Strategies to enhance transductional efficiency of adenoviral-based gene transfer to primary human fibroblasts and keratinocytes as a platform in dermal wounds

Genetically modified keratinocytes and fibroblasts are suitable for delivery of therapeutic genes capable of modifying the wound healing process. However, efficient gene delivery is a prerequisite for successful gene therapy of wounds. Whereas adenoviral vectors (Ads) exhibit superior levels of in vivo gene transfer, their transductional efficiency to cells resident within wounds may nonetheless be suboptimal, due to deficiency of the primary adenovirus receptor, coxsackie-adenovirus receptor (CAR). We explored CAR-independent transduction to fibroblasts and keratinocytes using a panel of CAR-independent fiber-modified Ads to determine enhancement of infectivity. These fiber-modified adenoviral vectors included Ad 3 knob (Ad5/3), canine Ad serotype 2 knob (Ad5CAV-2), RGD (Ad5.RGD), polylysine (Ad5.pK7), or both RGD and polylysine (Ad5.RGD.pK7). To evaluate whether transduction efficiencies of the fiber-modified adenoviral vectors correlated with the expression of their putative receptors on keratinocytes and fibroblasts, we analyzed the mRNA levels of CAR, alpha upsilon integrin, syndecan-1, and glypican-1 using quantitative polymerase chain reaction. Analysis of luciferase and green fluorescent protein transgene expression showed superior transduction efficiency of Ad5.pK7 in keratinocytes and Ad5.RGD.pK7 in fibroblasts. mRNA expression of alpha upsilon integrin, syndecan-1 and glypican-1 was significantly higher in primary fibroblasts than CAR. In keratinocytes, syndecan-1 expression was significantly higher than all the other receptors tested. Significant infectivity enhancement was achieved in keratinocytes and fibroblasts using fiber-modified adenoviral vectors. These strategies to enhance infectivity may help to achieve higher clinical efficacy of wound gene therapy.     

Avascular osteonecrosis after hyperthermia in children and adolescents with pelvic malignancies: a retrospective analysis of potential risk factors.

PURPOSE: In children with locally advanced or recurrent malignant tumours, prognosis can be improved by regional deep hyperthermia (RHT) in combination with platin-based chemotherapy. However, because of the increasing number of patients that achieve long-time remission with this therapy, it is necessary to evaluate long-term sequelae of thermochemotherapy. During the years 1993-2004 one has observed avascular osteonecrosis (AON) of the femoral head after RHT in seven children with pelvic germ cell tumours or rhabdomyosarcomas. METHODS: Although AON may develop in patients with malignancies treated with chemo- or radiotherapy alone, RHT might nevertheless contribute to the occurrence of AON. In order to determine potential risk factors for AON after RHT, this study analysed the relationship of AON to the patient's age, medical history and treatment parameters such as thermal dose equivalent and power output. RESULTS AND CONCLUSIONS: In the present study AON was associated with young age as well as intensity of hyperthermia indicated by high power levels that exceed 20 W per kg body weight and/or application of eight or more heat sessions as well as additional radiotherapy. Based on this observation, it was assumed that an optimized three dimensional thermal field modelling may be helpful to avoid hazardous temperatures in the femoral heads during RHT treatment and to reduce AON of the femoral heads.     

Documenting asthma severity: do we get it right?

