Characterization of a novel adeno-associated viral vector with preferential oligodendrocyte tropism.

No adeno-associated virus (AAV) capsid has been described in the literature to exhibit a primary oligodendrocyte tropism when a constitutive promoter drives gene expression, which is a significant barrier for efficient in vivo oligodendrocyte gene transfer. The vast majority of AAV vectors, such as AAV1, 2, 5, 6, 8 or 9, exhibit a dominant neuronal tropism in the central nervous system. However, a novel AAV capsid (Olig001) generated using capsid shuffling and directed evolution was recovered after rat intravenous delivery and subsequent capsid clone rescue, which exhibited a >95% tropism for striatal oligodendrocytes after rat intracranial infusion where a constitutive promoter drove gene expression. Olig001 contains a chimeric mixture of AAV1, 2, 6, 8 and 9, but unlike these parental serotypes after intravenous administration Olig001 has very low affinity for peripheral organs, especially the liver. Furthermore, in mixed glial cell cultures, Olig001 exhibits a 9-fold greater binding when compared with AAV8. This novel oligodendrocyte-preferring AAV vector exhibits characteristics that are a marked departure from previously described AAV serotypes.

The effect of mouse serum on lipid metabolism in embryonic chick limb-bud cells.

Dissociated embryonic chick limb cells will undergo terminal differentiation in culture. The addition of whole or delipidated mouse serum to the cultures will, however, inhibit the chondrogenic potential of the cells. The inhibitory serum readily stimulates increased incorporation of arachidonic acid and palmitic acid into triacylglycerol in the treated cells, while the incorporation of arachidonate into various phospholipids is significantly lowered. In contrast mouse serum has no effect on the incorporation of inorganic [32P]phosphate into phospholipid. We interpret the patterns of incorporation of these lipid substrates as indicating that mouse serum modulates the deacylation-reacylation cycle of phospholipids (Lands' cycle), which is primarily responsible for the incorporation of arachidonic acid. This finding suggests that there may be a decrease in membrane fluidity which might play a key role in cellular regulation and differentiation.

The inhibitory effects of pentamidine on biochemical events in human liver cells.

The present study was undertaken to determine the biochemical effects of pentamidine on an established human liver cell line (HEP-G2) and its drug resistant subclone (HEP-G2DR). The HEP-G2DR cells were included in the study in order to determine whether drug resistance played a role in any of the observed effects seen during and/or following pentamidine treatment. The results indicate that a 5 mum concentration of the drug significantly reduced the level of protein synthesis, as well as the continued expression of albumin, a tissue-specific product of both cell lines. Pentamidine also significantly inhibited DNA synthesis in HEP-G2 cells, but not in HEP-G2DR cells, during the first 24 hr. Conversely, only the resistant cells showed a significant recovery in both protein and albumin synthesis after the drug was removed (reversal). The significant recovery in these biochemical parameters was observed during the initial 24-hr reversal period, while a recovery in albumin secretion was not obtained in the HEP-G2 cells even after 72 hr. There was no apparent stimulation in the activity of various phase II drug metabolizing enzymes following pentamidine treatment. These findings suggest that resistance plays a role in the mechanism of action of pentamidine, since biochemical modulation of both cell lines is brought about by the drug, but only the resistant cells appear to recover fully following the removal of pentamidine from the culture medium.

Death in the nursing home: senescence, infection and other causes.

1. Clinical reasons for death in a skilled nursing home continue to show infectious causes as the leading etiology. 2. The concept of senescence is probably overlooked as a legitimate explanation for death in elderly patients. Senescence can be defined as a protracted and progressive decline of multiple body functions not attributable to one specific etiology and culminating in the patient's death. 3. The majority of deaths occurred within the first year after admission to the skilled nursing home setting. 4. The majority of deaths among the patients in this study occurred in the nursing home setting, not the hospital.

Lack of differentiation following mouse serum treatment of cultured chick limb myoblasts.

