Vinylpyrrolidone-co-(meth)acrylic acid inserts for ocular drug delivery: synthesis and evaluation

Copolymeric hydrogels constituting of vinylpyrrolidone and methacrylic or acrylic acid repeat units have been prepared and investigated for their ability to act as controlled release vehicles in ophthalmic drug delivery. The materials were synthesized by radical-induced polymerization in the presence of N,N'-methylenebisacrylamide crosslinker, and the influences of network composition and drug solubility upon the swelling properties, adhesion behavior, and drug release characteristics were studied. In vitro release experiments showed that some of these materials could be useful vehicles for the delivery of drugs such as pilocarpine or chloramphenicol, while in vivo studies, using the rabbit model, confirmed their high potential for the controlled ocular delivery of pilocarpine hydrochloride.     

A geometric model for evaluating the effects of inter-fraction rectal motion during prostate radiotherapy

A geometric model is presented which allows calculation of the dosimetric consequences of rectal motion in prostate radiotherapy. Variations in the position of the rectum are measured by repeat CT scanning during the courses of treatment of five patients. Dose distributions are calculated by applying the same conformal treatment plan to each imaged fraction and rectal dose-surface histograms produced. The 2D model allows isotropic expansion and contraction in the plane of each CT slice. By summing the dose to specific volume elements tracked by the model, composite dose distributions are produced that explicitly include measured inter-fraction motion for each patient. These are then used to estimate effective dose-surface histograms (DSHs) for the entire treatment. Results are presented showing the magnitudes of the measured target and rectal motion and showing the effects of this motion on the integral dose to the rectum. The possibility of using such information to calculate normal tissue complication probabilities (NTCP) is demonstrated and discussed.     

Transport, enzymatic activity, and stability of mutant sulfamidase (SGSH) identified in patients with mucopolysaccharidosis type III A

Mucopolysaccharidosis type IIIA (MPSIIIA) is an autosomal recessive lysosomal storage disease caused by mutations in the N-sulfoglucosamine sulfohydrolase gene (SGSH; encoding sulfamidase, also sulphamidase) leading to the lysosomal accumulation and urinary excretion of heparan sulfate. Considerable variation in the onset and severity of the clinical phenotype is observed. We report here on expression studies of four novel mutations: c.318C>A (p.Ser106Arg), c.488T>C (p.Leu163Pro), c.571G>A (p.Gly191Arg), and c.1207_1209delTAC (p.Tyr403del), and five previously known mutations: c.220C>T (p.Arg74Cys), c.697C>T (p.Arg233X), c.1297C>T (p.Arg433Trp), c.1026dupC (p.Leu343fsX158), and c.1135delG (p.Val379fsX33) identified in MPSIIIA patients. Transient expression of mutant sulfamidases in BHK or CHO cells revealed that all the mutants were enzymatically inactive with the exception of c.318C>A (p.Ser106Arg), which showed 3.3% activity of the expressed wild-type enzyme. Western blot analysis demonstrated that the amounts of expressed mutant sulfamidases were significantly reduced compared with cells expressing wild type. No polypeptides were immunodetectable in extracts of cells transfected with the cDNA carrying the c.697C>T (p.Arg233X) nonsense mutation. In vitro translation and pulse-chase experiments showed that rapid degradation rather than a decrease in synthesis is responsible for the low, steady-state level of the mutant proteins in cells. The amounts of secreted mutant precursor forms, the cellular stability, the proteolytic processing, and data from double-label immunofluorescence microscopy suggest that the degradation of the majority of newly synthesized c.220C>T (p.Arg74Cys), c.571G>A (p.Gly191Arg), c.1297C>T (p.Arg433Trp), c.1026dupC (p.Leu343fsX158), and c.1135delG (p.Val379fsX33) mutant proteins probably occurs in the ER, whereas c.488T>C (p.Leu163Pro) mutant protein showed instability in the lysosomes.     

