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K Crook

Publications and source records attributed to K Crook.

12 recordsLinked to original sources
PubMed2000

A New Self-Assembling System for Targeted Gene Delivery We thank the BBSRC Chiroptical Service for CD studies, Brian McManus (Optokem Instruments, Nercyws, Flintshire) for assistance with the light-scattering studies, Dr. Clive Roberts (Molecular Profiles Ltd.) for AFM studies, Professor J.-H. Fuhrhop for many discussions, Dr. I. S. Blagbrough for related synthetic work, the EPSRC for support, and the Royal Society for a Leverhulme Trust Senior Research Fellowship (D.P.).

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Journal Article↗
PubMed1998

Mouse beta defensin-1 is a functional homolog of human beta defensin-1.

Defensin are 3-4 kDa antimicrobial peptides of which three distinct families have been identified; alpha-defensin, beta-defensins, and insect defensins. Recent investigations have shown that beta-defensins are present in the human airways and may be relevant to the pathogenesis of cystic fibrosis (CF) lung disease. We report here the further characterization of a recently identified mouse beta-defensin gene, Defb1, sometimes referred to as mBD-1, which is homologous to the human airway beta defensin hBD-1. We report that Defb1 is expressed in a variety of tissues including the airways and, similar to hBD-1, is not upregulated by lipopolysaccharide (LPS). Defb1 was found to consist of two small exons separated by a 16-kb intron and cytogenetic, and physical mapping linked it to the alpha defensin gene cluster on mouse Chromosome (Chr) 8. Functional studies demonstrate that, like hBD-1, Defb1 demonstrates a salt-sensitive antimicrobial activity against Pseudomonas aeruginosa. Of relevance to CF lung disease is the fact that neither the hBD-1 nor the mBD-1 peptides are active against Burkholderia cepacia.

Amino Acid Sequence↗
PubMed1998

Inclusion of cholesterol in DOTAP transfection complexes increases the delivery of DNA to cells in vitro in the presence of serum.

The contrast between the relative efficiency of transfection by cationic lipid reagents in vitro and that in vivo is well recognised. One suggested reason for this is the presence of competing polyanionic surfaces in blood and other biological fluids, and even in vitro transfections have to be performed in low-serum medium. In this study we have shown that by preparing cationic lipid reagents based on DOTAP with cholesterol as a second constituent of the bilayer we can achieve significant levels of in vitro transfection in serum concentrations of up to 80% (DOTAP alone did not transfect at all in these conditions). In an effort to explain the behaviour of DOTAP/cholesterol mixes under these conditions, we examined the effect of serum on the transfection complex. We could detect protein bound to each type of cationic lipid complex, but there was no difference in the amount nor in the type of protein bound. DNA within either type of complex which were incubated with increasing amounts of serum remained resistant to digestion with DNase I, and there was no reduction in the condensation of the DNA as measured by ethidium bromide fluorescence. Finally, we measured the attachment and uptake into cells by the different complexes in the presence of serum and showed that more DOTAP-cholesterol than DOTAP complexes attach to and are taken up by cells in the presence of serum. We suggest that improved cell binding and uptake may be the main mechanism by which cholesterol acts to maintain transfection in the presence of serum.

Animals↗
PubMed1996

Enrichment of early fetal-liver hemopoietic stem cells of the rat using monoclonal antibodies against the transferrin receptor, Thy-1, and MRC-OX82.

Fetal livers from inbred rat fetuses at 14 days' gestation were dispersed into a single-cell suspension by physical disruption and collagenase digestion. Pluripotent stem cells were characterized and partially purified by a combination of monoclonal antibodies. These included CD71 (anti-transferrin receptor, MRC-OX26, used for rosetting), Cdw90 (anti-Thy-1, MRC-OX7), and the newly described MRC-OX82 (reacting with myeloiid cells in peritoneal exudate), employed in FACS sorting. Enrichment was monitored by long-term reconstitution of lethally irradiated congenic rats genetically distinguishable from the donor by an allelomorphic variant of the CD45 cell-surface antigen. At intervals from 3 months to 1 year, lymph-node cells and peritoneal exudate cells were biopsied for analysis by two-color flow cytometry--one color to determine donor origin, the other to identify Th cell (CD4+), Tc cell (CD8+), B cell (sIg+ or CD45RC+), neutrophil (OX82+ or OX43-), and macrophage (OX43+) compartments. The degree of chimaerism was taken as the read out of stem-cell activity. No significant differentials between lymph-node and peritoneal exudate chimaerisms were detected in any of the recipients; therefore, the enrichment procedure revealed only pluripotent cells, not stem cells of restricted potency. All recovered stem-cell activity was in the OX26(CD71)-negative, OX7(CDw90)-positive, OX82-positive fraction. In the optimum case, an enrichment of very roughly 200-fold in cell-for-cell activity was obtained. Rat bone-marrow colony-forming units in the spleen (CFUs-12) were found to lack the surface antigens recognized by the monoclonal antibodies CD53 (MRC-OX44), MRC-OX39, MRC-OX59, and 144.2.15. These would provide a strategy for their enrichment by depletion.