This investigation was conducted to assess the effects of mouse serum on chick skeletal muscle cell differentiation. In light of earlier findings of altered membrane phospholipid metabolism following mouse serum treatment of Friend erythroleukemic and chick chondrogenic cells, it was of interest to determine whether similar changes would modulate the fusion of mononucleated myoblasts, which is necessary for the formation of multinucleated skeletal muscle fibers. When mouse serum is added to low density cultures of enriched chick myoblasts shortly following cell attachment to the substratum, fusion is inhibited and neutral lipid accumulation ensues. There is an early inhibitory effect on DNA synthesis but not on protein synthesis. There is no increase in the uptake of 2-deoxyglucose following insulin stimulation of the cells, which suggests that while the cells are accumulating large amounts of lipid, they are not being converted into typical adipocytes. Finally, even in cultures of mouse serum-treated cells that undergo significant fusion, one observes thinner myotubes that do not spontaneously contract as do those of control cultures, as well as a disorganization of fluorescently stained actin and myosin myofilaments. These findings demonstrate that mouse serum acts in a dose-dependent manner, is not cytotoxic to the cells, but is capable of modulating normal developmental events of myoblasts as reported for other cell and tissue types.

Systemic anaphylaxis caused by parenteral spectinomycin.

Spectinomycin is commonly used for the treatment of uncomplicated gonorrhea, and is considered to be a safe drug. We have described a case of anaphylaxis characterized by peripheral vascular collapse after intramuscular administration of spectinomycin.

Development of the cyclic AMP response to parathyroid hormone and prostaglandin E2 in the embryonic chick limb.

The developing chick limb was studied to determine the ability of parathyroid hormone (PTH) and prostaglandin E2 (PGE2) to increase intracellular cyclic AMP (cAMP) during various stages of development. All developmental stages examined (stages 20-21, 24-25, and 26-28) responded to PGE2 when the cells were assayed immediately following the removal of the limbs from the embryos. In contrast, only stage 26-28 limb cells responded to PTH when assayed in a similar manner. The response to PTH was temporally correlated with the appearance of cartilage matrix in vivo. Undifferentiated limb cells were also cultured and assayed at various times for hormone responsiveness. Stage 24-25 high-density cell cultures responded initially to PGE2 but not to PTH. However, by 36 h and in all subsequent time intervals tested, the response to PTH was significantly greater than that to PGE2. The PTH receptor, in contrast to that of PGE2, was shown to be sensitive to trypsin treatment, but could be generated during subsequent cell culture. The majority of the hormone-responsive cells were found in cartilaginous regions of the limb, and were shown to respond to both hormones in a dose-dependent manner. The PTH-induced cAMP response was affected by low cell density and mouse serum, both of which significantly inhibit the chondrogenic potential of cultured limb cells. These findings are consistent with a temporal correlation between the development of the PTH response and chondrogenesis in vivo.

Immunoprecipitation assay of alpha-fetoprotein synthesis by cultured mouse hepatoma cells treated with estrogens and glucocorticoids.

This investigation was to study the biosynthesis of 3H-labeled alpha-fetoprotein (AFP) by cultured mouse hepatoma (HEPA-2) cells. Both the function and regulation of this oncodevelopmental gene are unknown. However, evidence indicates that mechanisms controlling the expression of AFP involve aspects of both normal embryonic development and neoplastic transformation. the secretion of AFP was analyzed during different phases of the growth cycle to provide information on AFP production using standard culture conditions. The highest rate of secretion occurred during the stationary phase, followed by the late logarithmic and early logarithmic phases of growth, respectively. The production of AFP was then determined following the addition of glucocorticoids and estrogens in an attempt to understand hormonal factors that may be involved. Studies utilizing estradiol-17 beta indicated that the secretion of AFP did not appear to be sensitive to this steroid even though sucrose density gradient analysis of HEPA-2 cytosol, for estrogenic receptors, revealed competitive binding moieties on the 8S and 4S regions of the gradient. In contrast, the secretion of the total complement of proteins, including AFP, was significantly stimulated by the glucocorticoids, dexamethasone and corticosterone. Analysis of HEPA-2 cytosol for glucocorticoid receptors revealed binding components in the 7S and 3-4S regions of the gradient. The 3H-dexamethasone binding appeared to be stereospecific since nonlabeled dexamethasone, but not nonlabeled estradiol-17 beta, effectively displaced the bound radioactivity. The glucocorticoid-binding component in HEPA-2 therefore displayed characteristics reported for glucocorticoid receptors in normal liver and other hepatomas.

