Lipofection of rabbit chondrocytes and long lasting expression of a lacZ reporter system in alginate beads

OBJECTIVE: Our aim was to investigate the maintenance of the transfection status of non-viral transfected chondrocytes in an alginate culture system. DESIGN: Chondrocytes harvested from rabbit knees were isolated by sequential digestion and cultivated in monolayer culture. At 60-70% cell density, chondrocytes were transfected with different transfection systems (FuGENE6, CaCl2, Lipofectin). A lac Z expression vector (pcDNA 3.1/Myc-His+ lacZ) was used as a reporter system. In order to improve transfection rates, hyaluronidase (4 U/ml) was used prior and during the transfection procedure. Thereafter, transfected cells were either kept in monolayer culture or embedded in alginate beads and kept in culture for up to the next 30 weeks. RESULTS: Transfection efficiency was maximal using FuGENE6TM/DNA at a ratio of 3:2 and hyaluronidase (4 U/ml). Transfection efficiency reached up to 40.8% (+/- 3.2%) after 36 h. In alginate beads lac Z positive cells declined to 8.5% +/- 3.3% after 4 weeks and to 4.6% +/- 3.2% after 12 weeks of culturing. After 30 weeks 3% of chondrocytes still expressed lac Z. In contrast, during culturing in monolayer, no lac Z expression was detectable after 4 weeks. Differentiation status of the chondrocytes was confirmed by histology and immunohistochemistry methods. CONCLUSIONS: After successful gene transfer to rabbit chondrocytes the alginate system made it possible to culture lipofected chondrocytes phenotypically stable. Genetically engineered chondrocytes express the lac Z reporter gene over a period of at least 30 weeks. This transfection and culture system provides a promising tool to further investigate the over-expression of growth factors and enzyme inhibitors.     

Pharmacokinetics of propofol infusions in critically ill neonates,infants, and children in an intensive care unit

BACKGROUND: Propofol is a commonly used anesthetic induction agent in pediatric anesthesia that, until recently, was used with caution as an intravenous infusion agent for sedation in pediatric intensive care. Few data have described propofol kinetics in critically ill children. METHODS: Twenty-one critically ill ventilated children aged 1 week to 12 yr were sedated with 4-6 mg. kg(-1).h(-1) of 2% propofol for up to 28 h, combined with a constant morphine infusion. Whole blood concentration of propofol was measured at steady state and for 24 h after infusion using high-performance liquid chromatography. RESULTS: A propofol infusion rate of 4 mg. kg(-1).h(-1) achieved adequate sedation scores in 17 of 20 patients. In 2 patients the dose was reduced because of hypotension, and 1 patient was withdrawn from the study because of a increasing metabolic acidosis. Mixed-effects population models were fitted to the blood propofol concentration data. The pharmacokinetics were best described by a three-compartment model. Weight was a significant covariate for all structural model parameters; Cl, Q2, Q3, V1, and V2 were proportional to weight. Estimates for these parameters were 30.2, 16.0, and 13.3 ml. kg(-1).min(-1) and 0.584 and 1.36 l/kg, respectively. The volume of the remaining peripheral compartment, V3, had a constant component (103 l) plus an additional weight-related component (5.67 l/kg). Values for Cl were reduced (typically by 26%) in children who had undergone cardiac surgery. CONCLUSIONS: Propofol kinetics are altered in very small babies and in children recovering from cardiac surgery. Increased peripheral distribution volume and reduced metabolic clearance following surgery causes prolonged elimination.     

Perinatal management of a preterm neonate affected by hyperprostaglandin E2 syndrome (HPS)

BACKGROUND: Neonates affected by hyperprostaglandin E(2) syndrome (HPS) present with severe polyuria. Both urinary losses as well as prostaglandin synthesis inhibitors may precipitate acute renal failure (ARF). AIM: Our goal was to maintain euvolaemia by replacement of urinary losses. PATIENT: Our patient was born prematurely with a family history typical of HPS. Urinary salt and water losses and PGE(2) excretion were determined in 2- to 4-h intervals. Salt and water were replaced accordingly. RESULTS: Within the first 48 h, urinary losses and PGE(2) increased continuously to 50 ml/kg/h and 374 ng/h/1.73 m(2), respectively. Following exposure to 0.05-0.5 mg/kg/d indomethacin, urinary output decreased steadily to 10-15/ml/kg/h.CONCLUSION: In euvolaemic preterm neonates with HPS and the need for excessive replacement of salt and water, inhibition of renal PGE(2) excretion with indomethacin effectively reduces polyuria and natriuresis without acutely compromising renal function.     

