Expression of human bone morphogenic protein 7 in primary rabbit periosteal cells: potential utility in gene therapy for osteochondral repair.

A commonly encountered problem in orthopedics is bone and cartilage tissue injury which heals incompletely or without full structural integrity. This necessitates development of improved methods for treatment of injuries which are not amenable to treatment using current therapies. An already large and growing number of growth factors which play significant roles in bone remodeling and repair have been identified in the past few years. It is well established that bone morphogenic proteins induce the production of new bone and cartilage. An efficient method of delivery of these growth factors by conventional pharmacological means has yet to be elucidated. We wished to evaluate the use of retroviral vector-mediated gene transfer to deliver genes of therapeutic relevance for bone and cartilage repair. To determine the feasibility of using amphotropically packaged retroviral vectors to transduce primary rabbit mesenchymal stem cells of periosteal origin, primary periosteal cells were isolated from New Zealand white rabbits, transduced in vitro with a retroviral vector bearing both the nuclear localized lacZ marker gene and the neo(r) gene, and selected in G418. We used a convenient model for analysis of in vivo stability of these cells which were seeded on to polymer scaffold grafts and implanted into rabbit femoral osteochondral defects. The nuclear localized beta-galactosidase protein was expressed in essentially 100% of selected cells in vitro and was observed in the experimental explants from animals after both 4 and 8 weeks in vivo, while cells transduced with a retroviral vector bearing only the neo(r) gene in negative control explants showed no blue staining. We extended our study by delivering a gene of therapeutic relevance, human bone morphogenic protein 7 (hBMP-7), to primary periosteal cells via retroviral vector. The hBMP-7 gene was cloned from human kidney 293 cell total RNA by RT-PCR into a retroviral vector under control of the CMV enhancer/promoter. Hydroxyapatite secretion, presumably caused by overexpression of hBMP-7, was observed on the surface of the transduced and selected periosteal cells, however, this level of expression was toxic to both PA317 producer and primary periosteal cells. Subsequently, the strong CMV enhancer/promoter driving the hBMP-7 gene was replaced in the retroviral vector by a weaker enhancer/promoter from the rat beta-actin gene. Nontoxic levels of expression of hBMP-7 were confirmed at both the RNA and protein levels in PA317 producer and primary periosteal cell lines and cell supernatants. This work demonstrates the feasibility of using a gene therapy approach in attempts to promote bone and cartilage tissue repair using gene-modified periosteal cells on grafts.     

A Secondary Analysis of Smoking Among Rural and Urban Youth Using the MTF Data Set

ABSTRACT: This paper compares rural and urban youth cigarette-smoking behavior using the Monitoring the Future data set, a national, probability-based, multi-stage sample design. Cigarette smoking was examined by region, race, and gender over time. Results indicated that rural White males smoked more ofen (30-day prevalence of 34%) than any other group. Urban Black males smoked the least (10%). Urban White females smoked more (30-day prevalence of 33%) than urban Black females (7%). In general, rural and urban Whites reported smoking at significantly higher rates than rural and urban Blacks. Smoking rates declined steadily for both rural and urban Black females and urban Black males over the study period. These data document distinctive patterns of tobacco use among the nation's youth, which can be used by program planners for specific targeted interventions.     

Investigating stress effect patterns in hospital staff nurses: Results of a cluster analysis

A comprehensive and reliable assessment of work stress, burnout, affective, and physical symptomatology was conducted with 260 hospital nurses. As previous attempts to categorize nursing stress and burnout by ward type have yielded inconsistent results, an alternative method for grouping nursing stress effects was sought. Cluster analysis was chosen as it offers a statistically sound means of delineating natural groupings within data. Sets of questionnaires measuring burnout, work stressors, and physical and emotional symptomatology were sent to all staff nurses at a large university hospital. Of 709 nurses employed there, a total of 260 nurses returned completed questionnaire packets. These nurses were separated into two equal groups using random sampling procedures. Cluster analysis of this data revealed groupings which were based on nursing stressors (particularly workload and conflict with physicians), social support, and patient loads. These cluster-analytic findings were replicated on both samples, and validated using data not used in the original cluster analysis. Results suggest that the effects of stress have more to do with the characteristics of the work environment and overall workload than with the degree of specialization on the unit. Results also suggest that intraprofessional conflict (i.e. with other nurses) is less psychologically damaging than is interprofessional conflict (i.e. conflict with physicians). Findings are discussed with respect to the burnout process and possible interventions.     

