ALBERT

Description: Human megakaryocytes and platelets contain counterparts of several plasma proteins. The origin of most of these alpha-granule proteins is unclear. Fibrinogen represents one of those molecules, being essential in hemostasis, thrombosis, and platelet aggregation. To study whether fibrinogen is endocytosed by megakaryocytes and packaged into alpha- granules or newly synthesized by these cells, we established a highly sensitive nested primer polymerase chain reaction for the detection of human fibrinogen gamma-chain mRNA. In enriched megakaryocyte fractions, as well as fluorescence-activated cell sorter-purified megakaryocytes from bone marrow samples of healthy volunteers, no fibrinogen gamma- chain mRNA could be detected, despite the presence of the corresponding fibrinogen gamma-chain DNA. We conclude that fibrinogen gamma-chain mRNA, as detectable by our amplification system, is missing in megakaryocytes. This finding suggests that fibrinogen might be acquired from plasma by endocytosis and sequestered in alpha-granules before reentering the circulation after platelet activation.

Description: The Philadelphia chromosome of chronic myelogenous leukemia (CML) patients is caused by a translocation of the c-abl gene from chromosome 9 to the breakpoint cluster region (bcr) on chromosome 22. A new bcr- abl mRNA is expressed in these cases. We have developed a modified polymerase chain reaction (PCR) for the detection of this mRNA. The method is extremely sensitive, reliable, and relatively fast. The analysis of peripheral blood or bone marrow cells from CML patients treated with chemotherapy shows that the two possible mRNAs are expressed in various combinations. Our results show that even after myeloablative therapy for bone marrow transplantation bcr-abl mRNAs are still expressed. Further studies, however, are necessary to determine the clinical relevance of a small number of persisting cells expressing the bcr-abl mRNA.

Description: The Philadelphia chromosome of chronic myelogenous leukemia (CML) patients is caused by a translocation of the c-abl gene from chromosome 9 to the breakpoint cluster region (bcr) on chromosome 22. A new bcr- abl mRNA is expressed in these cases. We have developed a modified polymerase chain reaction (PCR) for the detection of this mRNA. The method is extremely sensitive, reliable, and relatively fast. The analysis of peripheral blood or bone marrow cells from CML patients treated with chemotherapy shows that the two possible mRNAs are expressed in various combinations. Our results show that even after myeloablative therapy for bone marrow transplantation bcr-abl mRNAs are still expressed. Further studies, however, are necessary to determine the clinical relevance of a small number of persisting cells expressing the bcr-abl mRNA.

Description: Cytomegalovirus (CMV) disease can be effectively prevented in allogeneic hematopoietic stem cell transplant (HSCT) patients by ganciclovir (GCV) given as prophylaxis or preemptive therapy. Due to the low bioavailability of oral GCV capsules, GCV is usually administered intravenously (IV) to HSCT patients. Valganciclovir (VGCV) is the valine ester prodrug of ganciclovir. In healthy subjects, HIV-infected patients, and solid-organ transplants, the oral bioavailability of VGCV is about 60%, or 10-fold higher than oral GCV capsules. The bioavailability and total GCV exposure provided by oral VGCV relative to IV GCV in HSCT patients with gastrointestinal (GI) GHVD has not been established. METHODS: HSCT patients were eligible for the study if the following criteria were satisfied: 1) ≥16 years of age; 2) biopsy-proven GHVD of GI tract with nausea and/or diarrhea (300–1500 ml/day) or proven GVHD of skin or liver plus diarrhea with no other explanation; 3) no active CMV infection or disease; 4) neutrophil count ≥1000/μL; 5)creatinine clearance 〉60 ml/min. Following a standardized breakfast, eligible patients were randomized to receive a single dose of open-label study drug (900 mg of oral VGCV or 5 mg/kg of IV GCV). After a minimum 48 hr. washout period, patients were crossovered to alternate study drug. Blood for levels of GCV and VGCV were obtained predose and then over the 24 hours after dosing. Pharmacokinetic (PK) parameters were derived by noncompartmental methods. RESULTS: Data from 16 patients are currently available. Patient demographics include mean age 45 yrs (range 23 to 58 yrs); males 13, females 3; mean weight 80kg (range 52 to 107 kg); mean creatinine clearance 96 ml/min (range 62 to 184 ml/min). Median time after transplant for study was 303 days (range 102 to 988 days). Mean GCV PK parameters are summarized in the following table. Mean GCV Value (Coefficient of variation in %) Oral VGCV-900 mg IV GCV-5mg/kg Parameter N = 16 N = 16 AUC o -τ (μg•hr/mL) 43.58 (37) 46.74 (40) AUC o-∞ (μg•hr/mL) 46.03 (41) 48.89 (43) C(max) μg/mL) ( 6.45 (30) 12.53 (30) T max (hr) 3.13 (22) 0.97 (8) T½ (hr) 4.97 (31) 5.09 (29) GCV AUC values were similar, although maximum GCV concentrations were higher and acheived earlier with IV GCV. Terminal elimination half-life of GCV with oral VGCV and IV GCV were similar. After 900 mg of oral VGCV, mean plasma Cmax for VGCV was low (0.22 μg/mL), which is consistent with rapid and almost complete metabolism of VGCV to GCV. CONCLUSION: These preliminary results suggest that systemic exposure to GCV after 900 mg of oral VGCV is comparable to that achieved with IV GCV in HSCT patients with stable GI GVHD. Oral VGCV could be a useful alternative to IV GCV in certain HSCT patients requiring prophylaxis or preemptive therapy for CMV.