‘Abazali abazamayo’ (parents who keep on trying): mothers' responses to the Ububele Mother-Baby Home Visiting Programme

Early parent–infant home visiting interventions have been found to be effective in both developed and developing countries. However, there is a need to build an evidence base for these interventions in the South African context, to inform local early childhood development policy. The Ububele Mother-Baby Home Visiting Programme in Alexandra, Johannesburg, combines a psychoanalytic approach to thinking about parent–infant relationships and local infant care knowledge. Trained, local women offer a 14-visit intervention, which aims to support mothers, increase knowledge about the relational needs of infants, and increase maternal reflective function capacities. A mixed-methods approach was used to evaluate preliminary findings regarding mothers' responses to the programme and found significant increases in the mothers’ levels of perceived support, in levels of knowledge regarding the relational needs of infants and in maternal reflective function capacities. The supportive relationship between home visitors and mothers was highlighted as key to programme success.     

The Multiple Tasks Test as a predictor of falls in older adults

PURPOSE: Falls are among the most common and serious problems facing the elderly. The Berg Balance Scale (BBS) is the gold standard in measuring falls risk. With higher functioning elders, a ceiling effect is often evidenced using the BBS. The purpose of this study was to determine if the Multiple Tasks Test (MTT) when used in high functioning community dwelling elderly correlated with the BBS. Secondly, this study assessed the unidimensionality of the MTT. If a relationship existed between the performance of multiple tasks and the potential loss of balance resulting in falls, then the MTT would be more appropriate than the BBS at predicting falls in higher functioning individuals. METHODS: Twenty-two independent community dwelling older adults were tested using both the MTT and the BBS on the same day at a senior center. DATA ANALYSIS: Correlations between the BBS and the MTT ranged between -0.765 and -0.79. The results of the Guttman's scalability analysis were a plus percentage ratio of 0.75 and an index of reproducibility of 0.93. Scales with these values are regarded as having evidence of unidimensionality. SUMMARY: All of the MTT tests correlated with the BBS. In addition, the MTT was found to be unidimensional. CONCLUSION: Although the MTT correlated with the BBS and was found to be a good cumulative measure, it would benefit from continued study to examine quantifiability, validity and reliability. With further refinement and study, the MTT may be useful at discriminating fallers from non-fallers in high functioning older adults.     

Case report: diabetes flatbush - from ketoacidosis to non pharmacological treatment

A subgroup of patients presents diabetic ketoacidosis at the onset of diabetes mellitus (DM) but later is classified as type 2 DM based on the clinical follow-up. These individuals, most commonly obese of African or Hispanic origin, have negative auto-antibodies associated with type 1 DM, but frequently HLA class II DRB1*03 and/or DRB1*04 are detected. This peculiar subtype of DM is commonly referred to as diabetes flatbush. Here we report the case of a Caucasian patient that exhibited the described evolution and in whom it was possible to withdraw insulin therapy. The possible factors associated with this favorable development are also discussed.     

The experts speak. Studying the potential of tenofovir to prevent sexual transmission of HIV: first steps. Interview by Vicki Glaser

Melanie Thompson, M.D., is Founder and Principal Investigator of the AIDS Research Consortium of Atlanta (ARCA), one of the first non-profit community-based HIV/AIDS research centers in the United States and the primary resource for HIV clinical research in Georgia. Dr. Thompson is also a private practitioner in Atlanta and serves on the International AIDS Society- USA's Antiretroviral Guidelines Panel. She is the past chair of the National Institutes of Health Office of AIDS Research Therapeutics Research Working Group. Dr. Thompson has served as ARCA's principal investigator for more than 200 studies involving more than 12,000 patients. To date, ARCA's studies have contributed to the FDA approval of 21 treatments for HIV and its complications.