In vivo induction of CD4+ T cell responses by antigens covalently linked to synthetic microspheres does not require adjuvant

Macrophages, dendritic cells or B lymphocytes have been shownto play a major role in the presentation of soluble antigensto CD4⁺ T cells. In contrast, the capacity of these cells topresent particulate antigens such as bacterial or parasiticantigens to T cells remains controversial. To investigate thisquestion, well defined particulate antigens were prepared bycovalent linkage of proteins or peptides to 1 µm in diametersynthetic microspheres. The T cell immunogenicity of such particulateantigens was analyzed in vitro and in vivo. In vitro, a solubleprotein such as hen egg lysozyme (HEL) coupled to beads stimulateda strong proliferative T cell response of lymph node cells fromHEL-primed mice or of specific T cell hybridomas. HEL coupledto beads was presented to the specific T cell hybridomas bysplenocytes or by peritoneal macrophages, but not by lymphomaB cells. Immunization of mice with several different proteinantigens or with a synthetic peptide covalently linked to beadsinduced strong CD4⁺ T cell responses in the absence of adjuvant.The strong in vivo immunogenicity of proteins coupled to beadsdid not result from a non-specific adjuvant effect of beadssince covalent linkage of the antigen to beads was strictlyrequired to induce T cell responses in the absence of adjuvant.In vivo treatment by carrageenan showed that macrophages arerequired for the in vivo stimulation of T cell responses bythese particulate antigens. Thus, these results demonstratedthe role of phagocytic cells, especially macrophages, for invivo presentation of particulate antigens. These particulateantigens represent an interesting approach for the developmentof new vaccines, and for the in vivo analysis of the role ofvarious antigen presenting cells in T cell activation and differentiation.     

FACTORS RELATED TO PERINEAL TRAUMA IN CHILDBIRTH

We conducted an observational cohort study in three nurse-midwifery services to identify patient characteristics and clinical care measures related to perineal trauma at birth. Data were collected on all women who began care with a nurse-midwife in labor, using an adaptation of the Nurse-Midwifery Clinical Data Set (n = 3,049). Study variables included demographics, perineal management techniques and position for birth, and other intrapartum care and events. Univariate and multivariate analyses showed that episiotomy was strongly related to fetal bradycardia, prolonged second stage, ethnic status, and maternal education level. Warm compresses and flexion/counterpressure to slow delivery were protective. Spontaneous lacerations were influenced by these factors as well. The lateral position for birth was protective, and use of oils or lubricants and the lithotomy position increased lacerations. Multisite studies in nurse-midwifery practices may provide an ideal means of determining effective care measures in healthy populations.     

Exchangeable magnesium pool masses in healthy women: effects of magnesium supplementation.

BACKGROUND: Studying magnesium pools in the body with use of stable isotopes may be helpful for evaluating magnesium status. Data on the evaluation of magnesium pools in humans are scarce. OBJECTIVE: We undertook this study to evaluate the effects of a magnesium supplementation program on the size of the exchangeable body pools of magnesium and on classic indexes of magnesium status in healthy women with normal magnesium status. DESIGN: Ten healthy women participated in a kinetic study with magnesium stable isotopes before and after 8 wk of magnesium supplementation. Each woman received 3 supplements containing 5.08 mmol (122 mg) elemental Mg/d (366 mg/d). Before and at the end of the supplementation period, each woman received an intravenous injection of 1.67 mmol (40 mg) (25)Mg, and the plasma magnesium disappearance curve was followed for the next 7 d. Two methods were used to analyze the exchangeable pools of magnesium: 1) formal multicompartmental modeling and 2) a simplified estimation of the total mass of the rapidly exchangeable magnesium pool (EMgP). RESULTS: In these healthy women, exchangeable magnesium pools represented 11-12% of total body magnesium on the basis of multicompartmental analysis. The simplified estimation of EMgP overestimated the size of the exchangeable magnesium pools by approximately 45-50%. Eight weeks of magnesium supplementation did not significantly modify the size of the exchangeable magnesium pools, whereas urinary magnesium excretion was significantly higher after 8 wk of supplementation. CONCLUSION: Women with no clinical evidence of magnesium deficiency may not respond to short-term supplementation with increases in the mass of the exchangeable magnesium body pool or in magnesium turnover rates.