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1.  Treatment of Diabetes and Long-Term Survival After Insulin and Glucokinase Gene Therapy 
Diabetes  2013;62(5):1718-1729.
Diabetes is associated with severe secondary complications, largely caused by poor glycemic control. Treatment with exogenous insulin fails to prevent these complications completely, leading to significant morbidity and mortality. We previously demonstrated that it is possible to generate a “glucose sensor” in skeletal muscle through coexpression of glucokinase and insulin, increasing glucose uptake and correcting hyperglycemia in diabetic mice. Here, we demonstrate long-term efficacy of this approach in a large animal model of diabetes. A one-time intramuscular administration of adeno-associated viral vectors of serotype 1 encoding for glucokinase and insulin in diabetic dogs resulted in normalization of fasting glycemia, accelerated disposal of glucose after oral challenge, and no episodes of hypoglycemia during exercise for >4 years after gene transfer. This was associated with recovery of body weight, reduced glycosylated plasma proteins levels, and long-term survival without secondary complications. Conversely, exogenous insulin or gene transfer for insulin or glucokinase alone failed to achieve complete correction of diabetes, indicating that the synergistic action of insulin and glucokinase is needed for full therapeutic effect. This study provides the first proof-of-concept in a large animal model for a gene transfer approach to treat diabetes.
PMCID: PMC3636629  PMID: 23378612
2.  Nonviral-Mediated Hepatic Expression of IGF-I Increases Treg Levels and Suppresses Autoimmune Diabetes in Mice 
Diabetes  2013;62(2):551-560.
In type 1 diabetes, loss of tolerance to β-cell antigens results in T-cell–dependent autoimmune destruction of β cells. The abrogation of autoreactive T-cell responses is a prerequisite to achieve long-lasting correction of the disease. The liver has unique immunomodulatory properties and hepatic gene transfer results in tolerance induction and suppression of autoimmune diseases, in part by regulatory T-cell (Treg) activation. Hence, the liver could be manipulated to treat or prevent diabetes onset through expression of key genes. IGF-I may be an immunomodulatory candidate because it prevents autoimmune diabetes when expressed in β cells or subcutaneously injected. Here, we demonstrate that transient, plasmid-derived IGF-I expression in mouse liver suppressed autoimmune diabetes progression. Suppression was associated with decreased islet inflammation and β-cell apoptosis, increased β-cell replication, and normalized β-cell mass. Permanent protection depended on exogenous IGF-I expression in liver nonparenchymal cells and was associated with increased percentage of intrapancreatic Tregs. Importantly, Treg depletion completely abolished IGF-I-mediated protection confirming the therapeutic potential of these cells in autoimmune diabetes. This study demonstrates that a nonviral gene therapy combining the immunological properties of the liver and IGF-I could be beneficial in the treatment of the disease.
PMCID: PMC3554392  PMID: 23099863
3.  Macrophage Plasticity and the Role of Inflammation in Skeletal Muscle Repair 
Mediators of Inflammation  2013;2013:491497.
Effective repair of damaged tissues and organs requires the coordinated action of several cell types, including infiltrating inflammatory cells and resident cells. Recent findings have uncovered a central role for macrophages in the repair of skeletal muscle after acute damage. If damage persists, as in skeletal muscle pathologies such as Duchenne muscular dystrophy (DMD), macrophage infiltration perpetuates and leads to progressive fibrosis, thus exacerbating disease severity. Here we discuss how dynamic changes in macrophage populations and activation states in the damaged muscle tissue contribute to its efficient regeneration. We describe how ordered changes in macrophage polarization, from M1 to M2 subtypes, can differently affect muscle stem cell (satellite cell) functions. Finally, we also highlight some of the new mechanisms underlying macrophage plasticity and briefly discuss the emerging implications of lymphocytes and other inflammatory cell types in normal versus pathological muscle repair.
PMCID: PMC3572642  PMID: 23509419
4.  Duodenal rupture secondary to blunt trauma from a football 
Journal of Surgical Case Reports  2013;2013(1):rjs041.