The lack of an inhibitory effect of hyaluronate on chondrogenesis in chick limb-bud mesoderm cells grown in culture.

The effect of hyaluronate on chondrogenesis in cultures of chick limb-bud mesoderm cells, derived from stage 20--21, 23--24 and 26 embryos grown at different cell densities and in 3 different culture media, was studied. The results show that hyaluronate at a concentration of 500 microgram/ml, does not consistently produce an inhibition of chondrogenesis in cultures of stage 20--21, 23--24 or 26 limb-bud mesoderm cells in contrast to what has been reported by Toole et al. (1972). It was demonstrated that under optimal conditions, stage 26 cells grown in the absence of hyaluronate do not form as many cartilage colonies in culture as do cells from stage 20--21 or 23--24 embryos. It was determined that culture medium composed of Eagle's MEM supplemented with 7% horse serum, 3% fetal calf serum and 5% 10-day chick embryo extract supported chondrogenesis significantly better than Ham's F-12 supplemented with 10% fetal calf serum. Our results suggest that the inhibition of chondrogenesis by hyaluronate reported earlier is most likely due to the sub-optimal conditions of growth medium, cell density and embryonic stage than to the hyaluronate treatment.

Teaching interviewing skills to pharmacy residents.

An instructional program for first-year pharmacy residents in the use of interviewing techniques when assessing the drug therapy of patients is described. The objectives of this project were to (1) teach the appropriate use of interviewing skills and (2) observe the performance of pharmacy residents in assessing the drug therapy of ambulatory patients. Instructional methods included a written self-instructional packet, videotaped lecture and demonstration skits, group discussion, and role playing. Following these, during a four-week period, the residents interviewed patients in the outpatient pharmacy and reviewed their performance on videotape with the instructor. Two simulated patient case situations using a resident posing as a patient who provided predetermined information were used to measure student performance in interviewing patients. An improvement in the ability of the residents to employ the interview techniques and to assess the therapy of patients was noted.

Arachidonate metabolism during chondrogenesis in vitro.

Chick embryo limb bud mesenchyme cells undergoing chondrogenesis in vitro were labeled with [3H] arachidonic acid and [14C] palmitic acid, and stimulated by mechanical means to convert a portion of their incorporated [3H] to radiolabeled compounds which co-chromatographed with authentic prostaglandins in the appropriate thin layer chromatography system. Chondrogenesis was (1) inhibited by concentrations of indomethacin or eicosa-5,8,11,14-tetraynoic acid which inhibited conversion of [3H] to prostaglandinlike compounds; and (2) stimulated by prostaglandin E2. We interpret these data to mean that (1) cells undergoing chondrogenesis in vitro are able to metabolize endogenous arachidonic acid to prostaglandins, and (2) synthesis of prostaglandinlike compounds is requisite to chondrogenesis in vitro.

Respiratory failure in neuromuscular disease. Management in a respiratory intensive care unit.

Patients with neuromuscular disease frequently experience acute respiratory failure. Most require endotracheal intubation or tracheostomy and mechanical ventilation because of paralysis and inability to maintain adequate spontaneous respiration. The prognosis is usually excellent if ventilatory management is successful.

Coxsackievirus B5 infection and aseptic meningitis in neonates and children.

In metropolitan Washington, DC, an outbreak of aseptic meningitis in children was recognized in the summer and fall of 1972. Age-specific attack rates were highest in children less than 1 year of age. The incidence of cases showed two peaks: one in July and another in October. Coxsackievirus B5 was associated with cases occurring in July, August, and September, but was not implicated in the October cases. Seventy-six percent of the confirmed coxsackievirus B5 infections in aseptic meningitis patients occurred in infants less than 2 months old. Specific meningeal symptoms were less frequently observed in these young infants, although viral isolations were more common (13 of 15) compared to patients over 2 months of age (four of 19). Analysis of reported coxsackievirus B5 infections in Washington, DC, and the United States as a whole suggests a five- or six-year periodicity.