BACKGROUND: The 1997 National Asthma Education and Prevention Program (NAEPP) recommends a severity classification scheme to optimize the use of anti-inflammatory therapy for persistent asthma. Physician documentation of asthma severity is often used as a quality assurance measure. OBJECTIVE: To test the hypothesis that physician documentation of asthma severity is associated with appropriate use of anti-inflammatory therapy. DESIGN/METHODS: Setting: inner-city academic health center. First, we reviewed a consecutive sample of charts of scheduled pediatric patients. Then, we administered a structured parent survey regarding the child's asthma symptoms and current asthma therapy. We used NAEPP guidelines to classify patients' severity of asthma. The main outcome measure was appropriate use of anti-inflammatory therapy. Appropriate therapy was defined as: (1) mild persistent asthmatics using anti-inflammatory therapy, and (2) moderate-severe persistent asthmatics using inhaled steroids. Chart classification of asthma severity was compared with the NAEPP-applied classification. RESULTS: Of 784 charts, 214 (27%) were asthmatic. Of these, 176 (82%) were surveyed. The mean age was 7.4 years; 61% were males. Severity classification was documented in 77% of charts. Chart documentation differed significantly from survey classification for the same patients: (mild intermittent 54% vs. 40%, mild persistent 21% vs. 14%, moderate persistent 24% vs. 36%, severe persistent 1% vs. 10%; all p < .001). Correctly classified patients were more likely to be on appropriate therapy. CONCLUSIONS: Physicians underestimated the severity classification of asthmatic patients. Incorrect classification was associated with inappropriate asthma therapy. These findings have implications for the institution of asthma quality improvement programs.     

The principle of regulation in biology--from bone to eating behavior.

Cell physiology and molecular biology typically follow a reductionistic approach in science. In the last decade, molecular principles and pathogenetic factors involved in the development of many diseases have been successfully discovered. Therefore, early biological concepts based on systemic and cybernetic thoughts have been largely overshadowed by these more recent molecular and pathogenetic factors. This review highlights discoveries on bone development and hypothalamic controlled feeding and eating behavior with a cybernetic and systemic perspective. Interestingly, ancient ideas on bone development and hypothalamic function are still reasonable considerations to embed new molecular discoveries into a systemic concept of principles organizing nature.     

Insurance-mandated preoperative dietary counseling does not improve outcome and increases dropout rates in patients considering gastric bypass surgery for morbid obesity

BACKGROUND: Preoperative dietary counseling (PDC) before bariatric surgery is mandated by a growing number of insurance payers. Their claim is that PDC improves outcomes and postoperative compliance. We compared outcomes of GBP patients undergoing a mandatory 13 weeks of PDC (n = 72) to a contemporaneous group of patients with no such requirement (no-PDC; n = 252) who underwent operation between January 2000 and December 2002. METHODS: The PDC and no-PDC groups were characterized by similar male:female ratios (1:4 vs 1:4.6), mean age (42 years), mean body weight (324 lb vs 309 lb), and mean body mass index (BMI) (52 kg/m2 vs 50 kg/m2). The PDC group had a higher incidence of obstructive sleep apnea compared with the no-PDC group (41% vs 28%; P < .04) but otherwise the two groups had similar incidences of obesity-related comorbidities. The presurgery dropout rate was 50% higher in the PDC group than in the no-PDC group (28% vs 19%; P < .05). RESULTS: At 1 year follow-up, the no-PDC patients had a statistically greater percentage excess weight loss (67% vs 60%; P < .0001), lower BMI (32 vs 35; P < .015), and lower body weight (197 vs 218; P < .01). Resolution of major comorbidities, complication rates, 30-day postoperative mortality, and postoperative compliance with follow-up were similar in the two groups. CONCLUSIONS: The data demonstrate that insurance-mandated PDC is an obstacle to patient access for surgical treatment of severe obesity and has no impact on weight loss outcome or postsurgical compliance. PDC should be abandoned by the insurance industry.     

Statins are associated with a reduced infrarenal abdominal aortic aneurysm growth.