Duodenal rupture secondary to blunt trauma is a relatively uncommon event and is usually a result of a road traffic accident. As the duodenum is a retroperitoneal organ, delays in diagnosis can occur, as the patient may present with vague abdominal symptoms and other non-specific signs. Computed tomographic scanning is therefore a useful tool in the diagnosis of this condition. We present a 19-year-old girl who was hit in the abdomen with a football and subsequently had a duodenal rupture.
PMCID: PMC3578668  PMID: 24963935
5.  Notch3 and Hey-1 as Prognostic Biomarkers in Pancreatic Adenocarcinoma 
PLoS ONE  2012;7(12):e51119.
In order to achieve a better outcome for pancreatic cancer patients, reliable biomarkers are required which allow for improved diagnosis. These may emanate from a more detailed molecular understanding of the aggressive nature of this disease. Having previously reported that Notch3 activation appeared to be associated with more aggressive disease, we have now examined components of this pathway (Notch1, Notch3, Notch4, HES-1, HEY-1) in more detail in resectable (n = 42) and non-resectable (n = 50) tumours compared to uninvolved pancreas. All three Notch family members were significantly elevated in tumour tissue, compared to uninvolved pancreas, with expression maintained within matched lymph node metastases. Furthermore, significantly higher nuclear expression of Notch1, -3 and -4, HES-1, and HEY-1 (all p≤0.001) was noted in locally advanced and metastatic tumours compared to resectable cancers. In survival analyses, nuclear Notch3 and HEY-1 expression were significantly associated with reduced overall and disease-free survival following tumour resection with curative intent, with nuclear HEY-1 maintaining independent prognostic significance for both outcomes on multivariate analysis. These data further support a central role for Notch signalling in pancreatic cancer and suggest that nuclear expression of Notch3 and its target gene, HEY-1, merit validation in biomarker panels for diagnosis, prognosis and treatment efficacy. A peptide fragment of Notch3 was detected in plasma from patients with inoperable pancreatic cancer, but due to wide inter-individual variation, mean levels were not significantly different compared to age-matched controls.
PMCID: PMC3514220  PMID: 23226563
6.  Vestigial-like-2b (VITO-1b) and Tead-3a (Tef-5a) expression in zebrafish skeletal muscle, brain and notochord 
Gene expression patterns : GEP  2007;7(8):827-836.
The vestigial gene has been shown to control skeletal muscle formation in Drosophila and the related Vestigial-like 2 (Vgl-2) protein plays a similar role in mice. Vgl-family proteins are thought to regulate tissue-specific gene expression by binding to members of the broadly expressed Scalloped/Tef/TEAD transcription factor family. Zebrafish have at least four Vgl genes, including two Vgl-2s, and at least three TEAD genes, including two Tead3s. We describe the cloning and expression of one member from each family in the zebrafish. A novel gene, vgl-2b, with closest homology to mouse and human vgl-2, is expressed transiently in nascent notochord and in muscle fibres as they undergo terminal differentiation during somitogenesis. Muscle cells also express a TEAD-3 homologue, a possible partner of Vgl-2b, during myoblast differentiation and early fibre assembly. Tead3a is also expressed in rhombomeres, eye and epiphysis regions.
PMCID: PMC3360971  PMID: 17916448
muscle; adaxial; zebrafish; vestigial-like; transcription enhancer factor; TEAD domain
7.  Improving accuracy of medication identification in an older population using a medication bottle color symbol label system 
BMC Family Practice  2011;12:142.
The purpose of this pilot study was to evaluate and refine an adjuvant system of color-specific symbols that are added to medication bottles and to assess whether this system would increase the ability of patients 65 years of age or older in matching their medication to the indication for which it was prescribed.
This study was conducted in two phases, consisting of three focus groups of patients from a family medicine clinic (n = 25) and a pre-post medication identification test in a second group of patient participants (n = 100). Results of focus group discussions were used to refine the medication label symbols according to themes and messages identified through qualitative triangulation mechanisms and data analysis techniques. A pre-post medication identification test was conducted in the second phase of the study to assess differences between standard labeling alone and the addition of the refined color-specific symbols. The pre-post test examined the impact of the added labels on participants' ability to accurately match their medication to the indication for which it was prescribed when placed in front of participants and then at a distance of two feet.
