Prospects for insulin delivery by ex-vivo somatic cell gene therapy

The principle of insulin delivery by ex-vivo somatic cell gene therapy involves the removal of non-B-cell somatic cells (e.g. fibroblasts) from a diabetic patient, and genetically altering them in vitro to produce and secrete insulin. The cells can be grown in culture and returned to the donor as a source of insulin replacement. Cells modified in this way could be evaluated before implantation, and reserve stocks could be cryopreserved. By using the patient's own cells, the procedure should obviate the need for immunosuppression and overcome the problem of tissue supply, while avoiding a recurrence of cell destruction. Ex-vivo somatic cell gene therapy requires an accessible and robust cell type that is amenable to multiple transfections and subject to controlled proliferation. Special problems associated with the use of non-B-cell somatic cells include the processing of proinsulin to insulin, and the conferment of sensitivity to glucose-stimulated proinsulin biosynthesis and regulated insulin release. Preliminary studies using fibroblasts, pituitary cells, kidney (COS) cells and ovarian (CHO) cells suggest that these challenges could be met, and that ex-vivo somatic cell gene therapy offers a feasible approach to insulin replacement therapy.     

The membrane affinities of the aliphatic amino acid side chains in an alpha-helical context are independent of membrane immersion depth

Understanding, predicting, and designing the binding of peptides and proteins to bilayers require quantifying the intrinsic propensities of individual amino acid residues to bind membranes as a function of structural context and bilayer depth. A host-guest study was performed using the peptide host named helix5 in order to determine the membrane affinities of the aliphatic side chains both in an alpha-helical context and as a function of bilayer depth. Use of the alpha-helical host with a constrained geometry allowed the placement of guest sites at three different depths in bilayers and minimized secondary structural changes due to guest substitutions. Circular dichroism and electron paramagnetic resonance (EPR) were used to characterize the aqueous and bilayer-bound structures of the peptide variants. EPR was also used to measure the bilayer-water partition constants of the peptide variants, and the Delta DeltaGtr values (relative to Gly) of the aliphatic amino acid side chains were subsequently calculated. Surprisingly, the DeltaDeltaGtr values did not significantly vary as a function of the guest site depth in bilayers. In addition, the Delta DeltaGtr values determined in an alpha-helical context are reduced to approximately two-thirds of Delta DeltaGtr values determined in other studies for the bilayer-water and octanol-water partitioning of amino acid side chains in extended and unstructured hosts. Both the relative reduction in Delta DeltaGtr values in the context of an alpha-helical host and the invariance of Delta DeltaGtr values with respect to bilayer depth are consistent with the membrane affinities of the aliphatic residues being largely determined by the classical hydrophobic effect.     

Validation of highly polymorphic fluorescent multiplex short tandem repeat systems using two generations of DNA sequencers

Validation studies are a crucial requirement before implementation of new genetic typing systems for clinical diagnostics or forensic identity. Two different fluorescence-based multiplex DNA profiling systems composed of amelogenin, HumD21S11 and HumFGA (referred to as multiplex 1A), and HumD3S1358, HumD21S11 and HumFGA (multiplex 1B) have been evaluated for use in forensic identification using the Applied Biosystems Model 373A and Prism 377 DNA Sequencers, respectively. Experiments were aimed at defining the limit of target DNA required for reliable profiling, the level of degradation that would still permit amplification of the short tandem repeat (STR) loci examined, and the robustness of each locus in the multiplexes after samples were exposed to environmental insults. In addition, the specificity of the multiplexes was demonstrated using nonhuman DNAs. Forensically relevant samples such as cigarette butts, chewing gum, fingernails and envelope flaps were processed using both an organic extraction procedure and a QIAamp protocol. DNAs and resultant multiplex STR profiles were compared. The validation of the triplex STR systems was extended to include over 140 nonprobative casework specimens and was followed with a close monitoring of initial casework (over 300 exhibits). Our results document the robustness of these multiplex STR profiling systems which, when combined with other multiplex systems, could provide a power of discrimination of approximately 0.9999.     

Specific recognition of HLA-E, but not classical, HLA class I molecules by soluble CD94/NKG2A and NK cells

The CD94/NKG2 receptors expressed by subpopulations of NK cells and T cells have been implicated as receptors for a broad range of both classical and nonclassical HLA class I molecules. To examine the ligand specificity of CD94/NKG2 proteins, a soluble heterodimeric form of the receptor was produced and used in direct binding studies with cells expressing defined HLA class I/peptide complexes. We confirm that CD94/NKG2A specifically interacts with HLA-E and demonstrate that this interaction is dependent on the association of HLA-E with peptide. Moreover, no interaction between CD94/NKG2A and classical HLA class I molecules was observed, as assayed by direct binding of the soluble receptor or by functional assays using CD94/NKG2A+ NK cells. The role of the peptide associated with HLA-E in the interaction between HLA-E and CD94/NKG2A was also assessed. All class I leader sequence peptides tested bound to HLA-E and were recognized by CD94/NKG2A. However, amino acid variations in class I leader sequences affected the stability of HLA-E. Additionally, not all HLA-E/peptide complexes examined were recognized by CD94/NKG2A. Thus CD94/NKG2A recognition of HLA-E is controlled by peptide at two levels; first, peptide must stabilize HLA-E and promote cell surface expression, and second, the HLA-E/peptide complex must form the ligand for CD94/NKG2A.     

