Antitransferrin receptor immunotoxin inhibits proliferating human retinal pigment epithelial cells.

Cultured human retinal pigment epithelial cells were exposed to an immunotoxin composed of a monoclonal antibody, 454A12, directed against transferrin receptors conjugated to a toxin, recombinant ricin A chain. Exposure of proliferating human retinal pigment epithelial cells to the immunotoxin (0.1 to 10,000 ng/mL) caused a statistically significant (P less than .0001) decrease in the number of cells. This inhibitory effect was induced after an exposure to the immunotoxin as short as 5 minutes and was maximal after 24 hours of exposure. The diminution in cell number was dose dependent over the range from 0.1 to 100 ng/mL. Monoclonal antibody alone, recombinant ricin A chain alone, or an irrelevant immunotoxin, MOP21C monoclonal antibody-recombinant ricin A, did not diminish the number of cells. There was a marked decrease in DNA synthesis measured by nuclear tritiated thymidine incorporation that accompanied the immunotoxin-mediated decrease in cell number. Viable cells remaining after exposure to the immunotoxin (0.1 to 10,000 ng/mL) were morphologically abnormal; typically the cells had elongated spindle-shaped processes and had lost their normal cuboidal appearance. In contrast, cell number was not decreased in confluent human retinal pigment epithelial cells after treatment with maximal doses of immunotoxin. Morphologic changes similar to those seen in proliferating cells were observed in confluent cells exposed to more than 100 ng/mL of immunotoxin. The effect of the immunotoxin was species specific because large doses of immunotoxin did not reduce the number of viable cells in proliferating or confluent pig retinal pigment epithelial cells or cause observable morphologic changes in this cell type. Our results indicate that the immunotoxin selectively inhibited proliferating retinal pigment epithelial cells by receptor-mediated internalization of the antitransferrin receptor monoclonal antibody-recombinant ricin A chain conjugate.

Application of the Postconcussive Syndrome Questionnaire with medical and psychiatric outpatients.

The Postconcussive Syndrome Questionnaire (PCSQ; Lees-Haley, 1992) was previously found (Axelrod, Fox, Lees-Haley, Earnest, Dolezal-Wood, & Goldman, 1996) to produce four factors, named Psychological, Somatic, Cognitive, and Infrequency. These four factors of the questionnaire were evaluated across five groups of medical and psychiatric outpatients. The patients were from neurology, mental health, family practice, and internal medicine clinics as well as from a clinic that evaluated new patients to a health maintenance organization. Mental health patients had greater psychological symptoms and fewer health concerns than the other groups. Neurology patients differed from the other groups by having greater Infrequency symptoms. Patients who were referred for their screening evaluation or were seen by internal medicine had fewer overall symptoms than the other three patient groups. The data from this study provide support for the use of the PCSQ as a multifactorial self-report measure of symptom presentation.

Prostaglandin F2 alpha stimulates progesterone secretion by porcine luteal cells in vitro throughout the estrous cycle.

In this study we examined the stimulatory effects of PGF2 alpha on progesterone secretion by porcine luteal cells on different days of the estrous cycle, and the effects of PGF2 alpha, A23187 and PMA on progesterone secretion by isolated large and small luteal cells, in vitro. Corpora lutea were obtained from cycling pigs (days 6-16), collagenase dispersed and luteal cells incubated in medium 199 in the absence or presence of increasing doses of PGF2 alpha, A23187, and PMA. Progesterone concentrations in spent media were measured by RIA. PGF2 alpha stimulation of progesterone secretion by mixed luteal cells did not vary significantly throughout the estrous cycle. Progesterone secretion by large, but not small, luteal cells was increased (p < 0.05) in a dose-dependent fashion by PGF2 alpha. A23187 also caused a dose-dependent increase in progesterone secretion by large luteal cells but inhibited small luteal cells. Progesterone secretion by both large and small luteal cells was significantly increased by increasing doses of PMA. We conclude that the stimulatory response of luteal cells to PGF2 alpha in vitro did not correlate with PGF2 alpha receptor concentrations (not measured in this study), and we speculate that calcium/protein kinase C may be involved in mediating the stimulatory action of PGF2 alpha on luteal cell progesterone secretion.

Bone histomorphometry in adults with type IA osteogenesis imperfecta.

A histologic and histomorphometric analysis was performed on undecalcified bone from 8 adult patients, ages 34 to 64 years, with Type IA osteogenesis imperfecta. Complete histomorphometric data, including static and dynamic parameters of bone remodeling, could be generated on 6 patients, and partial data were obtained from the other 2 patients. Findings in some patients of reduced eroded surfaces and reduced osteoid surfaces suggested low bone turnover. Other findings included normal or slightly reduced labeled surfaces, slightly reduced bone formation rate, decreased cortical thickness, and decreased bone volume. Histologic examination results showed lamellar bone with mature cortical Haversian systems. Trabeculae showed qualitatively normal connectedness despite low trabecular volume. The finding of normal or reduced bone turnover in adults with Type IA osteogenesis imperfecta has not been reported. Earlier histomorphometric studies, performed without correlation with a specific age or phenotype, indicated high bone turnover. The present study suggests that future research should correlate histopathologic changes with specific phenotypes. The finding of normal or slightly reduced bone turnover in Type IA osteogenesis imperfecta may have important therapeutic implications for this phenotype.

A redundant nuclear protein binding site contributes to negative regulation of the mouse mammary tumor virus long terminal repeat.

The tissue specificity of mouse mammary tumor virus (MMTV) expression is controlled by regulatory elements in the MMTV long terminal repeat (LTR). These regulatory elements include the hormone response element, located approximately between -200 and -75, as well as binding sites for NF-1, Oct-1 (OTF-1), and mammary gland enhancer factors. Naturally occurring MMTV deletion variants isolated from T-cell and kidney tumors, transgenic-mouse experiments with MMTV LTR deletions, and transient transfection assays with LTR constructs indicate that there are additional transcription regulatory elements, including a negative regulatory element (NRE), located upstream of the hormone response element. To further define this regulatory region, we have constructed a series of BAL 31 deletion mutants in the MMTV LTR for use in transient transfection assays. These assays indicated that deletion of two regions (referred to as promoter-distal and -proximal NREs) between -637 and -201 elevated basal MMTV promoter activity in the absence of glucocorticoids. The region between -637 and -264 was surveyed for the presence of nuclear protein binding sites by gel retardation assays. Only one type of protein complex (referred to as NRE-binding protein or NBP) bound exclusively to sites that mapped to the promoter-distal and -proximal NREs identified by BAL 31 mutations. The promoter-proximal binding site was mapped further by linker substitution mutations and transfection assays. Mutations that mapped to a region containing an inverted repeat beginning at -287 relative to the start of transcription elevated basal expression of a reporter gene driven by the MMTV LTR. A 59-bp DNA fragment from the distal NRE also bound the NBP complex. Gel retardation assays showed that mutations within both inverted repeats of the proximal NRE eliminated NBP binding and mutations within single repeats altered NBP binding. Intriguingly, the NBP complex was detected in extracts from T cells and lung cells but was absent from mammary gland cells. These results suggest that a factor contributing to high-level expression of MMTV in the mammary gland is the lack of negative regulation by NBP.