Unexpected effects of FERM domain mutations on catalytic activity of Jak3: structural implication for Janus kinases.

Janus kinases comprise carboxyterminal kinase, pseudokinase, SH2-like, and N-terminal FERM domains. We identified three patient-derived mutations in the FERM domain of Jak3 and investigated the functional consequences of these mutations. These mutations inhibited receptor binding and also abrogated kinase activity, suggesting interactions between the FERM and kinase domains. In fact, the domains were found to physically associate, and coexpression of the FERM domain enhanced activity of the isolated kinase domain. Conversely, staurosporine, which alters kinase domain structure, disrupted receptor binding, even though the catalytic activity of Jak3 is dispensable for receptor binding. Thus, the Jak FERM domain appears to have two critical functions: receptor interaction and maintenance of kinase integrity.

Platelets and neutrophils from healthy term neonates exhibit increased levels of immunoglobulins.

Immune-mediated thrombocytopenia and neutropenia are not uncommon problems in the newborn period. Such cytopenias have been associated with increased levels of IgG, IgM, or both, in the serum, documented by indirect assay, and/or on the cell surface, documented by direct assay, and decreased cell survival. However, interpretation of measurement of platelet- or neutrophil-associated antibodies is problematic due to the lack of data from healthy neonates. In this study, both direct and indirect neutrophil- and platelet-associated IgG and IgM were measured in cord samples from 44 healthy, term neonates. These infants had increased amounts of direct platelet-associated IgG and direct neutrophil-associated IgM and IgG compared with adult controls. Serum samples from these healthy newborns manifested a significantly decreased level of IgM binding to target platelets compared with serum from healthy adult controls. There was not a significant difference in direct platelet-associated IgM, or indirect Ig to neutrophils or platelets. Complete blood counts drawn at 24 h of age were within normal limits in 34/35 infants studied. Moreover, there was not a statistical difference in platelet or neutrophil Ig studies between the newborns of multiparous and primigravida mothers. The physiologic consequences of the increased amounts of these Ig to the survival and function of platelets and neutrophils in neonates is unclear. However, these values must be considered for proper interpretation of platelet and neutrophil Ig measurements in newborns with cytopenias.

Flow cytometric analysis of residual white blood cell concentration and platelet activation antigens in double filtered platelet concentrates.

Reduction of contaminating leukocytes in platelet products by filtration has been shown to decrease the incidence of human leukocyte antigen (HLA) alloimmunization. Nonetheless, prevention is not complete when using current techniques, and a significant number of patients continue to exhibit clinical refractoriness and to produce alloantibodies. Interest in preventing HLA alloimmunization and other complications of white blood cell (WBC) contamination of transfused cellular products has resulted in ongoing efforts to increase the efficiency of leukodepletion filters. As the efficiency of these filters increases, more accurate and precise methods for counting extremely low numbers of WBCs must be instituted to ensure quality control. We have validated a simple, rapid flow cytometric assay for quantitating low numbers of WBCs in platelet products. The assay is sensitive to a lower limit of 0.1 WBC/microliter without concentration of the platelet product sample and has an excellent correlation (R2 = 1.00) between calculated and expected WBC concentration over a range of 0.1 to 100.0 WBC/microliter. (R2 values over the concentration ranges of 0.1 to 1.0 WBC/microliter and 1.0 to 10.0 WBC/microliter were 0.988 and 0.996, respectively.) The intraassay coefficients of variation at WBC concentrations of 50.4/microliter, 0.9/microliter, and 0.1/microliter were 4%, 8%, and 18%, respectively. The flow cytometric counting technique was applied, in concert with a Nageotte chamber manual counting method, to the enumeration of residual WBCs in 20 apheresis and random donor platelet concentrates filtered through two leukodepletion filters sterile docked in series. A greater than four log10 WBC reduction capability was demonstrated when utilizing this double filtration procedure, and its clinical applicability is underscored by data that showed no statistically significant change in expression of activation-specific platelet antigens before versus after filtration.