an increasingly important role as life-threatening complications in immunocompromised patients. Early application of antimycotic agents is an essential prerequisite for successful therapy. However, standardized diagnostic techniques permitting rapid, sensitive and, no less importantly, economic screening for the clinically relevant fungi have been lacking. We have developed two different real-time PCR systems for quantitative analysis of pathogenic fungi. The Pan-AC assay* permits in a single reaction the detection of all important Aspergillus and Candida species, which are responsible for the great majority of IFI in immunosuppressed individuals. In view of the increasing incidence of invasive infections caused by hitherto uncommon fungal species, new diagnostic tests with very broad specificity are required. We have therefore developed an additional two-reaction Pan-fungus assay*, which facilitates quantitative detection of a wide spectrum of fungal species (n>50), including also the newly emerging pathogenic fungi. The assays display high sensitivity and show no cross-reactivity with non-fungal pathogens or human DNA sequences. We have established an additional rapid molecular assay based on PCR fragment length analysis of a variable region in the fungal genome permitting rapid identification of the fungal species present, in order to facilitate selection of the most appropriate antifungal treatment. Correct identification of specific fungal pathogens including different Aspergillus, Candida and Fusarium species detected by the above assays in patients with febrile neutropenia has been confirmed by sequence analysis. The new assays are readily applicable to routine clinical diagnosis and provide a rapid and economic approach to the screening and monitoring of invasive fungal infections.
Early “Mini” Donor Lymphocyte Infusions after Reduced-Intensity Conditioning in Allogeneic Transplant of Highly Purified CD34+ Cells from HLA-Identical Donors in Pediatric Leukemia Patients.
Authors: Diaz Miguel A. , Gonzalez-Vicent Marta , Ramirez Manuel , Perez Antonio , Garcia-Castro Javier , Sevila Julian , Madero Luis
Published: October 2019

To maximize graft-versus-leukemia (GvL) effect while minimizing transplant-related morbidity and mortality (TRM), we designed a study of allogeneic CD34+ selected PBSC transplantation followed by donor lymphocyte infusion (DLI). PBSC CD34+ selection was performed with the CliniMACS device. Between June 2004 and July 2005, fifteen consecutive patients (5 females and 10 males) aged between 1–16 years (median 5 years) diagnosed of AML (6) and ALL (9) were conditioned with fludarabine 30 mg/m2/day x 5 days and melphalan 140 mg/m2/day x 1 day. Status at transplantation was 1st CR 10, 2nd CR 4 and 3th CR 1. GvHD prophylaxis consisted of CsA + short Mtx in 13 cases and CsA in 2. Patients were grafted with a median number of 6.9 x 106/Kg CD34+ cells (range: 1.5–30.9 x 106/Kg) and 15.6 x 103 CD3+ cells (range: 2–59.2 x 103/Kg). All patients engrafted. Median time to neutrophil and platelet engraftment was 12 days (range: 9–15 days) and 13 days (range: 9–42 days), respectively. DLI of 0.5 x 105/Kg CD3+ cells were scheduled on day +15, +30 and +60. A total of 40 DLI were performed. One patient had acute GvHD grade I after the 2nd DLI that resolved with topical steroid treatment and another one developed graft hypoplasia after first DLI resolved after G-CSF treatment. One patient died of a sepsis on day +15 (TRM 6% ± 5%). Two patients relapsed, one with an isolated CNS relapse (6 months postransplant) and the other with a medullar relapse (3 months). With a median follow-up of 6 months (range: 3–13 months) the disease free survival was 75% ± 13% and overall survival 87% ± 8%. The preliminary results of our transplantation strategy are encouraging. A large prospective study is needed to substantiate these results.
Immunoablative Therapy with Autologous Stem Cell Transplantation in the Treatment of Poor Risk Multiple Sclerosis.
Authors: Kozak Tomas , Havrdova Eva , Pitha Jiri , Mayerova Karolina , Evzen Gregora , Petr Kobylka , Sarka Vodvarkova , Jan Pachl , Marek Trneny , David Dolezil , Jiri Fiedler , Vladimir Koza
Published: October 2019

