Background. Ectopic fat deposition in the pancreas and its relationshipwith hepatic steatosis and insulin resistance have not been compared between patients with nonalcoholic fatty liver disease (NAFLD) and healthy controls.Aim. Using a novel magnetic resonance imaging (MRI) based biomarker, the proton-density-fat-fraction (MRI-PDFF),we compared pancreatic fat content in patients with biopsy-proven NAFLD to healthy controls and determined whether it isassociated with insulin resistance and liver fat content. Methods. This nested case-control study was derived from two prospective studies including43 patients with biopsy-proven NAFLD and 49 healthy controls who underwent biochemical testing and MRI.Results. Compared to healthy controls, patients with NAFLD had significantly higher pancreatic MRI-PDFF , and these results remained consistent in multivariable-adjusted modelsincluding age, sex, body mass index, and diabetes . We found a strong correlation betweenhepatic and pancreatic MRI-PDFF . Participants with increasedinsulin resistance determined by homeostatic-model-of-insulin-resistance (HOMA-IR) greater than 2.5 had higherpancreatic  and liver  MRI-PDFF.Conclusion. Patients with NAFLD have greater pancreatic fat than normal controls. Insulin resistance is associated with liver and pancreatic fat accumulation. It is well established that obesity, insulin resistance, and other components of metabolic syndrome play a role in the development and progression of nonalcoholic fatty liver disease (NAFLD) –3. Due t Similar to fat accumulation in the liver, obesity and metabolic syndrome result in ectopic fat deposition in other organ systems including skeletal muscles, the heart, and the pancreas. In the setting of metabolic syndrome, fat accumulation in the pancreas may lead to a similar process of inflammation, or “nonalcoholic steatopancreatitis” . Recent Although questions remain regarding the clinical implications of fat accumulation in the pancreas, fatty pancreas may become an increasingly relevant condition with the rising prevalence of NAFLD. Few studies have evaluated the relationship between hepatic steatosis and fat accumulation in the pancreas. A recent study by our group revealed that amongst patients with biopsy-proven NAFLD, histology-determined steatohepatitis and liver steatosis were associated with higher levels of magnetic resonance imaging (MRI) estimated fat content in the pancreas . This stIn this study we aim to determine whether pancreatic MRI-PDFF is greater in patients with NAFLD than healthy controls. We also explore the relationship between components of metabolic syndrome including insulin resistance and pancreatic fat content. These insights may help us understand whether pancreatic steatosis is a marker of metabolic syndrome and liver steatosis and whether it is a risk factor for progression of NAFLD.http://fattyliver.ucsd.edu/). 43 consecutive adult participants with biopsy-proven NAFLD and 49 consecutive health control participants underwent clinical research evaluation, physical examination, biochemical testing, and detailed MRI phenotyping. All patients with NAFLD were diagnosed by liver biopsy as well as exclusion of other causes of liver disease (as detailed in the following section). All patients provided written informed consent to participate in the study and the study was approved by the University of California San Diego Institutional Review Board. All patients underwent a standard history and physical exam, biochemical testing, and MRI examination at UCSD. They also all underwent an alcohol history assessment by completing the AUDIT and Skinner Lifetime Drinking questionnaires.This is a nested case-control study derived from two prospective studies of healthy individuals and patients with biopsy-proven NAFLD at the UCSD NAFLD Research Unit (Definition of NAFLD. (1) Fat accumulation in the liver (steatosis) involves at least 5% of hepatocytes on routine stains. (2) No evidence of other acute or chronic liver disease was found. (3) There was absence of regular or excessive use of alcohol within 2 years prior to entry.Inclusion criteria in the NAFLD cohort included (1) age greater than 18 years; (2) evidence of NAFLD (as described above) on liver biopsy as assessed by the NASH-CRN histologic scoring system [Inclusion criteria in the healthy (non-NAFLD) control group included (1) age greater than 18 years and (2) liver MRI-PDFF <5%. A threshold of 5% is consistent with magnetic resonance spectroscopy (MRS) determined NAFLD . We haveAfter meeting inclusion and exclusion criteria, patients underwent a routine history and physical exam in a research clinic. Body weight, height, and vital sign measurements were obtained and standard blood testing was performed, including measurement of ALT, AST, alkaline phosphatase, gamma-glutamyl transpeptidase (GGT), total bilirubin, direct bilirubin, albumin, fasting glucose and insulin, hemoglobin A1c (HbA1c), lipid panel, free fatty acids (FFA), and C-reactive protein (CRP). Homeostatic-model-of-insulin-resistance (HOMA-IR) was calculated as the product of fasting insulin and glucose divided by a correction factor of 405.In order to quantify pancreas and liver fat content, we