Immune responses to AAV in a phase I study for Canavan disease

BACKGROUND: Canavan disease is a rare leukodystrophy with no current treatment. rAAV-ASPA has been developed for gene delivery to the central nervous system (CNS) for Canavan disease. This study represents the first use of a viral vector in an attempt to ameliorate a neurodegenerative disorder. METHODS: Subjects received intracranial infusions via six cranial burr holes. Adeno-associated virus, serotype 2 (AAV2), mediated intraparenchymal delivery of the human aspartoacylase cDNA at a maximum dose of 1 x 10(12) vector genomes per subject. The immune response and safety profiles were monitored in the follow-up of ten subjects. RESULTS: Following rAAV2 administration, we found no evidence of AAV2 neutralizing antibody titers in serum for the majority of subjects tested (7/10). In a subset (3/10) of subjects, low to moderately high levels of AAV2 neutralizing antibody with respect to baseline were detected. In all subjects, there were minimal systemic signs of inflammation or immune stimulation. In subjects with catheter access to the brain lateral ventricle, cerebrospinal fluid was examined and there was a complete absence of neutralizing antibody titers with no overt signs of brain inflammation. CONCLUSIONS: rAAV2 vector administration to the human CNS appears well tolerated. The low levels of immune response to AAV2 detected in 3/10 subjects in this study suggest at this dose and with intraparenchymal administration this approach is relatively safe. Long-term monitoring of subjects and expansion to phase II/III will be necessary in order to make definitive statements on safety and efficacy.     

Cancer screening practices among primary care physicians serving Chinese Americans in San Francisco

Previous research has reported a lack of regular cancer screening among Chinese Americans. The overall objectives of this study were to use a mail survey of primary care physicians who served Chinese Americans in San Francisco to investigate: a) the attitudes, beliefs, and practices regarding breast, cervical, and colon cancer screening and b) factors influencing the use of these cancer screening tests. The sampling frame for our mail survey consisted of: a) primary care physicians affiliated with the Chinese Community Health Plan and b) primary care physicians with a Chinese surname listed in the Yellow Pages of the 1995 San Francisco Telephone Directory. A 5-minute, self-administered questionnaire was developed and mailed to 80 physicians, and 51 primary care physicians completed the survey. A majority reported performing regular clinical breast examinations (84%) and teaching their patients to do self-breast examinations (84%). However, the rate of performing Pap smears was only 61% and the rate of ordering annual mammograms for patients aged 50 and older was 63%. The rates of ordering annual fecal occult blood testing and sigmoidoscopy at regular intervals of three to five years among patients aged 50 and older were 69% and 20%, respectively. Barriers (patient-specific, provider-specific, and practice logistics) to using cancer screening tests were identified. The data presented in this study provide a basis for developing interventions to increase performance of regular cancer screening among primary care physicians serving Chinese Americans. Cancer screening rates may be improved by targeting the barriers to screening identified among these physicians. Strategies to help physicians overcome these barriers are discussed.     

Cationic antimicrobial peptides activate a two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa

The two-component regulatory system PhoP-PhoQ of Pseudomonas aeruginosa regulates resistance to cationic antimicrobial peptides, polymyxin B and aminoglycosides in response to low Mg2+ conditions. We have identified a second two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides. This system responds to limiting Mg2+, and is affected by a phoQ, but not a phoP mutation. Inactivation of the pmrB sensor kinase and pmrA response regulator greatly decreased the expression of the operon encoding pmrA-pmrB while expression of the response regulator pmrA in trans resulted in increased activation suggesting that the pmrA-pmrB operon is autoregulated. Interposon mutants in pmrB, pmrA, or in an intergenic region upstream of pmrA-pmrB exhibited two to 16-fold increased susceptibility to polymyxin B and cationic antimicrobial peptides. The pmrA-pmrB operon was also found to be activated by a number of cationic peptides including polymyxins B and E, cattle indolicidin and synthetic variants as well as LL-37, a component of human innate immunity, whereas peptides with the lowest minimum inhibitory concentrations tended to be the weakest inducers. Additionally, we showed that the putative LPS modification operon, PA3552-PA3559, was also induced by cationic peptides, but its expression was only partially dependent on the PmrA-PmrB system. The discovery that the PmrA-PmrB two-component system regulates resistance to cationic peptides and that both it and the putative LPS modification system are induced by cationic antimicrobial peptides has major implications for the development of these antibiotics as a therapy for P. aeruginosa infections.     

