Abdalla Awidi

Relationship of serum imatinib trough level and response in CML patients: Long term follow-up

One hundred and three patients with Philadelphia chromosome or BCR-ABL positive chronic myeloid leukemia (CML) in chronic phase who were on oral imatinib were included in this study. The study aimed to assess the relationship between imatinib trough serum levels and clinical outcome (as determined by molecular response) in Jordanian CML patients who have been on imatinib therapy for at least 12 months. The mean trough imatinib serum level in the group with complete molecular response (CMR) was 2891±856 ng/ml, the group with major molecular response (MMR) was 2337±434 ng/ml, the group with complete cytogenetic response (CCyR) was 1817±563 ng/ml, and the group without CCyR was 1723±673 ng/ml. A receiver operating characteristic (ROC) curve was constructed after dividing patient sample into two groups, those with MMR or better and those without MMR, in order to estimate a threshold for imatinib level that correlates with a favorable response (the former group), and analysis yielded a value of 2158 ng/ml.

Comparison of platelet aggregation using light transmission and multiple electrode aggregometry in Glanzmann thrombasthenia

We examined platelet aggregation in platelet-rich plasma (PRP) and in whole blood in nine patients with Thrombasthenia Glanzmann (TG). In PRP, aggregation was measured by monitoring the changes in light absorbance that occurred in response to adenosine 5-diphosphate (ADP), collagen and ristocetin. To measure platelet aggregation in whole blood, we used multiple electrode Impedance aggregometry using the same aggregating agents. In PRP, the patients platelets showed defective aggregation in response to ADP, collagen, epinephrine and partially to ristocetin in all patients. In whole blood, platelet aggregation in response to the same aggregating agents showed similar response and appeared to be very similar to that which occurred in PRP. Whole blood impedance aggregometry seems to give similar results to PRP light transmission aggregometry in patients with TG. Multiple electrode aggregometry (MEA) is faster and more convenient to use in these patients.

Study of mutations in Jordanian patients with haemophilia A: identification of five novel mutations

Summary.  Haemophilia A (HA) is an X‐linked recessive bleeding disorder caused by mutations in the factor VIII gene (F8), which encodes factor VIII (FVIII) protein, a plasma glycoprotein, that plays an important role in the blood coagulation cascade. In the present study, our aim was to identify F8 gene mutations in HA patients from Jordan. One hundred and seventy‐five HA patients from 42 unrelated families were included in this study. Among these patients, 117 (67%) had severe HA, 13 (7%) had moderate HA and 45 (26%) had mild HA. Severe patients were first tested for intron‐22 inversion using long range polymerase chain reaction (PCR), then negative patients were tested for intron‐1 inversion using PCR. Sequencing for the entire F8 gene was performed for all severe HA patients who were found negative for intron‐22 and ‐1 inversions and it was also performed for moderate and mild HA patients. HA causative mutations were identified in all patients. Intron‐22 and ‐1 inversions were detected in 52% and 2% of families respectively. Beside these two mutations, 19 different mutations were identified, which include 15 missense and four frameshift mutations. Five novel mutations were identified including one frameshift and four missense mutations. No large deletions or nonsense mutations were detected in patients who participated in this study. Only 17 patients with severe HA were found positive for FVIII inhibitors. The data presented will play an important role for genetic counselling and health care of HA patients in Jordan.

Safety and Efficacy of Autologous Intra-articular Platelet Lysates in Early and Intermediate Knee Osteoarthrosis in Humans: A Prospective Open-Label Study.

