David Dennison

Cytokines in Sickle Cell Disease

Abstract Sickle red cells express adhesion molecules including integrin 4β1, CD36, band 3 protein, sulfated glycolipid, Lutheran protein, phosphatidylserine and integrin-associated protein. The proadhesive sickle cells may bind to endothelial cell P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), CD36 and integrins leading to its activation. Monocytes also activate endothelium by releasing proinflammatory cytokines like tumor necrosis factor alpha (TNF-) and interleukin 1β (IL-1β). Sickle monocytes also express increased surface CD11b and cytoplasmic cytokines TNF and IL-1β indicating activated state. Polymorphonuclear leukocytes (PMNs) are also activated with reduced L-selectin expression, enhanced CD64 expression and elevated levels of sL-selectin, sCD16 and elastase resulting in increased adhesiveness to the endothelium. Platelets are also activated and secrete thrombospondin (TSP) and cytokine IL-1. They also form platelet- monocytes aggregates causing endothelial cell P-selectin expression. Endothelial cell activation by these multiple mechanisms leads to a loss of vascular integrity, expression of leukocyte adhesion molecules, change in the surface phenotype from antithrombotic to prothrombotic, excessive cytokine production and upregulation of HLA molecules. Furthermore, contraction of these activated endothelial cells leads to exposure of extracellular matrix proteins, such as TSP, laminin, and fibronectin and their participation in adhesive interactions with bridging molecules from the plasma such as von Willebrand factor (vWf) released from endothelial cells, ultimately culminating in vasoocclusion and local tissue ischemia, the pathognomonic basis of vasoocclusive crisis.

Imatinib in pregnancy

To the Editor: Chronic myeloid leukemia (CML) is a myeloproliferative disorder seen predominantly in adults and not infrequently discovered incidentally during pregnancy. It is characterized by the presence of Philadelphia chromosome in almost 95% of cases (1). The abnormal chromosome occurs as a result of reciprocal translocation between the long arm of chromosomes 9 and 22; t(9;22) (2, 3). This translocation leads to the formation of a fusion gene BCR-ABL, with increased production of abl-tyrosine kinase, a molecule that plays a critical role in cell proliferation and may thus play a significant role in the abnormal cell growth seen in CML (3, 4). The introduction of imatinib (Glivec; Novartis, UK), an inhibitor for abl-tyrosine kinase, has made a considerable impact on the outcome and quality of life of patients with CML (5). Although the drug is well tolerated by patients with few side effects, experience with this drug during pregnancy in humans is rather limited (6). The drug is found to be teratogenic in mice but not in rabbits and data on the human fetus is limited (6). Here we report the outcome of two patients with CML who became pregnant while on imatinib.

Randomized trial of two different conditioning regimens for bone marrow transplantation in thalassemia--the role of busulfan pharmacokinetics in determining outcome.

Summary:In total, 94 patients with homozygous beta thalassemia were randomized to two different conditioning regimens: busulfan 600 mg/m2+cyclophosphamide 200 mg/kg or busulfan 16 mg/kg+cyclophosphamide 200 mg/kg and antilymphocyte globulin (47 in each group), for bone marrow transplantation, to see whether increased myeloablation or increased immunosuppression would reduce rejection. Busulfan pharmacokinetics in determining outcome was evaluated. There was no significant difference in engraftment, graft-versus-host disease, rejection, and overall and disease-free survival in the two groups. Systemic exposure to busulfan was significantly higher in the 600 mg/m2 group, but in both groups there was a wide interindividual variation in the busulfan kinetics. Six patients rejected the graft, two in the busulfan 600 mg group and four in busulfan 16 mg group (P=0.677 CI −0.17, 0.07), but in five patients (pharmacokinetic data not available in one patient) who rejected the graft busulfan first dose trough level (Cmin-1) was below 150 ng/ml while it was above this level in the 66 of 68 patients with successful engraftment (P⩽0.001). This randomized trial shows that rejection is influenced by busulfan levels and suggests that monitoring of busulfan levels and dose adjustment based on first-dose kinetics may reduce the risk of rejection.

Allogeneic bone marrow transplantation in the developing world: experience from a center in India.

We describe our experience of setting up an allogeneic BMT program at the Christian Medical College Hospital, Vellore over a period of 13 years, from October 1986 to December 1999. Two hundred and twenty-one transplants were performed during this period in 214 patients, with seven patients undergoing second transplants. Indication for BMT were thalassemia major – 106 (48%), CML – 30, AML – 35, ALL – 10, SAA – 22, MDS – six and six for other miscellaneous disorders. The mean age of this patient cohort was 15.6 years (range 2–52). Graft-versus-host disease of grades III and IV was seen in 36 patients (17%) and this was the primary cause of death in 20 patients (9.2%). All patients and donors were CMV IgG positive. Sepsis was the primary cause of death in 16 patients (7.4%), 10 bacterial, four fungal and two viral. One hundred and ten of this series of patients are alive and disease free (50%) with a median follow-up of 24 months (range 2–116). These results are comparable to those achieved for patients with similar disease status in transplant units in the Western world and cost a mean of US

Special issues related to hematopoietic SCT in the Eastern Mediterranean region and the first regional activity report

15 000. Bone Marrow Transplantation (2001) 27, 785–790.

