ALBERT

Description: L-selectin mediates leukocyte tethering and rolling, the first step in a sequential process of leukocyte adhesion and migration. Additionally, L-selectin has important signaling roles perhaps contributing to leukocyte activation and integrin-mediated adhesion. Because chemokines are critically involved in leukocyte activation, we questioned whether L-selectin signaling affects chemokine receptor expression and function. We observed that whereas only 5% to 15% of freshly isolated lymphocytes expressed CXCR4 on the cell surface, intracellular CXCR4 was detectable in all cells. Engagement of L-selectin by antibody cross-linking or the L-selectin ligands fucoidan or sulfatide mobilized intracellular CXCR4 to significantly increase surface CXCR4 expression but did not affect CCR5, CCR7, or β2-integrin expression. L-selectin stimulation also inhibited stromal-derived factor 1 (SDF-1)–induced CXCR4 internalization. The combined effects of L-selectin on CXCR4 trafficking are likely important in markedly enhancing cell activation by SDF-1. Blockade of SDF-1–induced CXCR4 internalization resulted in enhanced actin polymerization on subsequent exposure to SDF-1. Physiologically more important, L-selectin stimulation increased SDF-1–induced lymphocyte adhesion and transendothelial migration, which were inhibited by anti–leukocyte function-associated antigen 1 antibodies, tyrosine kinase inhibitors, and pertussis toxin. To further corroborate the additive stimulating effects, L-selectin signaling and SDF-1 increased β2-integrin activation. Taken together, L-selectin–mediated signals specifically enhance CXCR4 expression and function, suggesting a novel mechanism for the modulation of lymphocyte activation during cell adhesion and transmigration.

Description: We recently described a subset of patients with a myeloproliferative variant of hypereosinophilic syndrome (MHES) characterized by elevated serum tryptase levels, increased atypical mast cells in the bone marrow, tissue fibrosis, and the presence of the fusion tyrosine kinase, FIP1L1-PDGFRα, which is a therapeutic target of imatinib mesylate. Seven patients with MHES were treated with imatinib mesylate (300-400 mg daily). Clinical improvement and resolution of eosinophilia was observed in all patients, although cardiac dysfunction, when present, was not altered by therapy. Reversal of bone marrow pathology, including increased cellularity, the presence of spindle-shaped mast cells, and myelofibrosis, was evident in all patients at 4 to 8 weeks following initiation of therapy. This was accompanied by a decrease in activated eosinophils and mast cells in the peripheral blood and bone marrow, respectively. Serum tryptase levels declined rapidly to normal levels in all patients and remained in the normal range throughout therapy. Molecular remission, with disappearance of detectable FIP1L1/PDGFRA (F/P) transcripts, was achieved in 5 of 6 patients tested. The lack of reversal of cardiac abnormalities and persistence of the F/P mutation in some patients suggests that early intervention with higher doses of imatinib mesylate may be desirable in the treatment of patients with MHES.

Description: Familial eosinophilia (FE) is an autosomal dominant disorder characterized by marked eosinophilia and progression to end organ damage in some, but not all, affected family members. To better define the pathogenesis of FE, 13 affected and 11 unaffected family members (NLs) underwent a detailed clinical evaluation at the National Institutes of Health (NIH). No clinical abnormalities were more frequent in the family members with FE compared with the NLs. There was, however, a decreased prevalence of asthma in family members with FE compared with unaffected family members. Eosinophil morphology as assessed by either light or transmission electron microscopy was normal in family members with and without FE. Although levels of eosinophil-derived neurotoxin (EDN) and major basic protein (MBP) were elevated in patients with FE compared with NL, levels of both granule proteins were lower than in nonfamilial hypereosinophilic syndrome (HES). Similarly, increased surface expression of the activation markers CD69, CD25, and HLA-DR was detected by flow cytometry on eosinophils from patients with FE compared with NL, albeit less than that seen in HES. These data suggest that, despite prolonged marked eosinophilia, FE can be distinguished from HES by a more benign clinical course that may be related to a relative lack of eosinophil activation.