Objective:To explore the safety and benefit from intra-articular autologous platelet lysate (PL) injection in early and intermediate knee osteoarthritis. Design:Open-label prospective study. Setting:Laboratory. Patients:Adult patients, aged 35 to 70 years, with a history of chronic pain or swelling on one or both knees and imaging findings (radiograph or magnetic resonance imaging) of degenerative changes in the joint of grade I or II on the Kellgren scale were included. Interventions:Autologous PL was given in the knee joint by percutaneous intra-articular route every 3 weeks for a total of 3 injections. Main Outcome Measures:Response was evaluated by nonnormalized Knee Osteoarthritis and Disability Outcome Score (KOOS). Results:There was a significant improvement in the 5 aspects evaluated at weeks 32 and 52 compared with baseline. Symptoms score significantly improved at weeks 32 and 52 from a mean of 11.1 at baseline to 9.0 (P < 0.0001) and 8.7 (P < 0.0001). Stiffness score significantly improved at weeks 32 and 52 from 2.2 at baseline to 1.7 (P < 0.022) and 1.6 (P < 0.016). Pain score improved at 32 weeks and at 52 weeks from a baseline of 14.2 to 9.8 (P < 0.0001) and 9.2 (P < 0.0001). Daily Living score improved from 25.0 to 18.7 at 32 weeks (P < 0.0001) and to 15.6 at 52 weeks (P < 0.0001). Sport score improved from 10.7 to 8.4 at 32 weeks (P < 0.0001) and to 8.1 at 52 weeks (P < 0.0001). Conclusions:Intra-articular PL significantly improved score of all aspects evaluated by KOOS. Platelet lysate seems to be a safe product. Clinical Relevance:To the best of our knowledge, this is the first clinical study addressing the use of autologous PL as a treatment measure for knee osteoarthrosis (KOA). There are no studies published regarding the treatment of KOA by intra-articular injections of PL. The previous studies were on the use of platelet-rich plasma (PRP) treatment for KOA. Platelet-rich plasma use has been in place for several years, however, a standardized protocol has not yet been established. Platelet lysate represents a safe, economical, easy to prepare, and easy to apply source of growth factors in the treatment of KOA. A head-to-head study is needed to compare PRP with PL in KOA.

Comparison of osteo/odontogenic differentiation of human adult dental pulp stem cells and stem cells from apical papilla in the presence of platelet lysate

INTRODUCTION Human dental pulp cells (DPSCs) and stem cells from apical papilla have been used for the repair of damaged tooth tissues. Human platelet lysate (PL) has been suggested as a substitute for fetal bovine serum (FBS) for large scale expansion of dental stem cells. However, biological effects and optimal concentrations of PL for proliferation and differentiation of human dental stem cells remain to be elucidated. METHODOLOGY DPSCs and SCAP cells were isolated from impacted third molars of young healthy donors, at the stage of root development and identified by markers using flow cytometry. For comparison the cells were cultured in media containing PL (1%, 5% and 10%) and FBS, with subsequent induction for osteogenic/odontogenic differentiation. The cultures were analyzed for; morphology, growth characteristics, mineralization potential (Alizarin Red method) and differentiation markers using ELISA and real time -polymerase chain reaction (qPCR). RESULTS The proliferation rates of DPSCs and SCAP significantly increased when cells were treated with 5% PL (7X doubling time) as compared to FBS. 5% PL also enhanced mineralized differentiation of DPSCs and SCAP, as indicated by the measurement of alkaline phosphatase activity, osteocalcin and osteopontin, calcium deposition and q-PCR. CONCLUSION Our findings suggest that using 5% platelet lysate, proliferation and osteo/odontogenesis of DPSCs and SCAP for a short period of time (15 days), was significantly improved. This may imply its use as an optimum concentration for expansion of dental stem cells in bone regeneration.

Factors that contribute to clopidogrel resistance in cardiovascular disease patients: environmental and genetic approach.

BACKGROUND AND OBJECTIVE Clopidogrel is a potent antiplatelet drug that reduces the risk of vascular events in patients with cardiovascular disease. However, several studies have shown that about a quarter of patients showed low or no response to clopidogrel therapy. In this study, factors that contribute to clopidogrel resistance were investigated in 270 cardiovascular disease patients from Jordan. PATIENTS AND METHODS Clopidogrel resistance was determined through platelet aggregation analysis using the Multiplate analyzer®. Genetic factors (CYP2C19*2 and PON1 Q192R) were examined using polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS The incidence of clopidogrel resistance among Jordanians is about 32%. Significant association between clopidogrel resistance and female gender, concomitant use of calcium channel blockers, and low HDL was found (p < 0.05). In addition, presence of CYP2C19*2 allele is strongly related to clopidogrel resistance (p < 0.001). However, lack of contribution to clipidogrel resistance was found for PON1 Q192R polymorphism, age, diabetes, hypertension, smoking and aspirin use (p > 0.05). CONCLUSION Several factors might contribute to clopidogrel resistance including gender, concomitant use of calcium channel blockers, HDL and CYP2C19*2 polymorphism.