used a previously described advanced chemical shift based gradient-echo MRI technique that estimates PDFF, which is a standardized and objective measure of fat content , 23, 24.2 to 400 mm2 in area were placed in each of the nine liver segments on the PDFF parametric maps. Similarly, pancreatic PDFF was measured by placing 1 to 2 ROIs of 100 mm2 each in the head, body, and tail of the pancreas in each slice of the PDFF parametric maps. These protocols have been described in prior studies [In order to estimate PDFF across the entire liver, 3 regions of interest (ROIs) of 300 mm studies , 20, 24.A single resident physician who was trained in this method of MRI analysis performed the measurements. The physician was blinded to clinical and histological data and was under the supervision of the radiology investigator (CS). These findings were cross-validated by an independent radiology investigator who was blinded to the prior pancreatic and liver fat fraction maps.t-test was used for comparison of continuous variables between the NAFLD and control groups, while the chi-square test was used for comparisons of categorical variables. A multivariable-adjusted linear regression model was used to compare liver MRI-PDFF and pancreatic MRI-PDFF between groups after adjusting for differences in age, sex, body-mass index (BMI), and diabetes between the groups. A Spearman correlation was performed to compare MRI-PDFF of the liver and pancreas amongst all patients. Sample size estimation: we hypothesized that MRI-PDFF of pancreas would positively correlate with MRI-PDFF of liver, and therefore, pancreatic fat content would be higher in participants with NAFLD versus normal controls. We would need a sample size of at least 40 to have an alpha of 0.05 with a power of 80% (or higher) requiring an effect size of 0.38 or higher. All the statistical analysis was performed using Excel and SPSS software packages . In all analyses, P-value <0.05 was considered statistically significant.The two-tailed P-value <0.001). As expected, NAFLD patients had a higher BMI (in kg/m2) than controls . Other metabolic parameters are also provided in Forty-three patients with biopsy confirmed NAFLD and 49 healthy controls were enrolled in this study between 1/2010 and 3/2013. Demographic and biochemical data for these patients are shown in P-value <0.001) as shown in P-value =0.03). As expected, MRI-estimated liver fat content was significantly greater in patients with NAFLD than healthy controls . MRI-PDFF of the liver had a significant correlation with MRI-PDFF of the pancreas  as shown in MRI-estimated pancreatic fat content was significantly greater in patients with NAFLD than healthy controls  and liver fat  than those below the median HOMA-IR (please see Insulin resistance determined by HOMA-IR varied from 0.4 to 63.0 (median = 2.5) amongst all patients. Participants with increased insulin resistance determined by HOMA-IR greater than 2.5 (above the median HOMA-IR) had higher MRI-PDFF estimated pancreatic fat  for comparison with participants who have NAFLD. Most prior noninvasive studies of pancreatic fat reviewed cohorts of obese or healthy individuals without known NAFLD. In addition, this study utilized an MRI technique that has been well validated to measure fat content in the liver and has been used previously to measure fat content in the pancreas. Although this was a nested case control study in which individual patients were not matched with healthy controls, the use of multivariate statistics allowed analysis of liver and pancreatic fat content between the two groups independent of differences noted between these groups. Despite this, we do acknowledge limitations of this study. As this is not a longitudinal study, we are unable to assess whether pancreatic fat affects progression of NAFLD. In addition, although the MRI technique used in this study has been well validated in the liver, it has not been validated in analysis of pancreatic tissue. Finally, only NAFLD patients had histologic analysis, precluding the ability to compare histology in NAFLD patients to healthy controls in this study. Due to the risks of liver biopsy, it was not ethically possible to pursue a liver biopsy on healthy control participants.Additional studies are needed in this area to further characterize the relationship between pancreatic and liver fat. Longitudinal studies should focus on the effect of pancreatic fat on progression of NAFLD and development of NASH and/or worsening of insulin resistance or fibrogenesis in the pancreas itself. In addition, the development of fatty pancreas independent of NAFLD should be evaluated as a biomarker of metabolic syndrome and a risk factor for the development and progression of NAFLD. Patients with biopsy-proven NAFLD have higher pancreatic fat content than healthy controls. In addition, there is a significant correlation between MRI-estimated pancreatic and liver fat content amongst patients with NAFLD and healthy controls. Increased insulin resistance determined by HOMA-IR is associated with increased liver and pancreatic fat content. Future studies are needed to determine the effect of pancreatic fat on development and progression of NAFLD.