The immunoablative therapy with hematopoietic stem cell transplantation is tested in patients with intractable form of autoimmune diseases including multiple sclerosis (MS). Thirty one patients with secondary rapidly progressive MS were included in the phase I/II clinical trial involving the high dose chemotherapy with autologous peripheral blood progenitor cell (PBPC) support. Twenty seven patients underwent high dose conditioning BEAM. T cell depletion in vitro was performed in 18 grafts. Nine patients with not purged graft received in vivo ATG 4mg/kg i.v. D+1, D+2 after transplantation. Median follow-up is 48 months (24–72). Median EDSS (Expanded Disability Status Scale) of grafted patients at the time of inclusion was 6.5 (5.0–7.5), median EDSS of grafted patients at the last follow up was 7,0 (5.5–10.0). Two patients out of 27 (7 %) remain improved significantly (by ≥ 1.0 point on EDSS), 3 patients (11 %) are improved not significantly (by 0.5 point). Five patients (19 %) are stable in their EDSS. Seven patients (26 %) gained their disability significantly (by ≥ 1.0 point on EDSS) despite the treatment, one of them died 31 months after the transplantation from disease progression (EDSS 10.0). Ten patients (37 %) worsened not significantly (by 0.5 point) on their EDSS. Patients who clearly stabilized their disability or remain improved represent 37 %. The increase of EDSS at the 48 months (m) of the follow up is significatnt (Wicoxon’s, repeated measure ANOVA, t-test), however, is not at 36, 60 and 72 m respectively. The development of disability between the group that was grafted with in vitro purged graft and the group with ATG i.v. was not significant (Wilcoxon’s, Mann-Whitney). 20 patients stabilized their MRI finding, in 2 patients decreased number and size of lesions, 5 patients worsened their MRI. No mortality has been observed in this cohort. However, the toxicity of the transplantation differed in each individual; two serious events involving respiratory tract have been observed. Changes in lymphocyte subsets in peripheral blood were followed before and after the treatment. The percentage and absolute count of CD4+ cells and CD4+CD45 as well as IRI were significantly decreased 12 months after the transplantation

Conclusion: Almost 40 % of patients with otherwise intractable rapidly progressive MS remained stable or improved in their disability with median follow up 48 months after immunoablative therapy. The value of this type of therapy should be investigated in the randomized trial. The study was sponsored by Grant IGA No NF/6560-3 from Ministry of Health of the Czech Republic.
Voriconazole Replacing Liposomal Amphotericin B as First-Line Therapy in Suspected or Proven Fungal Infection in Acute Leukemia Patients: A Retrospective Audit of Clinical and Financial Outcomes in a UK District Hospital.
Authors: Hybnerova Jindrinska , Bahn Amit , Pocock Christopher
Published: October 2019

Background: In most centres in the United Kingdom, systemic antifungal therapy (AFT) is used as third-line therapy for fever complicating profound, prolonged neutropenia (PPN) during the treatment of acute leukemia. Voriconazole has been recommended as first-line AFT at the Kent and Canterbury Hospital (KCH) since October 2002; liposomal amphotericin B was the previous treatment of choice. The aim of the audit was to identify numbers of episodes of PPN and the numbers of suspected fungal infections in a 2-year period following the policy change. Subsequently the clinical and financial outcomes were examined. In addition, the impact on cost of AFT following centralisation of leukemia treatment from two district hospitals onto one site was examined.

Methods: A retrospective audit was conducted on data from hematology inpatients undergoing remission induction or consolidation therapy for acute leukemia at KCH between January 2003 and December 2004. The costs of voriconazole and liposomal amphotericin B treatment from 2002 to 2004, and 8 months prior and post centralisation of inpatient care (April 2004, which increased the population from 400,000 to 600,000), were examined.

Results: 84 episodes of PPN were identified in 41 patients undergoing treatment for acute leukemia; mostly acute myeloid leukemia (AML). Itraconazole prophylaxis from d1 of therapy and GCSF from d+5 was used in the majority of cases. 18 cases of suspected or radiologically proven fungal infection were identified. High-resolution computed tomography of the chest was performed in 10 cases and suspicious lesions identified in three. Voriconazole was used as first-line therapy in 17/18 cases. In 7 cases, treatment was switched to liposomal amphotericin B. Reasons for switching were rising C-reactive protein (1 patient), persistent fever (2 patients), radiological progression (1 patient) and side effects (3 patients). Of the 3 patients with radiological evidence of fungal infection, two had a complete resolution (1 voriconazole, 1 voriconazole/liposomal amphotericin B) and 1 patient died of refractory leukemia. There was a fall in total antifungal spend from £263K in 2002/3 to £229K in 2003 and a further 68% fall to £73K in 2004. We suspect this was due to increasing adherence to the new antifungal protocol and to improved practices following centralisation: in the 8 months pre-centralisation the antifungal spend across all hospitals was £102K falling by 74% to £26K in the 8 months on the single site.