OBJECTIVE: To evaluate the effect of statins on aneurysm growth in a group of consecutive patients under surveillance for infrarenal aortic aneurysms (AAA). MATERIALS AND METHODS: All patients (59 statin users, 91 non-users) under surveillance between January 2002 and August 2005 with a follow-up for aneurysm growth of at least 12 months and a minimum of three diameter evaluations were retrospectively included in the analysis. Multiple regression analysis, weighted with the number of observations, was performed to test the influence of statins on AAA growth rate. RESULTS: During a median period of 3.1 (1.1-13.1) years the overall mean aneurysm growth rate was 2.95+/-2.8 mm/year. Statin users had a 1.16 mm/year lower AAA growth rate compared to non-users (95% CI 0.33-1.99 mm/year). Increased age was associated with a slower growth (-0.09 mm/year per year, p = 0.003). Female gender (+1.82 mm/year, p = 0.008) and aneurysm diameter (+0.06 mm/year per mm, p = 0.049) were associated with increased AAA growth. The use of non-steroidal anti-inflammatory drugs, chronic lung disease, or other cardiovascular risk factors were not independently associated with AAA growth. CONCLUSIONS: Statins appear to be associated with attenuation of AAA growth, irrespective of other known factors influencing aneurysm growth.     

Evaluation of polymerase chain reaction and adenosine deaminase assay for the diagnosis of tuberculous effusions in children.

AIM: To evaluate and compare the utility of polymerase chain reaction (PCR) for the diagnosis of tuberculous effusions in children. METHODS: PCR, adenosine deaminase (ADA) activity and absolute lymphocyte count (ALC) were evaluated in the fluid of 31 tuberculous (20 pleural, 8 ascites and 3 pericardial) and 24 non-tuberculous (10 transudtative ascites, 8 empyema thoracis, 3 malignant pleural and 3 pyopericardium) effusions. RESULTS: Fluid PCR for Mycobacterium tuberculosis was positive in 74% of tuberculous effusions, whereas it was falsely positive in 13% of the non-tuberculous group. The mean fluid ADA and ALC values were significantly higher in tuberculous effusions than in non-tuberculous effusions (p<0.001). The sensitivity and specificity of PCR, ADA (> or =38 IU/l) and ALC (> or =275/mm3) were 74% and 88%, 81% and 75%, and 90% and 83%, respectively, in diagnosing tuberculous effusions. The sensitivity of PCR, ADA and ALC was 100%, 100% and 88%, respectively, for confirmed tuberculous effusions. When the two tests were combined (either/or positive), the sensitivity increased (90-100%) at the expense of specificity. When both the tests were positive, then the specificity markedly increased (92-96%), but sensitivity of the tests decreased. CONCLUSION: Fluid PCR alone should not be relied on as a single test; rather, combined analysis with either ADA or ALC could be more useful in the diagnosis of tuberculous effusions in children.     

Pre-eclampsia is associated with sleep-disordered breathing and endothelial dysfunction.

Pre-eclamptic toxaemia (PET) may be associated with both endothelial dysfunction (ED) and sleep-disordered breathing (SDB). It was hypothesised that females with PET would demonstrate both SDB and ED, and that a correlation between these two would suggest a potential causative association. A total of 17 females with PET and 25 matched females with uncomplicated pregnancy were studied. They underwent a nocturnal ambulatory sleep study (using Watch_PAT100) and noninvasive evaluation of endothelial function utilising the reactive hyperaemia test (using Endo_PAT 2000). A higher ratio of post- to pre-occlusion pulse-wave amplitude (endothelial function index (EFI)) indicated better endothelial function. Females with PET had a significantly higher respiratory disturbance index (RDI) and lower EFI than controls (18.4+/-8.4 versus 8.3+/-1.3.h(-1), and 1.5+/-0.1 versus 1.8+/-0.1, respectively). Blood pressure significantly correlated with RDI and with EFI. EFI tended to correlate with RDI. In conclusion, these results suggest that both sleep-disordered breathing and endothelial dysfunction are more likely to occur in females with pre-eclamptic toxaemia than in females with uncomplicated pregnancies. The current authors speculate that respiratory disturbances contribute to the functional abnormality of the blood vessels seen in females with pre-eclamptic toxaemia, although causality cannot be determined based on this study.