Identification of atypical PML-RARA breakpoint in a patient with acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) of the M3 subtype is characterized by translocation t(15;17) that generates the PML-RARA fusion gene. Depending on the breakpoint position in the PML gene, 3 main fusion transcripts usually result. These breakpoints are bcr1 and bcr3 in introns 6 and 3, respectively, and bcr2 in exon 6. This report describes a rare atypical bcr2 breakpoint in a patient with morphological, cytogenetic and molecular features of APL. The presence of t(15;17) was first revealed by fluorescent in situ hybridization. Molecular analysis by reverse transcription polymerase chain reaction using primers for different PML-RARA junctions showed bands with different sizes compared with those generated from the three classical breakpoints, namely bcr1, bcr2 and bcr3. However, sequence analysis confirmed the presence of a bcr2 transcript with an atypical breakpoint within exon 6. The patient responded well to treatment and is now in complete remission. However, suggesting a favorable prognosis associated with such a rare transcript is difficult as more similar cases are needed to confirm such a conclusion. This article also stresses the importance of sequencing unusual polymerase chain reaction products to confirm their nature.

Rare inherited bleeding disorders secondary to coagulation factors in Jordan: a nine-year study.

This work reports on rare inherited coagulation factors defects which were seen in a developing country over a 9-year period. There were a total of 30 cases which fulfilled this diagnosis. Fibrinogen abnormalities were the most frequently encountered. There were 10 patients with afibrinogenemia, 2 with hypofibrinogenemia and 1 case with dysfibrinogenemia. Factor XI deficiency was found in 7 patients, factor V and VII deficiencies accounted for 3 cases each. Factor X and XIII deficiencies were found in 2 patients each. All these rare deficiencies accounted for 10% of all inherited bleeding disorders in the population studied over 9 years.

Physicochemical and Microstructural Characterization of Injectable Load-Bearing Calcium Phosphate Scaffold

Injectable load-bearing calcium phosphate scaffolds are synthesized using rod-like mannitol grains as porogen. These degradable injectable strong porous scaffolds, prepared by calcium phosphate cement, could represent a valid solution to achieve adequate porosity requirements while providing adequate support in load-bearing applications. The proposed process for preparing porous injectable scaffolds is as quick and versatile as conventional technologies. Using this method, porous CDHA-based calcium phosphate scaffolds with macropores sizes ranging from 70 to 300 μm, micropores ranging from 5 to 30 μm, and 30% open macroporosity were prepared. The setting time of the prepared scaffolds was 15 minutes. Also their compressive strength and e-modulus, 4.9 MPa and 400 MPa, respectively, were comparable with those of the cancellous bone. Finally, the bioactivity of the scaffolds was confirmed by cell growth with cytoplasmic extensions in the scaffolds in culture, demonstrating that the scaffold has a potential for MSC seeding and growth architecture. This combination of an interconnected macroporous structure with pore size suitable for the promotion of cell seeding and proliferation, plus adequate mechanical features, represents a porous scaffold which is a promising candidate for bone tissue engineering.

Selection and targeting of EpCAM protein by ssDNA aptamer

Aptamers are molecules that reveal highly complex and refined molecular recognition properties. These molecules are capable of binding with high affinity and selectivity to targets, ranging from small molecules to whole living cells. Several aptamers have been selected for targeting cellular proteins and they have also used in developing therapeutics and diagnostic strategies. Epithelial cell adhesion molecule (EpCAM) is considered as a cancer stem cell (CSC) biomarker and one of the most promising targets for aptamer selection against CSCs. In this study, we have developed a ssDNA aptamer with high affinity and selectivity of targeting the EpCAM protein extracellular domain. The SELEX technique was applied and the resulted sequences were tested on EpCAM-positive human gastric cancer cell line, KATO III, and the EpCAM-negative mouse embryonic fibroblast, NIH/3T3 cells. Ep1 aptamer was successfully isolated and showed selective binding on EpCAM-positive KATO III cells when compared to EpCAM-negative NIH/3T3 cells, as observed by the flow cytometry and the confocal imaging results. Additionally, the binding of Ep1 to EpCAM protein was assessed using mobility shifting assay and aptamers-protein docking. Furthermore, the binding affinity of Ep1 was measured against EpCAM protein using EpCAM-immobilized on magnetic beads and showed apparent affinity of 118 nM. The results of this study could suggest that Ep1 aptamer can bind specifically to the cellular EpCAM protein, making it an attractive ligand for targeted drug delivery and as an imaging agent for the identification of cancer cells.