Dependence of adaptation of the human vertical angular vestibulo-ocular reflex on gravity

We determined the spatial dependence of adaptive gain changes of the vertical angular vestibulo-ocular reflex (aVOR) on gravity in five human subjects. The gain was decreased for 1 h by sinusoidal oscillation in pitch about a spatial vertical axis in a subject-stationary surround with the head oriented left-side down. Gains were tested by sinusoidal oscillation about a spatial vertical axis while subjects were tilted in 15° increments from left- to right-side down positions through the upright. Changes in gain of the vertical component of the induced eye movements were expressed as a percentage of the preadapted values for the final analysis. Vertical aVOR gain changes were maximal in the position in which the gain had been adapted and declined progressively as subjects were moved from this position. Gain changes were plotted as a function of head orientation and fit with a sine function. The bias level of the fitted sines, i.e., the gravity-independent gain change, was –29±10% (SD). The gains varied around this bias as a function of head position by ±18±6%, which were the gravity-dependent gain changes. The gravity-dependent gain changes induced by only 1 h of adaptation persisted, gradually declining over several days. We conclude that there is a component of the vertical aVOR gain change in humans that is dependent on the head orientation in which the gain was adapted, and that this dependence can persist for substantial periods.     

Implications of pharmacogenetics for individualizing drug treatment and for study design

Adverse drug reactions and ineffective drug treatment are responsible for a large health care burden. Considerable variability in drug response makes the prediction of the individual reaction difficult. Pharmacogenetics can help to individualize drug treatment in accordance with the genetic make-up of the patient. Drug response is best understood as a complex interplay between pharmacokinetics, pharmacodynamics, and other disease-associated factors. There are a large number of genetic variants in the enzymes of phase I and phase II drug metabolism, in drug transporters, and drug targets, all of which account for differences in drug response. The polymorphisms in the cytochrome P450 enzyme system have been investigated most extensively. Genotype-based dose adjustment which should ensure "bioequivalent" drug concentrations in all patients has been derived from pharmacokinetic parameters, but this approach will have to be verified in prospective studies. Drug transport has recently been recognized as a further crucial determinant in pharmacokinetics. The effect of genetics on disease susceptibility and drug treatment has been studied quite extensively; however, hardly any of this progress is at present reflected in routine health care. The integration of pharmacogenetic factors in clinical trials requires novel considerations for study design and data interpretation. It is to be hoped that the new science bioinformatics will (a) help us identify the contribution of genetics to disease and treatment response and will (b) create data-processing devices which help the physician in the face of the enormously expanding scientific knowledge in selecting the best individually adapted treatment for the patient.     

Estrogen receptors in skeletal metabolism: lessons from genetically modified models of receptor function

Estrogens have long been known to be important for skeletal homeostasis, but their precise mechanisms of action in bone are still unclear. Mice with targeted deletions of the estrogen receptors alpha (ERalpha) and beta (ERbeta) have been generated by two research groups and several studies performed characterizing the phenotype of ERalpha knockout (ERKOalpha), ERbeta knockout (ERKObeta), or double deletion of ERalpha and ERbeta (DERKO) mice. Initial studies reported a reduction in bone mineral density in male ERKOalpha mice. More extensive analyses have been puzzling, likely because of compensatory mechanisms in ERKO mice. Furthermore, the existence of a third ER continues to be a potential explanation for some actions of estrogen in bone. Other rodent models, including the testicular feminized mouse and rat, the aromatase knockout mouse, and a rat with a dominant negative ER mutation, have added information regarding estrogen's actions in bone. This review summarizes many reports characterizing available rodent models with genetic alterations relevant to estrogen action. The sum of these reports suggests that the ERbeta is not highly protective in bone because loss of its function results in minimal alterations in the skeleton. Furthermore, loss of both the ERalpha and the ERbeta does not account for loss of estrogen action in bone, because the impact of DERKO is seemingly not as great as the impact of gonadectomy on the skeleton. Finally, through studies of ERKO mice and other rodent models of altered sex steroid action, it appears that estrogen may be more protective in the skeleton than androgens.     