Description: Bacterial contamination has represented the second leading cause for transfusion- associated mortality. The French hemovigilance study indicated that in neutropenic and immune depressed patients the mortality is almost 100%. With the approval of two different systems to perform quality control tests on leukocyte-reduced platelets from bacterial contamination, we instituted a process to culture each platelet unit after a 24 hour incubation hold at 22±2C. After this hold, an aliquot (4 mL) was removed from each product bag and cultured aerobically (28% using the Pall BDS and 72% using the BacT/Alert system). The assignment was predetermined by collection site. Following 24 hour incubation at 35C, the cultures were reviewed and those platelet products with no growth/negative results were released. The BacT/Alert bottles remained under incubation until product outdate 5 days post collection. Between September 2003 and June 2004, 69,161 products were cultured. The reference laboratories performed on both the product and culture bottles/pouches a gram stain, and culture on agar plate and in tryptic soy broth, reading after five days if no growth. To resolve any questions regarding sampling volume as a contribution to lack of identification of true positives, larger volumes (20 mL) of product associated with all positive culture bottles (20% of samples sent to the reference laboratory) were examined but did not change the discordant negative yield versus true positive yield of organisms. There were 12 true positives (growth in the culture system which could be reproduced on subculture with the same organism growing from the product and culture system by an independent microbiology laboratory). There were 153 false positives or discordant negatives (growth in the culture system, which did not grow in the product but did repeat on subculture of the culture bottle/pouch). Bacterial species identified in products included Staph coagulase neg 5, Staph aureus 2, Bacillus species 1, Strep Group A beta hemolytic 1, Diphtheroids 1, Enteric gram negative bacillus 1, Corynebacterium species 1, Propionbacterium acnes 1. (One product had more than one organism.) The discordant negatives included additionally Comamonas acidivorans 1 and Micrococcus species 1; however, 95% of the organisms cultured were recognized skin contaminant organisms. There were 2.5 times more false positives with the Pall BDS system than the BacT/Alert. (The BDS system has been superseded by the eBDS system as of July). Comparatively the number of true positives remained within the 95% CI for the two systems. These results identify a true positive frequency of 1 in 5763 (0.00017%) [95% CI 5618–5911], but a false positive rate of 1 in 453 (0.00221%). There were 4 delayed positive cultures (bottle developed growth after the 24 hour reading time); however, none of the associated transfused products resulted in a reported reaction. Culturing platelet products has identified a number of organisms, primarily skin organisms. These data support the prevalence of true positive bacterial contamination in Platelets, Pheresis products as previous models have reported.

Description: Genetic modification of hemopoietic progenitor cells ex vivo, followed by the infusion of the genetically modified cells into the human immunodeficiency virus-1 (HIV-1) infected donor, has been proposed as a treatment for HIV-1 infection. The current study was undertaken to evaluate the effect of hemopoietic stem cell mobilization and harvesting on HIV-1 replication in persons with HIV-1 infection. Eighteen HIV-1–infected persons received recombinant granulocyte colony-stimulating factor (G-CSF; Filgrastim) 10 μg/kg per day, for 7 days. On days 4 and 5, peripheral blood mononuclear cells were harvested by leukapheresis. The CD4+ lymphocyte count at entry was 〉500/μL for 6 subjects, 200 to 500/μL for 6 subjects, and 0.6 log10) above baseline between days 4 and 7 and returned to baseline by day 27. Significant increases of plasma HIV-1 RNA levels occurred in 5 subjects despite 3-drug antiretroviral therapy. Changes in CD4+ and CD34+ cells during mobilization and harvesting were similar in all subjects whether they had or did not have increased plasma HIV-1 RNA levels. Thus, mobilization and harvesting of bone marrow progenitor cells from persons infected with HIV-1 induced a transient increase in viral replication in some patients but was not associated with adverse effects. (Blood. 2000;95: 48-55)