Animals↗
PubMed1996

Plasmid DNA molecules complexed with cationic liposomes are protected from degradation by nucleases and shearing by aerosolisation.

The cationic liposome DOTAP was complexed with plasmid DNA encoding beta-galactosidase in various ratios. As the concentration of DOTAP increased, the DNA became increasingly refractory to staining with ethidium bromide, presumably because the DNA was becoming condensed and being encapsulated by the liposomes. Transfection by DNA-DOTAP complexes at all ratios tested was unaffected by treatment of the complexes with DNase I. This finding has relevance to clinical trials for gene therapy of cystic fibrosis, in which patients are normally removed from treatment with DNase before receiving administration of DNA. We additionally tested the effect of aerosolisation of the liposome-DNA complex and of the DNA alone on the efficiency of in vitro transfection. Aerosolised DNA complexed with fresh DOTAP led to much lower reporter gene expression in Cos 7 cells than non-aerosolised complex, since aerosolisation appeared to destroy almost all of the plasmid. However, complexing the plasmid before passage through the nebuliser did protect most of the DNA from degradation, as reflected in the levels of transfection obtained. These findings contribute towards an overall understanding of both how DNA-cationic liposome complexes are formed and their fate following administration in vivo.

Aerosols↗
PubMed1994

Endocytosis and presentation of liposome-associated antigens by B cells.

B cells have limited endocytic capacity and are reported to endocytose and present liposome-encapsulated antigens poorly. B cells also endocytose soluble antigens poorly, except those for which their surface immunoglobulin is specific, which are taken up and presented efficiently. We present results indicating that, in vitro, B cells endocytose small liposomes bearing antigen with affinity for their surface immunoglobulin. Antigen encapsulated in liposomes targeted by antibody specific for surface immunoglobulin is presented to T cells as efficiently as specific antigen in soluble form. These studies provide a rational basis for the design of liposomes optimized to stimulate T-dependent B-cell responses.

Animals↗
PubMed1994

Induction of T lymphocyte subsets and levels of interleukin-2 and interleukin-3 after infection with Trichinella spiralis are similar in mice of high- and low-responder phenotypes.

Various aspects of T lymphocyte responses in mice with high- and low-responder phenotypes to infection with the nematode Trichinella spiralis were compared. Mesenteric lymph node cells (MLNC) from infected NIH strain mice (high-responder) showed a much higher level of proliferation in response to parasite antigen than MLNC from infected C57.B10 mice (low-responder). Levels of CD4+ and CD8+ T lymphocytes in the MLN remained relatively constant in both strains, ruling out the possibility that the more rapid worm expulsion in NIH mice (day 6 post-infection, as compared to day 12-15 in B10 mice) was due to quantitative differences in levels of CD4+ T helper cells. To examine any variation in the recruitment of Th1 v Th2 cell subsets, the production of the cytokines interleukins-2, -3, and -4 (IL-2, -3, and -4) by MLNC in response to parasite antigen was measured throughout the course of infection. Amounts of IL-2 and IL-3 were broadly similar in both strains, while almost background levels of IL-4 were detected. However, NIH strain mice produced IL-2 and IL-3 more rapidly than B10 mice. These data support the conclusion from other authors that the response to T. spiralis is not dependent on the absolute levels of cytokines produced, but rather on timing of production and rapidity of recruitment of effector cells at the level of bone marrow precursors.

Animals↗
PubMed1990

Perceived family environments of depressed and anxious children: child's and maternal figure's perspectives.

This study examined perceived environment among families with a depressed, depressed and anxious, anxious, or normal child from the 4th to 7th grades. Fifty-one such children were classified according to criteria from the K-SADS and a set of self-ratings of depression and anxiety. Results showed that children in all three diagnostic groups, and to a lesser extent their mothers, experienced their families as more distressed on a host of dimensions relative to controls. In addition, significant differences were found between families with a depressed and anxious child and those with an anxious child. Discriminant function analyses revealed that 68.63% of the youngsters could be classified correctly into depressed and anxious groups on the basis of their family ratings alone.

Adolescent↗