Regular and prolonged exercise is associated with increased left ventricular wall thickness that can overlap with hypertrophic cardiomyopathy (HCM). Differentiating physiological from pathological hypertrophy has important implications, since HCM is the commonest cause of exercise-related sudden cardiac death in young individuals. Most deaths have been reported in intermittent 'start-stop' sports such as football (soccer) and basketball. The theory is that individuals with HCM are unable to augment stroke volume sufficiently to meet the demands of endurance sports and are accordingly 'selected-out' of participation in such events. We report the case of an ultra-endurance athlete with 25 years of > 50 km competitive running experience, with genetically confirmed HCM; thereby demonstrating that these can be two compatible entities. Regular physical exercise is associated with physiological increases in cardiac dimensions which may be reflected on the electrocardiogram (ECG). Differentiating a physiological or pathological remodelling mechanism is important, as significant cardiac enlargement may be an expression of underlying cardiac disease, placing the athlete at a greater risk of sudden cardiac death (SCD) . ApproxiDeaths from HCM are predominantly confined to intermittent 'start-stop' sports such as American football, basketball and soccer, with few cases reported in endurance sports. The postulated theory is that individuals with HCM are unable to augment cardiac output sufficiently to participate in intensive and prolonged endurance sports due to a combination of pronounced LVH, a non-compliant LV, exercise-induced LV outflow obstruction and microvascular ischemia. However, we report an ultra-endurance athlete with confirmed HCM capable of performing high-levels of aerobic ultra-endurance activity.A 44 year-old Caucasian male was evaluated in our centre for investigation of a cardiac murmur identified by his primary care physician. The individual was asymptomatic with no past medical history, medication history or family history. He was an ultra-marathon runner with over 25 years of competitive running history; currently participating in 3 ultra-marathon (> 50 km) events per year often involving challenging mountainous and frozen terrain. Resting blood pressure of 95/60 mmHg and physical examination was unremarkable apart from the presence of a soft ejection systolic murmur.The ECG demonstrated first-degree heart block, right axis deviation, voltage criteria for bi-atrial enlargement, LVH and significant repolarisation anomalies including ST-segment depression in leads II, III and AVF, and deep T-wave inversions in leads V5 and V6 Figure . Echocar-1/min-1. Heart rate (91% predicted maximum) and BP response (systolic BP rising from 98 mmHg to 168 mmHg at peak exertion) to exercise was normal and there was no evidence of cardiac dysrhythmias on exercise or on the 24-hour Holter monitor.Subsequent investigations included an exercise stress test with the athlete completing 21 minutes of the Bruce protocol (19.1 METS) corresponding to an oxygen consumption of 67 ml/kgThe abnormal resting ECG, asymmetric septal hypertrophy and non-dilated LV cavity raised suspicion of HCM. However, the normal indices of diastolic function and supra-normal functional capacity favoured 'athlete's heart'. Consequently, CMR was performed using standardised imaging protocols . AnalysiAccordingly, first-degree relatives were invited to our centre for cardiovascular screening, which demonstrated an abnormal ECG and echocardiogram consistent with HCM in the mother and sister of the index case. Genetic testing for known mutations encoding sarcomeric contractile proteins proved positive for a mutation in the MYBPC3 gene encoding myosin binding protein C  in MYBPC3 exon 22) in all three individuals.Previous cases of endurance athletes with HCM have been reported , howeverThe ECG presented here has numerous features compatible with cardiac adaptation to exercise, including voltage criteria for LVH, ST-segment depression, a prolonged PR interval and inverted T-waves ,12-17. HWhilst echocardiography demonstrated a LVWT is 14 mm at the septum, it is well established that a minority of Caucasian athletes (< 2%) also demonstrate physiological LVH between 13-16 mm ,8,18. HoDue to the abnormal CMR and the abnormal cardiovascular evaluation of first-degree relatives, familial disease was presumed and genetic testing confirmed a diagnosis of HCM. Variable expression of the disease is common, even amongst members of the same family sharing the same gene defect. Mutations in more than 13 genes encoding sarcomeric contractile proteins have been identified as a cause of HCM . Importa<0.05), compared with veteran controls. We also observed the presence of myocardial fibrosis in 6 (50%) of the veteran athletes, but no LGE in the age-matched veteran controls or young athletes. Importantly, the prevalence of LGE in veteran athletes was significantly associated with the number of years spent training (p < 0.001), number of competitive marathons (p < 0.001) and ultra-endurance (> 50 miles) marathons (p < 0.007) completed, suggesting a link between life-long endurance exercise and 'acquired' myocardial fibrosis that requires further investigation. However, the extent of LVH and fibrosis observed with these life-long veteran athletes was not as extensive as the athlete presented here, thus pointing towards a pathological mechanism.Recently, our laboratory recently examined the cardiac structure and function of a unique cohort of 12 asymptomatic truly life-long, competitive veteran endurance athletes (56 ± 6 yr), with 20 age-matched veteran controls (60 ± 5 yr) and 17 younger male endurance athletes (31 ± 5 yr) using LGE CMR . VeteranPre-participation screening data from Italy, incorporating the 12-lead ECG, suggests that the incidence of sudden death from HCM may be reduced through earlier identification and subsequent disqualification of affected athletes from competitive sport . GuideliSporting disqualification from all high-intensity ultra-endurance activity was discussed with the athlete together with ICD insertion. Personal and family genetic counselling together with clinical symptom education was given to the athlete regarding the risks of ultra-endurance exercise and the potential for SCD. The athlete and family members agreed for genetic testing, but the athlete refused an ICD. Accordingly, the athlete is required to undergo a comprehensive yearly cardiovascular examination. However, the athlete continues to compete in ultra-endurance running events despite knowing the risks posed by continued high intensity competition; and with 2 years of follow-up data he remains asymptomatic without any significant cardiac changes.This case study reports an asymptomatic male athlete with 25 years of ultra-endurance competition, with genetically confirmed HCM phenotypically manifesting with LVH, a small LV cavity together with repolarisation abnormalities suggestive of HCM. Despite documented asymmetric hypertrophy and focal myocardial fibrosis in the basal anteroseptal and inferoseptal walls, it is suspected that the athlete is able to run ultra-marathons due to a compliant LV with normal diastolic and systolic parameters, which is able to augment stroke volume. In conclusion, rare as they might be, a minority of HCM patients are capable of life-long careers in ultra-endurance exercise. This case also highlights the importance of systematic evaluation of all athletes with electrocardiographic features suggestive of a cardiomyopathy or ion channelopathy, with LGE CMR Figure , maximalWritten informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.HCM: Hypertrophic cardiomyopathy; SCD: Sudden cardiac death; LV: Left ventricle; LVH: Left ventricular hypertrophy; LVWT: Left ventricular wall thickness; CMR: Cardiac magnetic resonance.The authors declare that they have no competing interests.MGW, NC and SS designed the case, MGW, NC, MP, ROH and SS collected and analyzed the data, MGW and NC wrote the preliminary draft of the manuscript and all authors supplied comments and corrections, SS is the guarantor. All authors read and approved the final manuscript.Authors NC and MP are funded by a research grant from the charitable organisation 'Cardiac Risk in the Young' . SKP was supported by the NIHR Cardiovascular Biomedical Research Unit of the Royal Brompton and Harefield NHS Foundation Trust and Imperial College.