Conclusion: Since introducing voriconazole as first-line AFT, centralising inpatient services, and adopting common policies for antimicrobial prophylaxis, there has been considerable financial benefit with no increase in morbidity and/or mortality.
Estimating the Total Cost of Infused Iron Chelation Therapy.
Authors: Desrosiers Marie-Pierre , Payne Krista A. , Baladi Jean-Francois
Published: October 2019

Background: Patients suffering from β-thalassemia or sickle cell disease require on-going blood transfusions. Chronic transfusion, however, results in iron overload, which if not removed by iron chelation therapy (ICT), causes organ damage. Deferoxamine (DFO) is currently the standard of care for ICT, but many patients do not adhere to therapy possibly because of the need for almost daily infusions lasting 8 to 10 hours each.

Rationale: While the impact of current care on clinical and patient outcomes is generally understood, less is known about the total cost of DFO therapy.

Objectives: To identify a complete set of cost items to inform the development of an ICT related Resource Use Questionnaire (RUQ) for administration in an international cohort study of the actual cost of ICT in practice; and to obtain a preliminary, literature-based estimate of total annual per patient costs of ICT.

Methods: A search of the literature (EMB Reviews; Scirus and Ovid Medline (1996+); PubMed (1995+) was performed using the following key words: thalassemia, sickle cell disease, myelodysplastic syndrome, cost, iron chelation, Desferal, deferoxamine, resource use, reimbursement and compliance. Cost items were extracted from eligible studies to create an aggregated, composite set of ICT-related variables to which unit costs (2004/2005 USD) were applied.

Results: Of 396 abstracts obtained, all but 96 were excluded because ICT cost data were lacking. Of those retained, only 4 studies (1 Israël;1 US;2 UK) reported ICT-related costs (1 lifetime;3 annual). Cost variables differed markedly among studies each focusing on some specific aspect. The application of unit costs to the composite list of ICT-related variables and associated resource use profiles reveal that total annual per patient ICT costs may be as high as $7,487 to $15,836 (£4,191 to £8,865) depending on age. The cost of DFO accounts for only 16%–31% of these estimated total costs, with the balance accounted for by other annual ancillary expenditures such as equipment and supplies, monitoring, and home health care services. Total costs could well be underestimated given that component lifetime costs such as DFO treatment complications, the clinical sequelae of poor adherence to DFO, and the indirect costs of lost productivity were not included. Cost estimates will be supplemented and validated at the time of abstract presentation by the resource use and unit cost data generated by the RUQ employed in the aforementioned international cohort study.

Conclusions: Estimated total costs of ICT are substantial and well exceed the cost of DFO alone. A paucity of published data related to the total costs of ICT underscores the need for additional ICT cost data from actual practice to better understand the economic impact of novel ICT agents.
SGN-35, an Anti-CD30 Antibody-Drug Conjugate, Exhibits Potent Antitumor Activity for the Treatment of CD30+ Malignancies.
Authors: Hamblett Kevin J. , Barton Jeremy , Cerveny Charles G. , Andreyka Jamie B. , Kissler Kim M. , Okeley Nicole M. , Stone Ivan , Sutherland May K. , Sun Michael M. , Senter Peter D. , Wahl Alan F. , Ihle Nathan C.
Published: October 2019

