C. I. Edvard Smith

The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases.

X-linked agammaglobulinaemia (XLA) is a human immunodeficiency caused by failure of pre-B cells in the bone marrow to develop into circulating mature B cells. A novel gene has been isolated which maps to the XLA locus, is expressed in B cells, and shows mutations in families with the disorder. The gene is a member of the src family of proto-oncogenes which encode protein-tyrosine kinases. This is, to our knowledge, the first evidence that mutations in a src-related gene are involved in human genetic disease.

A peptide nucleic acid–nuclear localization signal fusion that mediates nuclear transport of DNA

We have combined a peptide nucleic acid (PNA) with the SV40 core nuclear localization signal (NLS), to create a bifunctional PNA–NLS peptide. The PNA–NLS peptide increased the nuclear uptake of oligonucleotides and enhanced the transfection efficacy of plasmids. Gene expression from an enhanced green fluorescent protein plasmid and a lacZ plasmid was preserved when hybridized to PNA–NLS. In combination with the transfection agent polyethyleneimine, we have improved both the nuclear translocation of fluorescence-marked oligonucleotides, and the efficacy of plasmid transfection, up to eightfold. The technique obviates the use of cumbersome coupling procedures of the vector due to DNA–PNA duplex formation or displacement of the antisense plasmid DNA strand by a PNA molecule.

Bruton's tyrosine kinase (Btk): function, regulation, and transformation with special emphasis on the PH domain.

Summary:  Bruton’s agammaglobulinemia tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase important in B‐lymphocyte development, differentiation, and signaling. Btk is a member of the Tec family of kinases. Mutations in the Btk gene lead to X‐linked agammaglobulinemia (XLA) in humans and X‐linked immunodeficiency (Xid) in mice. Activation of Btk triggers a cascade of signaling events that culminates in the generation of calcium mobilization and fluxes, cytoskeletal rearrangements, and transcriptional regulation involving nuclear factor‐κB (NF‐κB) and nuclear factor of activated T cells (NFAT). In B cells, NF‐κB was shown to bind to the Btk promoter and induce transcription, whereas the B‐cell receptor‐dependent NF‐κB signaling pathway requires functional Btk. Moreover, Btk activation is tightly regulated by a plethora of other signaling proteins including protein kinase C (PKC), Sab/SH3BP5, and caveolin‐1. For example, the prolyl isomerase Pin1 negatively regulates Btk by decreasing tyrosine phosphorylation and steady state levels of Btk. It is intriguing that PKC and Pin1, both of which are negative regulators, bind to the pleckstrin homology domain of Btk. To this end, we describe here novel mutations in the pleckstrin homology domain investigated for their transforming capacity. In particular, we show that the mutant D43R behaves similar to E41K, already known to possess such activity.

Subcutaneous immunoglobulin replacement in patients with primary antibody deficiencies: safety and costs

Immunoglobulins (IgG) as replacement therapy in primary antibody deficiencies can be given as intramuscular injections, or as intravenous or subcutaneous infusions. Our aims were to obtain information on the frequency of adverse systemic reactions during subcutaneous therapy, the occurrence and intensity of tissue reactions at the infusion sites, and serum IgG changes. Furthermore, we compared costs between the different replacement regimes. Our study included 165 patients (69 women, 96 men, aged 13-76 years) with primary hypogammaglobulinaemia or IgG-subclass deficiencies. Data were compiled from questionnaires filled in by the patients and from their medical records. 33,168 subcutaneous infusions (27,030 in home therapy) had been given. 106 (of which 16 were at home) adverse systemic reactions (100 mild, 6 moderate) were recorded in 28 patients (17%). No severe or anaphylactoid reactions occurred. Despite large immunoglobulin volumes given during 434 patient years (28,480 infusions), no signs have been found that indicate the transmission of hepatitis virus. Transient tissue reactions occurred at the infusion sites but were not troublesome to most patients and we found significant increases in mean serum IgG. The use of subcutaneous instead of intravenous infusions at home would reduce the yearly cost per patient for the health-care sector by US

Early and prolonged intravenous immunoglobulin replacement therapy in childhood agammaglobulinemia: A retrospective survey of 31 patients☆☆☆

10,100 in Sweden alone. We conclude that subcutaneous administration of IgG is a safe and convenient method of providing immunoglobulins. We were able to reach serum IgG concentrations similar to those by the intravenous therapy and we found that the method could also be used successfully in patients with previous severe or anaphylactoid reactions to intramuscular injections.

Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo

OBJECTIVE To evaluate the outcome of children who received prolonged intravenous immunoglobulin (IVIg) replacement therapy early in life for X-linked agammaglobulinemia (XLA). STUDY DESIGN We performed a retrospective study of the clinical features and outcome of patients with genetic and/or immunologic results consistent with XLA. Patients receiving IVIg replacement therapy within 3 months of the diagnosis and for at least 4 years between 1982 and 1997 were included. RESULTS Thirty-one patients began receiving IVIg replacement therapy at a median age of 24 months and were followed up for a median time of 123 months. IVIg was given at doses >0.25 g/kg every 3 weeks, and mean individual residual IgG levels ranged from 500 to 1140 mg/dL (median, 700 mg/dL). During IVIg replacement, the incidence of bacterial infections requiring hospitalization fell from 0.40 to 0.06 per patient per year (P <. 001). However, viral or unidentified infections still developed, including enteroviral meningoencephalitis (n = 3) causing death in one patient, exudative enteropathy (n = 3), and aseptic arthritis (n = 1). At last follow-up, 30 patients were alive at a median age of 144 months (range, 58 to 253 months). Among 23 patients who were evaluated by respiratory function tests and computed tomography, 3 had an obstructive syndrome, 6 had bronchiectasis, and 20 had chronic sinusitis. CONCLUSION Early IVIg replacement therapy achieving residual IgG levels >500 mg/dL is effective in preventing severe acute bacterial infections and pulmonary insufficiency. More intensive therapy may be required to fully prevent the onset of bronchiectasis, chronic sinusitis, and nonbacterial infections, particularly enteroviral infections, in all cases.

Bruton's tyrosine kinase: cell biology, sequence conservation, mutation spectrum, siRNA modifications, and expression profiling

While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential.

HOME TREATMENT OF HYPOGAMMAGLOBULINAEMIA WITH SUBCUTANEOUS GAMMAGLOBULIN BY RAPID INFUSION

Summary:  Brutons tyrosine kinase (Btk) is encoded by the gene that when mutated causes the primary immunodeficiency disease X‐linked agammaglobulinemia (XLA) in humans and X‐linked immunodeficiency (Xid) in mice. Btk is a member of the Tec family of protein tyrosine kinases (PTKs) and plays a vital, but diverse, modulatory role in many cellular processes. Mutations affecting Btk block B‐lymphocyte development. Btk is conserved among species, and in this review, we present the sequence of the full‐length rat Btk and find it to be analogous to the mouse Btk sequence. We have also analyzed the wealth of information compiled in the mutation database for XLA (BTKbase), representing 554 unique molecular events in 823 families and demonstrate that only selected amino acids are sensitive to replacement (P < 0.001). Although genotype–phenotype correlations have not been established in XLA, based on these findings, we hypothesize that this relationship indeed exists. Using short interfering‐RNA technology, we have previously generated active constructs downregulating Btk expression. However, application of recently established guidelines to enhance or decrease the activity was not successful, demonstrating the importance of the primary sequence. We also review the outcome of expression profiling, comparing B lymphocytes from XLA‐, Xid‐, and Btk‐knockout (KO) donors to healthy controls. Finally, in spite of a few genes differing in expression between Xid‐ and Btk‐KO mice, in vivo competition between cells expressing either mutation shows that there is no selective survival advantage of cells carrying one genetic defect over the other. We conclusively demonstrate that for the R28C‐missense mutant (Xid), there is no biologically relevant residual activity or any dominant negative effect versus other proteins.

Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting

Intramuscular and intravenous gammaglobulin treatment for hypogammaglobulinaemia is often associated with systemic adverse reactions in some patients. Subcutaneous infusions of gammaglobulin are usually given at a slow rate. To assess the safety of home treatment with subcutaneous gammaglobulin, rapid infusions (34-40 ml/h) given by small portable pumps were used to treat twenty-five patients with hypogammaglobulinaemia. Fifteen patients had previously had adverse reactions to intramuscular or intravenous gammaglobulin treatment. After the patients had been taught how to use the pumps during 6 months of treatment in hospital, in which they initially received 100 mg of an intramuscular gammaglobulin preparation/kg per week, they went on to use the pumps at home or at work. So far, the patients have given themselves 3232 rapid subcutaneous infusions (2308 in home therapy). A median pre-infusion serum IgG concentration of 8.1 g/l resulted after 6 months of treatment. There were only 30 (0.93%) mild systemic adverse reactions; there were fewer reactions with subcutaneous gammaglobulin than with previously given intramuscular injections (n = 21, p less than 0.001) or intravenous infusions (n = 9, p less than 0.001) in this group of patients. Overall, the patients spent 0.2 days a year in hospital due to respiratory tract infections. The findings show that the method for subcutaneous administration is very easy to learn and is appreciated by the patients; moreover, the infusions can be given much faster than previously reported without any pronounced local reaction.

Cells release subpopulations of exosomes with distinct molecular and biological properties.

Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in a diverse range of biological processes. For future therapeutic applications and for EV biology research in general, understanding the in vivo fate of EVs is of utmost importance. Here we studied biodistribution of EVs in mice after systemic delivery. EVs were isolated from 3 different mouse cell sources, including dendritic cells (DCs) derived from bone marrow, and labelled with a near-infrared lipophilic dye. Xenotransplantation of EVs was further carried out for cross-species comparison. The reliability of the labelling technique was confirmed by sucrose gradient fractionation, organ perfusion and further supported by immunohistochemical staining using CD63-EGFP probed vesicles. While vesicles accumulated mainly in liver, spleen, gastrointestinal tract and lungs, differences related to EV cell origin were detected. EVs accumulated in the tumour tissue of tumour-bearing mice and, after introduction of the rabies virus glycoprotein-targeting moiety, they were found more readily in acetylcholine-receptor-rich organs. In addition, the route of administration and the dose of injected EVs influenced the biodistribution pattern. This is the first extensive biodistribution investigation of EVs comparing the impact of several different variables, the results of which have implications for the design and feasibility of therapeutic studies using EVs.