We present a case report of secondary vaginal cancer after complete hysterectomy due to myoma that was complicated by low-grade cervical intraepithelial neoplasia (CIN I) and human papilloma virus (HPV) infection. After complete hysterectomy, the HPV DNA level in the vaginal tissue was obviously increased, and vaginal cancer was diagnosed 6 months later. We conclude that HPV infection can cause vaginal cancer after complete hysterectomy in cases complicated by CIN. Therefore, HPV should be regularly assessed during the postoperative follow-up period. HPV viral subtypes can also cause other cancers, including vulval, vaginal, anal, penile and oropharyngeal carcinomas.2 Women over 60 years of age who have CIN lesions are at an elevated risk, and should be followed closely. Follow-up should continue even after a hysterectomy.3 Here, we present a case report of secondary vaginal cancer after complete hysterectomy due to myoma, which was complicated by low-grade cervical intraepithelial neoplasia (CIN I) and human papilloma virus (HPV) infection. A review of the relevant literature is presented to increase awareness of the importance of monitoring HPV infection.The incidence of vaginal intraepithelial neoplasia (VAIN) and vaginal cancer is very low, at 0.2-0.3 and 0.42 per 100,000, respectively, in the USA.A 62-year-old para 2, gravida 2 married patient was admitted to our unit due to intermittent vaginal bleeding over the preceding year. Four months prior to admission, she had undergone a thin-prep cytological test (TCT) which revealed low-grade squamous intraepithelial lesions (LSILs) of the cervix . Her hybThe patient was found to have uterine fibroids, and these were treated surgically. After anesthesia was instituted, a vaginal conventional hysterectomy was performed. Histopathological examination of the resected uterus revealed uterine fibroids, endometrial atrophy, chronic cervical inflammation, squamous cell hyperplasia, localized low-grade CIN I, and interstitial gland tumor-like hyperplasia . The shoThe patient was reviewed in clinic 6 months post-operatively. A vaginal cytology test by the TCT method showed dyskaryosis, with grade III high-grade squamous vaginal intraepithelial neoplasia . The res2 Brinton et al found that low education, low income, previous abnormal Pap smears, genital warts and a history of hysterectomy were important risk factors for VAIN and vaginal cancer; other risk factors included a current or past history of CIN, immunosuppression, previous radiotherapy and exposure to diethylstilbestrol.4VAIN and vaginal cancers are rare diseases with unclear epidemiologies. Conclusive evidence has demonstrated that a large number of cervical cancers occur due to infection with HPV.5 The outcomes of conservative treatment with cervical cone excision and total hysterectomy are similar. Therefore, to reduce the increased risk of injury associated with more extensive surgery, a hysterectomy for CIN II or CIN III is not usually indicated.5 We report the case of an elderly women with persistent HPV infection and CIN I who presented with vaginal bleeding secondary to uterine fibroids. She accepted the surgical intervention of hysterectomy with the aim of preventing future cervical cancer. Due to the patient’s age, and the high HPV viral load, we investigated her postoperatively with testing of vaginal exfoliated cells for HC2-HPV DNA. Vaginal cancer was identified after six months; with timely treatment, and her condition was well-controlled. Therefore, we advocate that patients with CIN, including those who have undergone hysterectomy, should undergo cervical conization and routine HC2-HPV DNA testing.A history of hysterectomy for CIN is a known risk factor for secondary VAIN; the incidence of VAIN ranges between 0.9%–6.8% after hysterectomy for CIN.5 recommended that cervical and vaginal colposcopy should be performed prior to hysterectomy due to CIN. The histology of exfoliated vaginal wall cells should be followed-up postoperatively for at least 18 months with samples taken every six months. If the histology at 18 months is negative for carcinoma, then follow-up can be discontinued.6 After treatment, colposcopy should be re-examined each year for up to 4 years after surgery. Patients with CIN I should be followed with annual colposcopy inspections for 2 years while a 10 year follow-up is recommended for patients with CIN II-III. This policy of post-treatment cytological surveillance ensures early detection of treatment failure and repeat conservative treatment as necessary for those who have a preserved uterus with cervical conization. However, some scholars believe that vaginal cancer does not develop after the hysterectomy, but the primary cancer in the vagina was already present prior to surgery. Therefore, a full examination should be conducted prior to the hysterectomy to prevent a missed diagnosis. Schockaert et al2 HC2-HPV-DNA testing is crucial for the successful overall outcome of conservative management of cervical CIN or in those who undergo total hysterectomy. Therefore, for resections due to CIN by cervical cone or hysterectomy patients, follow-up should include the HC2-HPV-DNA test and TCT, with HC2-HPV-DNA testing given higher priority. If the HC2-HPV-DNA test is positive, she should undergo cytology and, if abnormal cytology is present, colposcopy can then assist with the early detection of disease recurrence. If colposcopy is negative for HC2-HPV-DNA on the initial examination, repeated HC2-HPV-DNA testing and cytology inspection at 12 months will be adequate to discern disease recurrence. We believe that persistent HPV infection is the most salient risk factor for the recurrence of cervical CIN, from which VAIN and vaginal cancer can develop. The decision to commence antiviral therapy and the choice of therapy are therefore important questions that require accurate diagnostic tools and follow-up.In conclusion, HPV infection is the cause of cervical cancer, vaginal cancer, vulvar cancer, VAIN and other diseases. HPV testing has a powerful negative predictive value; if patients are followed-up for 6 months, a negative HC2-HPV-DNA test indicates that the risk of CIN recurrence is very small.