Production of serum antibodies that recognise epitopes located on the R3 region of Escherichia coli core lipopolysaccharides by patients infected with strains of enterohaemorrhagic E. coli

Antibody-antigen cross-reactions were examined with sera from patients with Escherichia coli O157 infection and lipopolysaccharide (LPS) purified from a range of enterohaemorrhagic E. coli (EHEC) including those belonging to serogroups O26, O103, O111, O145 and O157. Six of 10 patients infected with an O157 EHEC produced serum antibodies that cross-reacted with common LPS-core epitopes, which were expressed by 23 of 33 strains of EHEC examined. These common LPS-core epitopes were also present on strains of E. coli O26 which did not produce verocytotoxin. These cross-reacting antibodies did not influence the basic immunoblotting procedures used for the routine serodiagnosis of infections with E. coli O157.     

Distal Proctocolitis and n-3 Polyunsaturated Fatty Acids (n-3 PUFAs): The Mucosal Effect In Situ

It has been postulated that patients with ulcerative colitis (UC) have altered reactivity of gut-associated lymphoid tissue. In such cases there is intense infiltration of the mucosa with immune competent cells and associated tissue damage. We have shown previously that the dietary supplementation with the n-3 polyunsaturated fatty acids (n-3 PUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) results in significant systemic immune suppression. The aim of this study, therefore, was to evaluate the in situ effect of n-3 PUFAs on distal proctocolitis. Each patient received either fish oil extract (EPA 3.2 g, DHA 2.4 g) (n = 9) or sunflower oil (n = 9) daily in a double blind manner for six months. Monthly assessment included: (1) disease activity using clinical, sigmoidoscopic, and histological scores and (2) immunohistochemical analysis (immunoglobulins, CD profiles) of rectal biopsy specimens (before and after six months supplementation) using monoclonal antibodies and quantitative computer-assisted video image analysis. Prior to receiving supplementation, patients with proctocolitis (n = 18) showed significantly higher numbers of cells expressing CD3 (pan T cells) and HLA-DR and IgM containing cells compared with non-colitic controls (n = 8). Six months supplementation with n-3 PUFAs resulted in significant reduction in the number of cells expressing CD3 and HLA and the percentage of cells containing IgM. There was no significant change in the CD20 nor the percentage of IgG or IgA containing cells in either group of patients with procto-colitis. In patients receiving n-3 PUFA supplementation, there was improvement in the disease activity and histological scores, compared with pretreatment evaluation. This study has demonstrated both evidence of suppression of in situ immune reactivity and concurrent reduction in disease activity in patients with proctocolitis receiving n-3 PUFA supplementation. This may have important implication for therapy in patients with ulcerative colitis.     

Analysis of the mechanism of lipoprotein(a) assembly

We have assessed the ability of a battery of purified recombinant apolipoprotein(a) (r-apo(a)) derivatives to bind to immobilized low-density lipoprotein (LDL) by ELISA. Removal of the apo(a) kringle IV type 8 and type 9 sequences dramatically reduced apo(a) binding to LDL. The binding of apo(a) to LDL was effectively inhibited by arginine, lysine, the lysine analogue ε-aminocaproic acid and proline; comparable inhibition was observed using the 17K and KIV_5–8 r-apo(a) derivatives, suggesting a direct role for sequences contained in the latter species in mediating the initial non-covalent interactions which precede specific disulfide bond formation. We also determined that r-apo(a) binds directly to a synthetic apoB peptide spanning amino acid residues 3732–3745; this interaction appeared to be mediated by sequences present in apo(a) kringle IV types 8 and 9, and could be inhibited by arginine, lysine and proline. The results of this study indicate that the efficiency of Lp(a) assembly is a direct function of the initial non-covalent interactions between apo(a) and LDL; in addition, these studies suggest that Cys3734 in apoB mediates covalent linkage with apo(a) by virtue of the ability of the apoB sequences surrounding this residue to directly interact with apo(a) KIV type 9.