Geoff Cleghorn

The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events.

BackgroundAlthough lentiviral vectors have been widely used for in vitro and in vivo gene therapy researches, there have been few studies systematically examining various conditions that may affect the determination of the number of viable vector particles in a vector preparation and the use of Multiplicity of Infection (MOI) as a parameter for the prediction of gene transfer events.MethodsLentiviral vectors encoding a marker gene were packaged and supernatants concentrated. The number of viable vector particles was determined by in vitro transduction and fluorescent microscopy and FACs analyses. Various factors that may affect the transduction process, such as vector inoculum volume, target cell number and type, vector decay, variable vector – target cell contact and adsorption periods were studied. MOI between 0–32 was assessed on commonly used cell lines as well as a new cell line.ResultsWe demonstrated that the resulting values of lentiviral vector titre varied with changes of conditions in the transduction process, including inoculum volume of the vector, the type and number of target cells, vector stability and the length of period of the vector adsorption to target cells. Vector inoculum and the number of target cells determine the frequencies of gene transfer event, although not proportionally. Vector exposure time to target cells also influenced transduction results. Varying these parameters resulted in a greater than 50-fold differences in the vector titre from the same vector stock. Commonly used cell lines in vector titration were less sensitive to lentiviral vector-mediated gene transfer than a new cell line, FRL 19. Within 0–32 of MOI used transducing four different cell lines, the higher the MOI applied, the higher the efficiency of gene transfer obtained.ConclusionSeveral variables in the transduction process affected in in vitro vector titration and resulted in vastly different values from the same vector stock, thus complicating the use of MOI for predicting gene transfer events. Commonly used target cell lines underestimated vector titre. However, within a certain range of MOI, it is possible that, if strictly controlled conditions are observed in the vector titration process, including the use of a sensitive cell line, such as FRL 19 for vector titration, lentivector-mediated gene transfer events could be predicted.

Nutritional status of children with cystic fibrosis measured by total body potassium as a marker of body cell mass: Lack of sensitivity of anthropometric measures ☆ ☆☆ ★

OBJECTIVE To investigate measures aimed at defining the nutritional status of cystic fibrosis (CF) populations, this study compared standard anthropometric measurements and total body potassium (TBK) as indicators of malnutrition. METHODS Height, weight, and TBK measurements of 226 children with CF from Royal Childrens Hospital, Brisbane, Australia, were analyzed. Z scores for height for age, weight for age, and weight for height were analyzed by means of the National Centre for Health Statistics reference. TBK was measured by means of whole body counting and compared with predicted TBK for age. Two criteria were evaluated with respect to malnutrition: (1) a z score < -2.0 and (2) a TBK for age <80% of predicted. RESULTS Males and females with CF had lower mean height-for-age and weight-for-age z scores than the National Centre for Health Statistics reference (P <.01), but mean weight-for-height z score was not significantly different. There were no significant gender differences. According to anthropometry, only 7.5% of this population were underweight and 7.6% were stunted. However, with TBK as an indicator of nutritional status, 29.9% of males and 22.0% of females were malnourished. CONCLUSION There are large differences in the percentage of patients with CF identified as malnourished depending on whether anthropometry or body composition data are used as the nutritional indicator. At an individual level, weight-based indicators are not sensitive indicators of suboptimal nutritional status in CF, significantly underestimating the extent of malnutrition. Current recommendations in which anthropometry is used as the indicator of malnutrition in CF should be revised.

Successful topical tacrolimus (FK506) therapy in a child with pyoderma gangrenosum

The inflammatory skin disease pyoderma gangrenosum is characterized by destructive ulceration, typically occurring on the calves and thighs and less commonly on the buttocks and face. Lesions vary in size and may be multiple, often rapidly ulcerating to form deep painful wounds. Ulcers characteristically have ragged purple edges that overhang. In many patients a concomitant condition can be identified such as inflammatory bowel disease, rheumatoid arthritis, chronic autoimmune hepatitis, and various hematologic and solid tumours (1,2). Treatment of these ulcers in the past has been disappointing. The large lesions usually run a chronic course and heal very slowly, with traditional dressings often in combination with systemic steroids or immunosuppressants. Since 1998, a small number of case have been reported of adults with pyoderma gangrenosum whose lesions heal with the use of topical tacrolimus (FK506) (2–4). We report, to the best of our knowledge, the first successful treatment of a child with pyoderma gangrenosum using topical tacrolimus.

Growth patterns in preterm infants born appropriate for gestational age.

Aim:  This study aimed to document the growth patterns of a contemporary cohort of preterm infants born appropriate for gestational age (AGA). It was hypothesised that preterm AGA (PT‐AGA) infants would display poorer growth than full‐term AGA (FT‐AGA) infants.

Physical activity cost in children following an acquired brain injury: a comparative study

BACKGROUND Alterations in energy expenditure during activity post head injury has not been investigated due primarily to the difficulty of measurement. OBJECTIVE The aim of this study was to compare energy expenditure during activity and body composition of children following acquired brain injury (ABI) with data from a group of normal controls. DESIGN Energy expenditure was measured using the Cosmed K4b(2) in a group of 15 children with ABI and a group of 67 normal children during rest and when walking and running. Mean number of steps taken per 3 min run was also recorded and body composition was measured. RESULTS The energy expended during walking was not significantly different between both groups. A significant difference was found between the two groups in the energy expended during running and also for the number of steps taken as children with ABI took significantly less steps than the normal controls during a 3 min run. CONCLUSIONS Children with ABI exert more energy per activity than healthy controls when controlled for velocity or distance. However, they expend less energy to walk and run when they are free to choose their own desirable, comfortable pace than normal controls.

Value of Total Body Potassium in Assessing the Nutritional Status of Children with End‐Stage Liver Disease

Abstract: Malnutrition is a common problem in children with end‐stage liver disease (ESLD), and accurate assessment of nutritional status is essential in managing these children. In a retrospective study, we compared nutritional assessment by anthropometry with that by body composition. We analyzed all consecutive measurements of total body potassium (TBK, n= 186) of children less than 3 years old with ESLD awaiting transplantation found in our database. The TBK values obtained by whole body counting of 40K were compared with reference TBK values of healthy children. The prevalence of malnutrition, as assessed by weight (weight Z score <−2) was 28%, which was significantly lower (chi‐square test, p < 0.0001) than the prevalence of malnutrition (76%) assessed by TBK (<90% of expected TBK for age). These results demonstrated that body weight underestimated the nutritional deficit and stressed the importance of measuring body composition as part of assessing nutritional status of children with ESLD.

Development of Height-weight Based Equation for Assessment of Body Composition in Sri Lankan Children

ObjectiveTo develop a height and weight based equation to estimate total body water (TBW) in Sri Lankan children.MethodsCross sectional descriptive study done involving 5–15 year old healthy children. Height and weight were measured. TBW was assessed using isotope dilution method (D2O) and fat free mass (FFM) calculated. Multiple regression analysis was used to develop prediction equation and validated using PRESS statistical technique. Height, weight and sex code (male=1; female=0) were used as prediction variables.ResultsThis study provides height and weight equation for the prediction of TBW in Sri Lankan children. To the best of our knowledge there are no published height weight prediction equations validated on South Asian populations.ConclusionResults of this study need to be affirmed by more studies on other closely related populations by using multicomponent body composition.