The development of efficient gene delivery methods is essential if MSCs are to be of benefit using such an approach. Achieving high levels of transgene expression for the required period of time, without adversely affecting cell viability and differentiation capacity, is crucial. In the present study, we investigate lentiviral vector-mediated genetic modification of rat bone-marrow derived MSCs and examine any functional effect of such genetic modification in an in vivo condition of ischaemia was modelled in vitro by separation into its associated constituent parts i.e. hypoxia, serum and glucose deprivation, in which the effect of therapeutic gene over-expression on MSC survival was investigated.Transduction efficiency and transgene persistence of second and third generation rHIV-1 based lentiviral vectors were tested using reporter gene constructs. Use of the rHIV-pWPT-EF1-α-GFP-W vector was optimised in terms of dose, toxicity, cell species, and storage. The The second generation lentiviral vector rHIV-pWPT-EF1-α-GFP-W, was the most efficient and provided the most durable transgene expression of the vectors tested. Transduction with this vector did not adversely affect MSC morphology, viability or differentiation potential, and transgene expression levels were unaffected by cryopreservation of transduced cells. Over-expression of HSP70 resulted in enhanced MSC survival and increased resistance to apoptosis in conditions of hypoxia and ischaemia. MSC differentiation capacity was significantly reduced after oxygen deprivation, but was preserved with HSP70 over-expression.in vitro gene delivery to MSCs and suggest that lentiviral vector transduction can facilitate sustained therapeutic gene expression, providing an efficient tool for ex vivo MSC modification. Furthermore, lentiviral mediated over-expression of therapeutic genes in MSCs may provide protection in an ischaemic environment and enable MSCs to function in a regenerative manner, in part through maintaining the ability to differentiate. This finding may have considerable significance in improving the efficacy of MSC-based therapies.Collectively, these data validate the use of lentiviral vectors for efficient  Mesenchymal stem cells (MSCs) represent a significant area of interest in the field of cell therapy. They are a multipotent, self-renewing cell population isolated from bone marrow, capable of differentiation into cells of different lineages including chondrocytes, osteoblasts and adipocytes . The invin vivo environment into which they will be transplanted. Conditions of hypoxia, ischaemia and oxidative stress are associated with the infarct and its surrounding environment. In vitro studies have shown that, in the absence of oxygen, MSCs are capable of survival for a limited time [in vivo. In this respect, we hypothesize that increasing MSC survival capacity by genetic modification could afford the MSC a longer time in which to mediate repair and recovery both by direct and paracrine means, thereby enhancing their in vivo therapeutic value.Current cell-based therapeutic approaches for cardiovascular injury and disease are promising, although not without their shortcomings. MSC transplantation to the infarcted heart aims to promote repair and regeneration of the damaged cardiac tissue. Although several studies have proved somewhat successful , it may ted time . A more in vitro modification methods for rat MSCs. It was reported that lentiviral vectors demonstrated excellent transduction of rat MSCs in contrast to adenovirus, AAV, lipid transfection and electroporation [MSCs have been modified by various methods including lipid transfection and viral transduction. It is necessary to tailor the vector type chosen for MSC modification to the specific applications for which the cells are to be used. Lipid transfection of MSCs results in relatively low numbers of transgene expressing cells, approximately 30-40% maximum . Modificporation . Accuratex vivo stem cell modification.Lentiviral vectors have long since been established as having the ability to transduce a broad range of cell types efficiently, including non-dividing, senescent and terminally differentiated cells ,13. Lentin vitro model of ischaemia and its components, the main goal being to determine whether lentivirus vector-mediated over-expression of the pro-survival proteins catalase, SOD1, SOD3 and HSP70 can facilitate increased MSC survival.The present study aims to assess lentivirus vector-mediated genetic modification of MSCs, specifically transduction efficiency, durability of transgene expression and transgene or promoter "silencing" of three different lentiviral vector constructs. In addition, the effect of lentiviral vector transduction on MSC viability and differentiation capacity was examined. The effects of cell cryopreservation, cell passage number and species on transgene expression were also assessed. Further to establishing an optimal vector system for the modification of MSCs, a second objective of this study was to gain an understanding of how MSCs react to and influence post-ischaemic recovery, and to ascertain if this can be enhanced by induced therapeutic antioxidant or anti-apoptotic gene over-expression. This series of experiments included an evaluation of the survival and differentiation potential of MSCs in an All reagents utilised were from Sigma-Aldrich  unless otherwise stated.MluI and SalI restriction sites and lentiviral vectors expressing these transgenes were produced and vector titre calculated as described above.The present study compared second and third generation self-inactivating (SIN) human immunodeficiency