Participants appreciated the addition of a visual aid on existing medication labels because it would not be necessary to learn a completely new system of labeling, and generally found the colors and symbols used in the proposed labeling system easy to understand and relevant. Concerns were raised about space constraints on medication bottles, having too much information on the bottle, and having to remember what the colors meant. Symbols and colors were modified if they were found unclear or inappropriate by focus group participants. Pre-post medication identification test results in a second set of participants demonstrated that the addition of the symbol label significantly improved the ability of participants to match their medication to the appropriate medical indication at a distance of two feet (p < 0.001) and approached significant improvement when placed directly in front of participants (p = 0.07).
The proposed medication symbol label system provides a promising adjunct to national efforts in addressing the issue of medication misuse in the home through the improvement of medication labeling. Further research is necessary to determine the effectiveness of the labeling system in real-world settings.
PMCID: PMC3282670  PMID: 22206490
Medication labeling; medication errors; medication adherence
8.  Laparoscopic splenectomy: a personal series of 140 consecutive cases 
Laparoscopic splenectomy has emerged as a safe and effective treatment for a variety of haematological conditions. The objective was to review the results from a large personal series from the perspective of outcomes according to operative time, conversion to open operation, complications and mortality. The application of laparoscopic splenectomy to cases of splenomegaly without hand assistance is examined.
Patients and Methods
A retrospective review of 140 patients undergoing laparoscopic splenectomy at a single university hospital by one surgeon during 1994-2006. Case notes were reviewed and data collected on operative time, conversion to open procedure, morbidity and mortality. Particular reference was made towards the results of cases of splenomegaly.
In total 140 laparoscopic splenectomies were performed with a complication rate of 15% and no mortality. The median operative time was 100 min and conversion to open procedure was necessary in 2.1%. Conversion for cases of splenomegaly was only 5.7%. The median hospital stay was 3 days.
Laparoscopic splenectomy is a safe procedure with acceptable morbidity. A laparoscopic approach for splenomegaly is feasible.
PMCID: PMC3180312  PMID: 20487598
Laparoscopic splenectomy; Splenomegaly; Idiopathic thrombocytopenic purpura; Complications
9.  Aberrant repair and fibrosis development in skeletal muscle 
Skeletal Muscle  2011;1:21.
The repair process of damaged tissue involves the coordinated activities of several cell types in response to local and systemic signals. Following acute tissue injury, infiltrating inflammatory cells and resident stem cells orchestrate their activities to restore tissue homeostasis. However, during chronic tissue damage, such as in muscular dystrophies, the inflammatory-cell infiltration and fibroblast activation persists, while the reparative capacity of stem cells (satellite cells) is attenuated. Abnormal dystrophic muscle repair and its end stage, fibrosis, represent the final common pathway of virtually all chronic neurodegenerative muscular diseases. As our understanding of the pathogenesis of muscle fibrosis has progressed, it has become evident that the muscle provides a useful model for the regulation of tissue repair by the local microenvironment, showing interplay among muscle-specific stem cells, inflammatory cells, fibroblasts and extracellular matrix components of the mammalian wound-healing response. This article reviews the emerging findings of the mechanisms that underlie normal versus aberrant muscle-tissue repair.
PMCID: PMC3156644  PMID: 21798099
10.  A systematic review of tests for lymph node status in primary endometrial cancer 
BMC Women's Health  2008;8:8.
The lymph node status of a patient is a key determinate in staging, prognosis and adjuvant treatment of endometrial cancer. Despite this, the potential additional morbidity associated with lymphadenectomy makes its role controversial. This study systematically reviews the accuracy literature on sentinel node biopsy; ultra sound scanning, magnetic resonance imaging (MRI) and computer tomography (CT) for determining lymph node status in endometrial cancer.
Relevant articles were identified form MEDLINE (1966–2006), EMBASE (1980–2006), MEDION, the Cochrane library, hand searching of reference lists from primary articles and reviews, conference abstracts and contact with experts in the field. The review included 18 relevant primary studies (693 women). Data was extracted for study characteristics and quality. Bivariate random-effect model meta-analysis was used to estimate diagnostic accuracy of the various index tests.