Hodgkin/Reed-Sternberg (H-RS) and anaplastic large cell lymphoma (ALCL) cells express high levels of the cell surface receptor CD30, a member of the TNF receptor superfamily. To augment the antitumor activity of anti-CD30 based therapies, a novel synthetic anti-mitotic agent, monomethyl auristatin E (MMAE), has been conjugated to the anti-CD30 antibody cAC10. MMAE was synthetically modified to include a maleimide for conjugation to an antibody and a protease cleavable Val-Cit peptide linker to release the drug from the antibody. The in vitro and in vivo activity of SGN-35, the resulting antibody-drug conjugate, is outlined. SGN-35 has an average of four MMAE molecules per antibody. Cell surface binding, internalization and intracellular release of MMAE were monitored by immunofluoresence and isotope labeling methods. The H-RS cell line L540cy translocates SGN-35 to lysosomes within 16 hours. Analysis of the cellular contents demonstrated that MMAE is rapidly and efficiently liberated from the antibody, and MMAE accumulates inside the cells. After treating L540cy cells with 200 ng/mL of SGN-35 (6 nM MMAE), the intracellular concentration of MMAE reaches 800 nM after 24 hours, rising to nearly 1000 nM after 72 hours. From 24 to 72 hours, about 70% of the intracellular drug is liberated MMAE, while 30% is still bound to the antibody. In vitro potency of SGN-35 was measured by incubating various concentrations of SGN-35 with CD30+ tumor cells and measuring cell viability in culture. SGN-35 had potent, selective in vitro activity with IC50s ranging from 4 to 35 ng/mL for CD30+ cell lines, and an IC50 > 1000 ng/mL for the CD30− line WSU-NHL. This demonstrates that the conjugation of the novel cytotoxic agent MMAE to an antibody can be accomplished without compromising the antibody’s specific binding and internalization. SCID mice were implanted with L540cy and treated with intravenous doses of SGN-35. In mice bearing L540cy xenografts, complete tumor regressions were achieved at doses as low as 2 mg/kg q4dx4. SGN-35 is also highly effective in treating mice with disseminated L540cy implants, at well tolerated doses. Based on the potent cytotoxicity, efficacy in xenograft models, and tolerability, SGN-35 is being advanced toward clinical development in CD30-positive hematologic malignancies.
Very Low Prevalence of Hyperhomocysteinemia among US Children with Personal/Family History of Thrombosis. Towards Reconsideration of the Recommendation for Routine Testing.
Authors: Manco-Johnson Marilyn J. , Goldenberg Neil A. , Stabler Sally
Published: October 2019

Background: Comprehensive thrombophilia laboratory testing is recommended for the evaluation of children with thrombosis by the Perinatal and Pediatric Subcommittee of the International Society on Thrombosis and Haemostasis (ISTH). The C677T thermolabile variant of the methylene tetrahydrofolate reductase (MTHFR) gene is highly prevalent in Caucasians, carried in heterozygous form in 45% and homozygously in 10%. The thrombogenicity of the MTHFR C667T mutation is mediated via elevation in plasma homocysteine concentration. However, in the U.S., folate fortification of food products may circumvent the impact of this mutation upon plasma homocysteine.

Objective: To determine the prevalence of hyperhomocysteinemia among U.S. children with a personal history of thrombosis (thrombosis group) or a first-degree family history of thrombosis or thrombophilia (familial risk group).

Methods: All children comprising the two study groups had been referred to the Thrombosis/Thrombophilia Program at The Children’s Hospital, Denver, and were consecutively recruited for participation in collection of laboratory and clinical data for this research. Plasma homocysteine, performed clinically as part of the ISTH recommended thrombophilia laboratory assessment, was determined by gas chromatograph mass spectroscopy.

Results: Homocysteine concentration was normally distributed within the study population, and did not vary with age or differ significantly between the two study groups. Table 1 displays the results relative to age and study group. In 100% of subjects, homocysteine level was either low or normal with respect to the previously-established reference range for healthy individuals.

Conclusions: Despite a high historical prevalence of the MTHFR C677T mutation in Caucasians, the prevalence of hyperhomocysteinemia among U.S. children with thrombosis or a familial risk of thrombosis is extremely low, being absent among nearly 200 children studied. Because other mutations that do result in hyperhomocysteinemia and thrombosis occur rarely, homocysteine testing as part of a comprehensive thrombophilia assessment in U.S. children should be targeted to those patients in whom metabolic disease is suspected clinically.

Plasma Homocysteine Levels (UMl/L) in Children. Category Total < 1 year 1 - < 7 years 7 - < 13 years 13 - < 24 years + Thrombosis − Thrombosis N 193 25 48 44 76 128 65 Mean 5.6 4.4 5.0 5.3 6.6 5.6 5.6 Median 5.6 4.8 5.0 5.3 6.2 5.8 5.4 Std. Dev. 2.4 2.2 2.3 2.6 2.1 2.6 1.9