virus-1-based (HIV-1), VSV-G pseudotyped lentiviral vectors. Tat-dependent second generation lentiviral vectors consisted of rHIV-pWPT-EF1-α-GFP-W and rHIV-cPPT-CMV-GFP-W. The associated second generation packaging system production plasmids were provided by Didier Trono . Vectors were produced by standard transient transfection of a three-plasmid system into producer cells. Briefly, packaging plasmid ps-PAX2.2 (Addgene plasmid 12260), envelope plasmid pMD2.G (Addgene plasmid 12259) and either pWPT-GFP (Addgene plasmid 12255) or cPPT-CMV-GFP expression plasmids were transfected into HEK293 cells using Jet PEI transfection reagent  according to manufacturer's instructions. Culture medium was replaced 16 h post-transfection. Vector-containing supernatants were collected 48 h and 72 h post-transfection, filtered, pooled and concentrated by ultracentrifugation at 27,000 × g for 3 h. Lentivirus vector titre was determined by a quantitative real-time PCR based method, which detected stably integrated virus sequences in target HeLa cells and was expressed as transducing units per ml (TU/ml). Tat-independent third generation lentivirus vector, PPT-PGK-GFP-WPRE, containing the WPRE element and polypurine tract with the GFP transgene under the control of the PGK promoter was produced by Genethon  using four-plasmid transfection of 293T cells. Human Catalase, HSP70, SOD1 and SOD3 genes were cloned into the lentiviral expression plasmid pWPT using 2 flasks at a density of 5.7 × 105 cells per cm2. After 5 days, non-adherent red blood cells were removed by a PBS wash and cells were re-fed with 30 ml of complete medium per flask. When MSC colonies had reached 80-90% confluence, cells were detached with 0.25% trypsin(w/v)/1 mM EDTA solution, re-plated at 5.7 × 103 cells per cm2 with subsequent subculture, usually at 4-6 day intervals.Marrow was obtained from donors after informed consent and all procedures were approved by the Clinical Research Ethics Committee at University College Hospital . Briefly, 30 ml heparin-treated bone marrow aspirates were acquired in separate 6 ml aliquots from the iliac crest of healthy donors. Aliquots were pooled, washed in PBS and cell pellets were resuspended in 10 ml of complete human MSC medium, which consisted of low glucose DMEM, 10% (v/v) FBS and 1% penicillin/streptomycin mix. Cells were seeded into 175 cm6 cells per cm2. Non-adherent cells were removed after 3 days and cells were re-fed with complete rMSC medium, with additional media changes every 3-4 days. After approximately 8 days or when cell cultures reached confluence, cells were detached with 0.25% trypsin(w/v)/1 mM EDTA solution and re-plated at 5.7 × 103 cells per cm2, with subsequent passage when they again reached confluence.All procedures involving animals were performed in accordance with the ethical regulations of the National University of Ireland . MSCs were isolated from the bone marrow of 8-12 weeks old male Sprague Dawley rats  as previously described -16. BrieThe ability to differentiate down the adipogenic, osteogenic and chondrogenic lineages, following isolation procedures described, was routinely tested in-house. According to criteria recommended by the International Society for Cellular Therapy (ISCT), an MSC deemed acceptable for laboratory based investigations and pre-clinical studies is defined by its adherence to plastic, differentiation capacity and cell surface markers . Isolated MSCs were routinely characterised in-house by flow cytometry for their cell surface markers, as previously described [5 cells per well, grown overnight and transduced with lentiviral vectors in a minimal volume of medium. MSCs were harvested for analysis, at the earliest, 96 h post-transduction. For the 28-day transgene analysis experiments, cells were harvested at 7 d intervals, at which point each cell population per well was split, using one half to maintain the cells in culture and the other for GFP expression analysis (see below). For assessment of transgene silencing, transduced cell populations were cultured in medium containing the histone deacetylase (HDAC) inhibitor, trichostatin A  and/or the DNA methyltransferase inhibitor 5-azacytidine  for the 28-day period, harvesting at 7 d intervals. MSCs were harvested by trypsinisation, fixed in 4% paraformaldehyde and resuspended in PBS. Percentage GFP-positive cells and their mean fluorescence intensity (MFI) was determined using Guava Express Plus analysis on a GuavaEasyCyte™ flow cytometer . Total GFP was calculated by multiplication of the percentage GFP positive cells by the MFI.MSCs were seeded in 6-well plates at 1 × 10Rat MSCs were transduced by incubation with lentiviral vectors at a multiplicity of infection (MOI) of 100 for 18 hours. Transgene expression was measured 72 h post-transduction by immunofluorescent staining and Western blot analysis , ischaemia (O2 at 0.5% in serum and glucose free DMEM) and complete O2 and glucose deprivation by inhibition of glycolysis (ischaemia+2-deoxyglucose) in an in vivo 400 hypoxia chamber . 