A longitudinal study of the relationships between haemostatic, lipid, and oestradiol changes during normal human pregnancy

BACKGROUND: Chronic leukemia is a disease characterized by the malignant proliferation of immunologically incompetent lymphocytes. The knowledge of open heart surgery in patients with this disorder is limited. METHODS: Twelve patients with chronic lymphocytic leukemia underwent open heart surgery (nine coronary artery bypass grafting (CABG), two aortic valve replacement (AVR), one CABG and AVR) from September 1991 to September 1996. There were nine males and three females with a mean age of 68 years (41-81 years). Staging was assigned according to the Rai Classification. There were seven Stage 0, two Stage I, zero Stage II, one Stage III and two Stage IV patients. Cardiopulmonary bypass (CPB) was performed using standard techniques of cannulation, moderate hypothermia and antegrade/retrograde cardioplegia. RESULTS: Hospital mortality occurred in two (17%) patients. Both patients died of sepsis. Hospital morbidity occurred in seven (58%) patients. The most common complications were infections. Five patients were found to have other malignancies (basal cell, laryngeal, prostate, bladder and breast cancers). Transfusion of blood products was required in eight (67%) patients. The average length of stay was 15 days (7-50 days). Follow-up was complete. Late mortality occurred in four patients at a mean of 7 months (1-18 months). All deaths were non-cardiac related (ruptured AAA, kidney failure, respiratory failure and sepsis). Six patients remain alive at a mean of 25 months (1-48 months). CONCLUSION: Hospital mortality and morbidity in patients with chronic lymphocytic leukemia undergoing open heart surgery are high. Infection is the leading cause of hospital death, as well as the most common complication. The majority of patients receive blood products during the course of their hospitalization. Late mortality is high and non-cardiac related. Based on these findings, a re-definition of the aims, goals and expectations of open heart surgery in patients with chronic leukemia is necessary. Suggestions in management are presented.     

An analysis of group versus individual child health supervision

This study compares the effectiveness of group health supervision (three or four families counseled simultaneously) with traditional visits in conveying knowledge of child health and development, increasing perceived maternal support, and mitigating maternal depression. Subjects were recruited from a predominantly white, middle-class, suburban/rural pediatric practice. Twenty-five families were allocated to group health supervision and 25 to individual visits. A questionnaire covering knowledge of child health and development (CHDQ), the Maternal Social Support Index (MSSI), and the Center for Epidemiologic Studies Depression Scale (CESD) were administered to both groups before their 2-month and after their 10-month visits. A subset of these charts was reviewed for problem visits between 2 and 6 months. As compared with families having traditional visits, families who received the group intervention did at least as well in acquiring knowledge of child care and development and, although not statistically significant, tended to recover from postpartum depression faster and deal better with minor illnesses. The investigators found group child health supervision to be a pleasant and effective method of health care delivery.     

Clinical experience of trabeculotomy for the surgical treatment of aniridic glaucoma

OBJECTIVE: The purpose of this study is to determine the efficacy of initial trabeculotomy in the patient with aniridic glaucoma. DESIGN: Clinical charts were reviewed. PARTICIPANTS: Twenty-nine eyes of 16 patients with aniridia were studied. INTERVENTION: Glaucoma surgery was performed. As an initial procedure, trabeculotomy was performed in 12 eyes, other surgery was performed in 17 eyes (trabeculectomy, 5; goniotomy, 5; other, 7). MAIN OUTCOME MEASURES: Success was defined as an intraocular pressure (IOP) of 21 mmHg or lower, and no further surgery was performed. RESULTS: Ten (83%) of 12 eyes obtained IOP control after first (6 eyes) or second (4 eyes) trabeculotomy with a mean follow-up period of 9.5 years. Five eyes maintained visual acuity of 20/40 to 20/200. No serious complications were found after trabeculotomy. Three (18%) of 17 eyes were controlled with the first glaucoma surgery other than trabeculotomy (goniotomy, trabeculectomy, trabeculectomy combined with trabeculotomy, and Molteno implant). Good IOP control was obtained in 8 (47%) of 17 eyes after several surgeries with a mean follow-up period of 10.4 years. Four of 17 eyes became phthisical. CONCLUSION: This study suggests that trabeculotomy is the preferred initial operation for uncontrolled glaucoma with aniridia.