A model of corrective gene transfer in X-linked ichthyosis

Single gene recessive genetic skin disorders offer attractive prototypes for the development of therapeutic cutaneous gene delivery. We have utilized X-linked ichthyosis (XLI), characterized by loss of function of the steroid sulfatase arylsulfatase C (STS), to develop a model of corrective gene delivery to human skin in vivo. A new retroviral expression vector was produced and utilized to effect STS gene transfer to primary keratinocytes from XLI patients. Transduction was associated with restoration of full-length STS protein expression as well as steroid sulfatase enzymatic activity in proportion to the number of proviral integrations in XLI cells. Transduced and uncorrected XLI keratinocytes, along with normal controls, were then grafted onto immunodeficient mice to regenerate full thickness human epidermis. Unmodified XLI keratinocytes regenerated a hyperkeratotic epidermis lacking STS expression with defective skin barrier function, effectively recapitulating the human disease in vivo. Transduced XLI keratinocytes from the same patients, however, regenerated epidermis histologically indistinguishable from that formed by keratinocytes from patients with normal skin. Transduced XLI epidermis demonstrated STS expression in vivo by immunostaining as well as a normalization of histologic appearance at 5 weeks post-grafting. In addition, transduced XLI epidermis demonstrated a return of barrier function parameters to normal. These findings demonstrate corrective gene delivery in human XLI patient skin tissue at both molecular and functional levels and provide a model of human cutaneous gene therapy.      

Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal: lack of genotype/phenotype correlations

It has previously been shown that, in the heterozygous state, mutations in the SOX9 gene cause campomelic dysplasia (CD) and the often associated autosomal XY sex reversal. In 12 CD patients, 10 novel mutations and one recurrent mutation were characterized in one SOX9 allele each, and in one case, no mutation was found. Four missense mutations are all located within the high mobility group (HMG) domain. They either reduce or abolish the DNA-binding ability of the mutant SOX9 proteins. Among the five nonsense and three frameshift mutations identified, two leave the C-terminal transactivation (TA) domain encompassing residues 402-509 of SOX9 partly or almost completely intact. When tested in cell transfection experiments, the recurrent nonsense mutation Y440X, found in two patients who survived for four and more than 9 years, respectively, exhibits some residual transactivation ability. In contrast, a frameshift mutation extending the protein by 70 residues at codon 507, found in a patient who died shortly after birth, showed no transactivation. This is apparently due to instability of the mutant SOX9 protein as demonstrated by Western blotting. Amino acid substitutions and nonsense mutations are found in patients with and without XY sex reversal, indicating that sex reversal in CD is subject to variable penetrance. Finally, none of 18 female patients with XY gonadal dysgenesis (Swyer syndrome) showed an altered SOX9 banding pattern in SSCP assays, providing evidence that SOX9 mutations do not usually result in XY sex reversal without skeletal malformations.      

Intracellular survival of Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease being increasingly recognized as an important cause of morbidity and mortality in many regions of the world. Several features of melioidosis suggest that B. pseudomallei is a facultative intracellular pathogen. This study was designed to assess the ability of B. pseudomallei to invade and survive in eukaryotic cells. We have shown that B. pseudomallei has the capacity to invade cultured cell lines, including HeLa, CHO, A549, and Vero cells. We have demonstrated intracellular survival of B. pseudomallei in professional phagocytic cells, including rat alveolar macrophages. B pseudomallei was localized inside vacuoles in human monocyte-like U937 cells, a histiocytic lymphoma cell line with phagocytic properties. Additionally, electron microscopic visualization of B. pseudomallei-infected HeLa cells and polymorphonuclear leukocytes confirmed the presence of intracellular bacteria within membrane-bound vacuoles. B. pseudomallei was found to be resistant to the cationic peptide protamine and to purified human defensin HNP-1.     

Palliation in cyanotic congenital heart disease. Fifteen years' experience of various shunt procedures

During the past 15 years, 143 systemic pulmonary shunt procedures have been performed in 117 patients. These have been evaluated for their clinical effectiveness, the need for a repeat operation and the mortality; particular attention was paid to the Teflon shunt. Variations were found in shunt performance, depending on the primary defect, the type of shunt that was employed and the year of operation. The overall shunt patency after three years was 77% (85% with the Teflon shunt). Although, in our total experience, mortality at 30 days was 12%, with 16% late deaths, "modified Blalock" (Teflon) shunts had only a 5% hospital mortality and a 5% late mortality within three years. Pulmonary atresia, without a ventricular septal defect, is often insufficiently palliated by a shunt alone. Ten of 82 patients with variations of the tetralogy complex died within 30 days of operation, and a further 11 died in the late follow-up period. Six of these 21 shunts were patent at autopsy. Less common defects, such as univentricular heart, transposition and double-outlet right ventricular connections, that are associated with pulmonary stenosis had no early mortality but led to four late deaths among 27 patients. Two of the four patients had patent shunts. Results in the early part of this experience were less than acceptable owing to inferior shunting techniques, postoperative management errors and, particularly, inadequate follow-up surveillance. With correction of these factors we find that the modified Blalock shunt provides very good early and late mortality results, with excellent clinical palliation and patency rates.     

Domain communication in the dynamical structure of human immunodeficiency virus 1 protease.

A dynamical model for the structure of the human immunodeficiency virus 1 (HIV-1) protease dimer in aqueous solution has been developed on the basis of molecular dynamics simulation. The model provides an accurate account of the crystal geometry and also a prediction of the structural reorganization expected to occur in the protein in aqueous solution compared to the crystalline environment. Analysis of the results by means of dynamical cross-correlation coefficients for atomic displacements indicates that domain-domain communication is present in the protein in the form of a molecular "cantilever" and is likely to be involved in enzyme function at the molecular level. The dynamical structure also suggests information that may ultimately be useful in understanding and further development of specific inhibitors of HIV-1 protease.