2-deoxyglucose (2DG) is a glycolytic inhibitor, which prevents hexokinase phosphorylation of glucose. Addition of this inhibitor provided an in vitro model of complete glucose deprivation. For these experiments, MSCs were seeded and transduced under normal culture conditions until 72 h post transduction when cells were optimally expressing transgene. The appropriate media for each condition  was placed in the hypoxia chamber for a minimum of 3 hours to deplete the oxygen levels to the required 0.5%. Depleted media was added to MSCs, which were treated with the appropriate conditions for the required period of time.To mimic the MSC viability was determined by standard MTT assay as described previously . Apoptos5 cell per well in 6-well plates. Once cells had reached confluence, adipogenic differentiation was induced by three 72 h cycles of adipogenic induction media . Following each round of induction, cells were maintained in maintenance medium for 24 h (10 μg/ml insulin in complete high glucose medium), and for 5-7 days after final induction. Cells were fixed in 10% formalin and differentiated cells were identified by Oil Red O stain for lipid vacuoles. Images of 10 random fields for each well were acquired and differentiated cells containing stained lipid vacuoles per microscope field were counted. Oil Red O stain was also quantified by extraction of the stain into 99% isopropanol and absorbance measurement on a Wallac plate reader at 490 nm. Non-induced MSCs, maintained in normal growth medium, were included as negative controls for the adipogenesis assay.MSCs (both transduced and non-transduced) were seeded at 2 × 10n = 3) of three independent experiments. Statistical analyses were carried out on data sets using SigmaStat for Windows, version 3.5. One-way analysis of variance (ANOVA) was used to determine statistical significance between groups, followed by multiple paired comparisons for normally distributed data (Tukey Test). Non-normally distributed data were analysed by ANOVA on ranks using either Dunn's Test or Kruskal-Wallis. Paired t-tests were used for pairwise comparisons. Values of p < 0.05 were considered statistically significant .Data are presented as mean ± standard deviation , was achieved in rat MSCs transduced with rHIV-pWPT-EF1-α-GFP-W vector, as expected. The percentage of GFP-positive MSCs increased from ~17% at MOI 1 to ~98% at MOI 100 Figure . The effMSC viability was detected by MTT assay after exposure to hypoxic conditions Figure . MSC viaThe effect of HSP70 over-expression on hypoxia-induced apoptosis was also assessed. Classical signs of apoptotic cell death were visible with respect to nuclear morphology, which included misshapen or "bean-shaped" nuclear membranes, intense fluorescence indicative of nuclear condensation, nuclear fragmentation and disassembly into apoptotic bodies Figure . The perNot unexpectedly, MSCs were more sensitive to conditions of ischaemia than hypoxia Figure . After 1in vivo scenario of ischaemia where cells may not be able to use glycolysis for ATP level maintenance, MSCs were treated with 1 mM 2DG in addition to the conditions of ischaemia previously described. Addition of 2DG exacerbated the observed effects on MSC survival in terms of viability and apoptosis. After 12 hours of treatment, viability of unmodified MSCs had decreased below 40%, falling to less than 10% at the latest time point examined . There was no apparent effect of exogenous HSP70 expression at any later time points. Analysis of nuclear morphology showed that increased numbers of apoptotic cells were present with ischaemia + 2DG exposure compared to the previous treatments Figure . Numbersin vitro models of ischaemia produced fewer lipid vesicles compared to normoxic MSCs. However, HSP70 over-expression reduced this inhibitory effect of oxygen, serum and glucose deprivation on MSC adipogenesis. This was in terms of numbers of differentiated cells  and genes relating to transcriptional and post-transcriptional functions (tat and rev). These vectors are produced with a 3 plasmid packaging system. The latest third generation vectors have further HIV-1 genes deleted leaving only gag, pol and rev and are produced using a four-plasmid set. This vector generation has been designed using a chimeric LTR  ensuring transcription in the absence of tat. As a result, this vector system possesses a higher safety profile and is more suitable for clinical applications. Vector generation and promoter type will undoubtedly affect transduction levels - low or short-lived transgene expression levels, cytotoxicity or transgene silencing may prompt the choice of an alternative vector type or model. Consequently, a full investigation into the ex vivo transduction potential of MSCs derived from the same species is essential. This series of experiments set out to assess and optimise lentiviral vector transduction efficiency in rat MSCs, and also to examine any functional effect of genetic modification in an in vitro model of ischaemia.Numerous studies have utilised genetically engineered MSCs, which have been modified by various gene therapy vectors including adenovirus ,19, AAV in vitro transduction of MSCs. Modification with this particular vector facilitated sustained, persistent and high-level transgene expression in target rat MSCs, possibly due to various factors, the most likely being promoter type. Several in vitro studies have compared promoters in similar lentiviral vector constructs and have determined the Ef1-α promoter to be superior to other promoters tested and mediate high transgene expression levels [ex vivo transplantation in various preclinical models, where long term gene expression is required. To this end, this second generation VSV-G rHIV-pWPT-EF1-α-GFP-W lentiviral vector was chosen for all further MSC modification in this study.Gene silencing can present problems when using gene delivery as certain vectors and promoters have been shown to be susceptible to transcriptional silencing in particular host cell types. It is widely accepted that retroviruses are silenced in stem cells -28, howen levels . This fiper se. Indeed, several studies have similarly observed that MSC transduction with lentivirus does not negatively