Description: The bcl-2 oncogene blocks programmed cell death (apoptosis). Epstein- Barr virus (EBV) can immortalize B lymphocytes into continuously growing lymphoblastoid cell lines (LCL) by the coordinate expression of at least 9 latent genes (EBV nuclear antigen [EBNA] 1–6, latent membrane protein [LMP], and terminal proteins [TP] 1 and 2). We analyzed transcription and expression of bcl-2 and latent EBV genes in Burkitt's lymphoma (BL) cell lines with a germinal center phenotype (group I) as well as activated BL cell lines (group III) and LCLs. We found high expression of bcl-2 as well as the full spectrum of latent EBV genes in LCLs and activated group III BL cell lines. Group I BL cells expressed little or no bcl-2, EBNA-2, and LMP. Superinfection with nondefective EBV or an EBNA-2-defective virus as well as transfection with EBNA-2- or LMP-carrying vectors into the EBV-negative cell lines RAMOS, DG75, U698, or BJAB induced upregulation of bcl-2 expression. The strongest effect on bcl-2 was obtained by transfection with LMP, or infection with the nondefective virus. No change of bcl-2 expression was observed with EBNA-1. Our data indicate that the immortalization capacity of EBV and the growth advantage of EBV- positive compared with EBV-negative BL cells in vitro may predominantly be mediated via induction of bcl-2 and the main effectors are EBNA-2 and LMP.

Description: Human megakaryocytes and platelets contain counterparts of several plasma proteins. The origin of most of these alpha-granule proteins is unclear. Fibrinogen represents one of those molecules, being essential in hemostasis, thrombosis, and platelet aggregation. To study whether fibrinogen is endocytosed by megakaryocytes and packaged into alpha- granules or newly synthesized by these cells, we established a highly sensitive nested primer polymerase chain reaction for the detection of human fibrinogen gamma-chain mRNA. In enriched megakaryocyte fractions, as well as fluorescence-activated cell sorter-purified megakaryocytes from bone marrow samples of healthy volunteers, no fibrinogen gamma- chain mRNA could be detected, despite the presence of the corresponding fibrinogen gamma-chain DNA. We conclude that fibrinogen gamma-chain mRNA, as detectable by our amplification system, is missing in megakaryocytes. This finding suggests that fibrinogen might be acquired from plasma by endocytosis and sequestered in alpha-granules before reentering the circulation after platelet activation.

Description: The bcl-2 oncogene blocks programmed cell death (apoptosis). Epstein- Barr virus (EBV) can immortalize B lymphocytes into continuously growing lymphoblastoid cell lines (LCL) by the coordinate expression of at least 9 latent genes (EBV nuclear antigen [EBNA] 1–6, latent membrane protein [LMP], and terminal proteins [TP] 1 and 2). We analyzed transcription and expression of bcl-2 and latent EBV genes in Burkitt's lymphoma (BL) cell lines with a germinal center phenotype (group I) as well as activated BL cell lines (group III) and LCLs. We found high expression of bcl-2 as well as the full spectrum of latent EBV genes in LCLs and activated group III BL cell lines. Group I BL cells expressed little or no bcl-2, EBNA-2, and LMP. Superinfection with nondefective EBV or an EBNA-2-defective virus as well as transfection with EBNA-2- or LMP-carrying vectors into the EBV-negative cell lines RAMOS, DG75, U698, or BJAB induced upregulation of bcl-2 expression. The strongest effect on bcl-2 was obtained by transfection with LMP, or infection with the nondefective virus. No change of bcl-2 expression was observed with EBNA-1. Our data indicate that the immortalization capacity of EBV and the growth advantage of EBV- positive compared with EBV-negative BL cells in vitro may predominantly be mediated via induction of bcl-2 and the main effectors are EBNA-2 and LMP.