Pharmacokinetics of oral busulphan in children with beta thalassaemia major undergoing allogeneic bone marrow transplantation

Although several centers are now performing allogeneic hematopoietic SCT (HSCT) in the Eastern Mediterranean (EM) region, the availability is still limited. Special issues including compatible donor availability and potential for alternative donor programs are discussed. In comparison to Europe and North America, differences in patterns of diseases and pre-HSCT general status, particularly for patients with BM failure, are described. Other differences including high sero-positivity for CMV, hepatitis B and C infection, and specific observations about GVHD and its relation to genetically homogeneous communities are also discussed. We report that a total of 17 HSCT programs (performing five or more HSCTs annually) exist in 9 countries of the EM region. Only six programs are currently reporting to European Group for Blood and Marrow Transplantation or Center for International Blood and Marrow Transplantation Research. A total of 7617 HSCTs have been performed by these programs including 5701 allogeneic HSCTs. The area has low-HSCT team density (1.56 teams per 10 million inhabitants vs 14.43 in Europe) and very low-HSCT team distribution (0.27 teams per 10 000 sq km area vs <1–6 teams in Europe). Gross national income per capita had no clear association with low-HSCT activity. Much improvement in infrastructure and formation of an EM regional HSCT registry are needed.

Quantification of busulfan in plasma by gas chromatography-mass spectrometry following derivatization with tetrafluorothiophenol

The pharmacokinetics of busulphan were studied in 23 thalassaemic children undergoing BMT. Patients received busulphan at a dose of either 16 mg/kg with cyclophosphamide and ATG (Group A) or 600 mg/m2 (with cyclophosphamide alone) (Group B) in 16 divided doses every 6 h over 4 days. Busulphan levels were analyzed by a modified GC-MS method. The dose of busulphan/kg for patients in group B was 64% (range 56–71%) higher than that for patients in group A. The mean AUC, Css, Cmax and MRV were significantly higher in group B as compared with group A for both doses 1 and 13. There was no significant difference in Vd/F, T1/2 and Kel between the two groups. A significant decrease in AUC and Css was found between 1st and 13th doses in group B, but not in group A. The Cl/F values in group A were significantly higher than those in group B after dose 1, but not after dose 13. No increase in toxicity due to the higher dose of busulphan was noted. We conclude that busulphan at 600 mg/m2 results in much higher systemic exposure to the drug as compared to 16 mg/kg, without increase in toxicity in children with beta thalassaemia major.

Etiology of diarrhea in patients undergoing allogeneic bone marrow transplantation in South India.

A specific and highly sensitive method has been developed for the determination of busulfan in plasma by gas chromatography-mass spectrometry using a deuterium-labeled busulfan (busulfan-d8) as internal standard. Plasma containing busulfan and busulfan-d8 were extracted with ethyl acetate and derivatized with 2,3,5,6-tetrafluorothiophenol prior to the monitoring of specific ions. The limit of quantification of the assay was 20 ng/ml and the calibration curve was linear over the range of 10 to 2000 ng/ml of derivatized busulfan. This method was in good agreement with the GC-MS assay using derivatization with sodium iodide and measuring diiodobutane. In addition, a pharmacokinetic study of busulfan was conducted in six children. The apparent oral clearance was 5.7+/-1.9 ml/kg/min and the volume of distribution was 1.0+/-0.4 l/kg and were similar to those previously reported in pediatric patients.

High-performance liquid chromatographic method for quantification of busulfan in plasma after derivatization by tetrafluorothiophenol

Background. No studies so far have examined enteric infections in patients undergoing bone marrow transplantation (BMT) in developing countries where asymptomatic carriage and colonization with enteric pathogens is frequent. Methods. A prospective study followed 65 patients who underwent BMT in South India between 1995 and 1998. Patients were screened for enteric pathogens before transplantation, weekly during the first 4 weeks after transplantation, and during all episodes of diarrhea. Results. Enteric pathogens were found in 60% of patients before or after transplantation. Pretransplantation screening revealed asymptomatic excretion of enteric pathogens in 29% (19/65). Forty-eight percent of patients undergoing BMT developed diarrhea. Diarrhea was mainly of noninfectious origin in the first 20 days after transplantation. More than 20 days after transplantation, the major causes of diarrhea were graft-versus-host disease and infection. Parasitic infections other than Cryptosporidium did not contribute significantly to morbidity in the pre- and posttransplantation period. Rotavirus and adenoviruses were found in approximately 12% and 5% of subjects, respectively. Bacterial infections in the posttransplantation period were found to be more common in India than in developed countries. Clostridium difficile-associated diarrhea was seen in the posttransplantation period but not before transplantation. Enterotoxigenic and enterohemorrhagic Escherichia coli caused symptomatic infections in the posttransplantation period, but the association of other classes of diarrheogenic E. coli with diarrhea was doubtful. Conclusions. There was significantly higher mortality (P <0.01) in patients with symptomatic or asymptomatic gastrointestinal infections caused by bacteria than in patients with parasitic or viral infections or without enteric infections.

Primary immunodeficiency diseases in oman: five years' experience at sultan qaboos university hospital.

A high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of busulfan in plasma. Busulfan was extracted in toluene, derivatized by 2,3,5,6-tetrafluorothiophenol to obtain di-TFTP-butane, the derivatization product was then re-extracted in toluene and injected into the HPLC system with ultraviolet detection (wavelength: 275 nm). Recovery from extraction was 80%, the limit of quantification was 50 ng/ml and linearity ranged from 50 to 2000 ng/ml. In addition, forty-two samples obtained from pediatric patients treated with busulfan were analyzed by the HPLC and GC-MS assays based on the same derivatization procedure. The correlation between the di-TFTP-butane concentrations was highly significant (p<0.0001), demonstrating that the two methods were in good agreement.