Identification of subsets of proliferating low Thy 1 cells in thymus cortex and medulla

Subsets of proliferating thymocytes were identified in the normal mouse thymus by in vivo labeling with [³H]TdR and by cell separation according to relative amounts of Thy 1 antigen. In order to resolve apparent discrepancies in the literature, parenteral and topical application of [³H]TdR were compared as labeling methods for dividing thymocytes, and limited complement lysis and fluorescence-activated cell sorting were compared as separation principles for high Thy 1 and low Thy 1 thymocyte subsets. The separated cells were further characterized by immunofluorescence for terminal deoxynucleotidyltransferase (TdT), which normally is restricted to cortical thymocytes, and for H2 alloantigens, which are preponderant on medullary thymocytes. Four subsets of proliferating cortical thymocytes were identified after application of [³H]TdR to the thymus capsule. The major subset, which comprised about 92% of dividing cortical thymocytes, had a high Thy 1, low H2 phenotype. Most were also TdT + ve. The three minor subsets of proliferating cortical thymocytes each had a low Thy 1 phenotype, but differed according to H2 and TdT markers. Systemic injection of [³H]TdR also labeled the above subsets of dividing cortical thymocytes, but in addition it detected a subset of proliferating low Thy 1, low H2, TdT — ve cells in the thymus medulla. The latter subset comprised about one-third of the pool of proliferating low Thy 1 cells. In their aggregate the four subsets of low Thy 1 cells constituted approximately 13% of total proliferating thymocytes and 1.1% of total thymocytes. The identification of discrete subsets of proliferating low Thy 1 cells in the thymus cortex as well as in the thymus medulla is compatible with the hypotheses that all thymocytes are descended from low Thy 1 precursors and that separate precursor cell subsets exist for cortical and medullary thymocytes.     

Frameshift mutagenesis by ultraviolet light effects of broth and caffeine in the post-irradiation plating medium

Ultraviolet-induced back mutation yields were studied in the frameshift strain of Salmonella typhimurium, LT2 hisC3076. The numbers and frequencies per 10⁸ survivors of small and large revertant colonies were found to be affected significantly by plating density, but it was possible to detect a considerable enhancement of mutation frequency when broth (2.5%, v/v) was present in the post-irradiation plating medium. Caffeine also significantly enhanced the yields of UV-induced frameshift mutations, but not of γ-induced frameshifts, indicating that the UV-induced pre-mutational lesions which lead to frameshift mutations may be treated in a similar way by the excision-repair system to those which lead to base-pair substitutions.     

Changes in growth,appetite, food conversion efficiency and body composition in mice selected for high post-weaning weight gain on restricted feeding

Summary This study aimed to test the hypothesis that if animals were fed the same amount over the same time period, selection of the fastest growers would result in a change in the partitioning of metabolisable energy toward more protein and less fat deposition. Two mouse lines (S1 and S2) were selected for high 5 to 9 week weight gain corrected to mean 5 week weight. Appetite variation between mice was eliminated by feeding a fixed amount to each mouse daily. After 6 generations of selection, the lines were compared with an unselected control (C) on restricted and ad libitum levels of feeding for growth rate, appetite, food conversion efficiency and chemical body composition.Realised heritabilities of 5 to 9 week gain were 0.36+ 0.05 and 0.19±0.04 for S1 and S2 respectively. Nine week weights were increased by an average of 13% on both feeding levels. Most of this increase, particularly in S2, occurred before 5 weeks and was therefore outside the period of measurement used in selection. On ad libitum feeding, selection increased food intake per unit time by 6% but there was no increase per unit body weight. Food conversion efficiency (gain/food) increased by 12%. Compared with controls at 9 weeks, 3% more of the body weights of selected mice was fat and 1% less was protein. These differences were reduced but were still in the same direction when comparisons were made at the same body weight. Thus the expected change in energy partitioning toward greater protein and less fat deposition in the S lines did not occur.It was concluded that the increased growth and energy retention in the S lines was brought about by a reduction in maintenance requirement. To achieve the desired change in energy partitioning using a similar selection scheme, higher levels of dietary protein should be fed, and some measure of protein deposition rather than growth rate used as the selection criterion.     

Measuring the Lamellarity of Giant Lipid Vesicles with Differential Interference Contrast Microscopy

Giant unilamellar vesicles are a widely utilized model membrane system, providing free-standing bilayers unaffected by support-induced artifacts. To measure the lamellarity of such vesicles, fluorescence microscopy is one commonly utilized technique, but it has the inherent disadvantages of requiring lipid staining, thereby affecting the intrinsic physical and chemical properties of the vesicles, and it requires a calibration by statistical analysis of a vesicle ensemble. Herein we present what we believe to be a novel label-free optical method to determine the lamellarity of giant vesicles based on quantitative differential interference contrast (qDIC) microscopy. The method is validated by comparison with fluorescence microscopy on a statistically significant number of vesicles, showing correlated quantization of the lamellarity. Importantly, qDIC requires neither sample-dependent calibration nor sample staining, and thus can measure the lamellarity of any giant vesicle without additional preparation or interference with subsequent investigations. Furthermore, qDIC requires only a microscope equipped with differential interference contrast and a digital camera.