MRI (pooled positive LR 26.7, 95% CI 10.6 – 67.6 and negative LR 0.29 95% CI 0.17 – 0.49) and successful sentinel node biopsy (pooled positive LR 18.9 95% CI 6.7 – 53.2 and negative LR 0.22, 95% CI 0.1 – 0.48) were the most accurate tests. CT was not as accurate a test (pooled positive LR 3.8, 95% CI 2.0 – 7.3 and negative LR of 0.62, 95% CI 0.45 – 0.86. There was only one study that reported the use of ultrasound scanning.
MRI and sentinel node biopsy have shown similar diagnostic accuracy in confirming lymph node status among women with primary endometrial cancer than CT scanning, although the comparisons made are indirect and hence subject to bias. MRI should be used in preference, in light of the ASTEC trial, because of its non invasive nature.
PMCID: PMC2409306  PMID: 18457596
11.  Diagnostic accuracy of tests for lymph node status in primary cervical cancer: a systematic review and meta-analysis 
Lymph node status is the key to determining the prognosis and treatment of cervical cancer. However, it cannot be assessed clinically, and testing for nodal metastasis is controversial. We sought to systematically review the diagnostic accuracy literature on sentinel node biopsy, positron emission tomography, magnetic resonance imaging and computed tomography to evaluate the accuracy of each index test in determining lymph node status in patients with cervical cancer.
We searched MEDLINE (1966–2006), EMBASE (1980–2006), Medion (1980–2006) and the Cochrane library (Issue 2, 2006) for relevant articles. We also manually searched the reference lists from primary articles and reviews, and we contacted experts in the field for conference abstracts and unpublished studies. We performed random-effects meta-analysis of accuracy indices, and we performed meta-regression analysis to test the effect of study quality on diagnostic accuracy and to identify other sources of heterogeneity.
We included 72 relevant primary studies, involving a total of 5042 women, in our analysis. We found that, in determining lymph node status, sentinel node biopsy had a pooled positive likelihood ratio of 40.8 (95% confidence interval [CI] 24.6–67.6) and a pooled negative likelihood ratio of 0.18 (95% CI 0.14–0.24). The pooled positive likelihood ratios (and 95% CI) were 15.3 (7.9–29.6) for positron emission tomography, 6.4 (4.9–8.3) for magnetic resonance imaging and 4.3 (3.0–6.2) for computed tomography. The pooled negative likelihood ratios (and 95% CIs) were 0.27 (0.11–0.66) for positron emission tomography, 0.50 (0.39–0.64) for magnetic resonance imaging and 0.58 (0.48–0.70) for computed tomography. Using a 27% pretest probability of lymph node metastasis among all cases (regardless of stage), we found that a positive sentinel node biopsy result increased post-test probability to 94% (95% CI 90%–96%), whereas a positive finding on positron emission tomography increased it to 85% (75%–92%).
Sentinel node biopsy has greater accuracy in determining lymph node status among women with primary cervical cancer than current commonly used imaging methods.
PMCID: PMC2267838  PMID: 18362381
12.  Movies of cellular and sub-cellular motion by digital holographic microscopy 
Many biological specimens, such as living cells and their intracellular components, often exhibit very little amplitude contrast, making it difficult for conventional bright field microscopes to distinguish them from their surroundings. To overcome this problem phase contrast techniques such as Zernike, Normarsky and dark-field microscopies have been developed to improve specimen visibility without chemically or physically altering them by the process of staining. These techniques have proven to be invaluable tools for studying living cells and furthering scientific understanding of fundamental cellular processes such as mitosis. However a drawback of these techniques is that direct quantitative phase imaging is not possible. Quantitative phase imaging is important because it enables determination of either the refractive index or optical thickness variations from the measured optical path length with sub-wavelength accuracy.
Digital holography is an emergent phase contrast technique that offers an excellent approach in obtaining both qualitative and quantitative phase information from the hologram. A CCD camera is used to record a hologram onto a computer and numerical methods are subsequently applied to reconstruct the hologram to enable direct access to both phase and amplitude information. Another attractive feature of digital holography is the ability to focus on multiple focal planes from a single hologram, emulating the focusing control of a conventional microscope.
A modified Mach-Zender off-axis setup in transmission is used to record and reconstruct a number of holographic amplitude and phase images of cellular and sub-cellular features.
Both cellular and sub-cellular features are imaged with sub-micron, diffraction-limited resolution. Movies of holographic amplitude and phase images of living microbes and cells are created from a series of holograms and reconstructed with numerically adjustable focus, so that the moving object can be accurately tracked with a reconstruction rate of 300ms for each hologram. The holographic movies show paramecium swimming among other microbes as well as displaying some of their intracellular processes. A time lapse movie is also shown for fibroblast cells in the process of migration.
Digital holography and movies of digital holography are seen to be useful new tools for visualization of dynamic processes in biological microscopy. Phase imaging digital holography is a promising technique in terms of the lack of coherent noise and the precision with which the optical thickness of a sample can be profiled, which can lead to images with an axial resolution of a few nanometres.
PMCID: PMC1448199  PMID: 16556319
13.  Analyses of the differentiation potential of satellite cells from myoD-/-, mdx, and PMP22 C22 mice 
Sporadic and sometimes contradictory studies have indicated changes in satellite cell behaviour associated with the progressive nature of human Duchenne muscular dystrophy (DMD). Satellite cell proliferation and number are reportedly altered in DMD and the mdx mouse model. We recently found that satellite cells in MSVski transgenic mice, a muscle hypertrophy model showing progressive muscle degeneration, display a severe ageing-related differentiation defect in vitro. We tested the hypothesis that similar changes contribute to the gradual loss of muscle function with age in mdx and PMP22 mice, a model of human motor and sensory neuropathy type 1A (HMSN1A).
Single extensor digitorum longus muscle fibres were cultured from mdx and PMP22 mice and age- and genetic background-matched controls. Mice at several ages were compared with regard to the differentiation of satellite cells, assayed as the proportion of desmin-expressing cells that accumulated sarcomeric myosin heavy chain.
Satellite cells of 2 month, 6 month, and 12 month old mdx mice were capable of differentiating to a similar extent to age-matched wild type control animals in an in vitro proliferation/differentiation model. Strikingly, differentiation efficiency in individual 6 month and 12 month old mdx animals varies to a much higher extent than in age-matched controls, younger mdx animals, or PMP22 mice. In contrast, differentiation of myoblasts from all myoD null mice assayed was severely impaired in this assay system. The defect in satellite cell differentiation that occurs in some mdx animals arises from a delay in differentiation that is not overcome by IGF-1 treatment at any phase of cultivation.
Overall, a defect in satellite cell differentiation above that arising through normal ageing does not occur in mdx or PMP22 mouse models of human disease. Nonetheless, the impaired differentiation of satellite cells from some mdx animals suggests that additional factors, environmental or epigenetic, may lead to deteriorating muscle repair through poor differentiation of satellite cells in genetically predisposed individuals.
PMCID: PMC1079863  PMID: 15762989
14.  Management guidelines for women with normal colposcopy after low grade cervical abnormalities: population study 
BMJ : British Medical Journal  2000;320(7251):1693-1696.
To develop an evidence based protocol for the follow up of women with low grade cervical abnormalities for whom treatment is not immediately indicated.
Population outcome study.
Colposcopy clinic of an inner city teaching hospital.
566 women with low grade cytological abnormalities who were not treated at a first visit to the colposcopy clinic, followed up for a total of 881 years.
Main outcome measures
Resolution of abnormalities, persistence of disease, and treated disease.
Abnormalities resolved in 306 (54.1%) women, whereas 138 (24.4%) had persistent disease and 122 (21.5%) were subsequently treated. Colposcopic opinion, smear test results, age, smoking history, and number of pregnancies were all significantly related to outcome. Logistic regression analysis produced a model that correctly identified 70% of women whose abnormalities resolved. Only 23 of 295 women (7.8%) with a normal cervix on colposcopy and a smear without dyskaryosis at a first visit were treated by the end of the observation period.
Women referred with low grade cytological abnormalities who have a normal cervix on colposcopy and a negative or borderline repeat smear test result may be discharged from the colposcopy clinic. We propose a follow up protocol that could safely avoid unnecessary visits to a clinic.
PMCID: PMC27411  PMID: 10864542

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