affect osteogenic, chondrogenic or adipogenic differentiation, and that transgene expression is retained in the differentiated cells [In addition to assessment of transduction efficiency, we subsequently sought to examine the survival and differentiation potential of MSCs modified with this vector. As expected, a dose-response effect was achieved by transducing cells with increasing MOI, with levels approaching 100% transduction being achieved at MOI 100. In addition, we examined any toxic effect that transduction with this vector may have on cell viability and found that lentiviral vector modification of rat MSCs had no significant negative effects. Levels of cell death did not surpass ~5% of total cell population when transduced with the rHIV-pWPT-EF1-α-GFP-W vector at a high MOI of 100. This observed low level of cell death at the highest MOI measured may be in part due to a toxic effect of transgene rather than toxicity of the vector itself. GFP expression has previously been documented as being toxic in various cell types . This laed cells ,35. ThisThis study also investigated the effect of MSC transduction at subsequent passages and demonstrated that MSC passage number is important and affects vector transduction efficiency in terms of transgene expression levels. MSC transduction was more efficient at early passage numbers and was significantly decreased at later passage numbers. This is of importance for future experiments, as transduction at earlier passage numbers ultimately results in higher levels of transgene expression. It was also determined that cryopreservation of transduced MSCs had no negative effect on transgene expression levels. This is of particular relevance to preclinical and human trials, from a logistical perspective, as it is standard practice to harvest cells, transduce, expand and freeze, ready for therapeutic use. Other gene therapy vectors are unsuitable in this respect for MSC gene delivery, as with certain vectors e.g. adenovirus, continued and sustained transgene expression is not achievable . As precin vitro model of ischaemia, a condition of particular relevance to MSC transplantation, and several putative pro-survival genes. In our laboratory, Mylotte et al. demonstrated that MSCs tolerated in vitro conditions of hypoxia and ischaemia better than cardiomyocytes and were more vulnerable to ischaemia induced death rather than that of hypoxia [In our investigation of lentivirus transduction of MSCs, we also sought to demonstrate a functional effect mediated by lentivirus vector-mediated genetic modification. For this, we primarily focused on an  hypoxia . This suin vitro representation of the post-MI state, consisting of hypoxia and nutrient deprivation. Here, we found that MSCs tolerated hypoxia relatively well up to 72 h, after which survival was adversely affected in terms of cell morphology, viability, cell numbers and apoptosis. At all time points measured, exposure to ischaemia significantly reduced MSC numbers and viability and significantly increased apoptosis. Addition of the glycolytic inhibitor, 2DG, has previously been used to mimic the inhibition of glycolysis that may occur during in vivo episodes of ischaemia [In the present study, we assessed any benefit of induced therapeutic gene expression to MSCs, in an schaemia ,38. Thusin vivo ischaemia components; hypoxia, serum deprivation and glucose deprivation. As higher levels of MSC death occurred in the models that incorporated serum and glucose deprivation as well as hypoxia, it is reasonable to conclude that MSCs are more vulnerable to apoptosis as a result of nutrient and survival growth factor withdrawal rather than hypoxia alone. With this in mind, rendering the MSC more resistant to apoptosis by any means would prove beneficial for the use of transplanted MSCs in the treatment of MI.All treatments triggered cell death by apoptosis in a time dependent manner, as identified by morphology  and caspase-3 activity. Hypoxia activated apoptotic cell death at 72 h whereas, under ischaemic conditions, apoptosis occurred earlier at 48 h as well as at 72 h. Caspase activation detected in oxygen and serum-deprived MSCs correlated with time points where the highest cell death levels were observed, suggesting that MSC death is caspase dependent. These results were consistent with findings from other researchers that have observed MSCs to be relatively resistant to hypoxia to a certain point, but sensitive to ischaemia ± 2DG ,39. The in vitro models of hypoxia and ischaemia has been previously reported [Following preliminary analysis of treatment effect, MSCs were transduced to over-express high levels of the pro-survival genes catalase, HSP27, HSP70, SOD1 and SOD3. The ability of the modified MSCs to tolerate the treatment conditions of conditions of hypoxia, ischaemia and ischaemia + 2DG, compared with wild-type MSC was subsequently examined. Transduced MSC groups were observed to be more resistant to all treatments compared to control GFP and unmodified MSC groups in terms of survival. Cell survival was assessed initially by MTT viability assay so that the most effective gene could be focused on for further study. Of the genes tested, HSP70 over-expression significantly increased MSC viability in conditions of hypoxia, ischaemia and ischaemia + 2DG. In order to ascertain the manner of cell death occurring as a result of hypoxia and ischaemia, analysis of nuclear morphology and caspase induction was performed. The occurrence of apoptosis in response to reported ,39. We fin vitro hypoxia exposure [et al. demonstrated optimal MSC function at a reduced oxygen concentration of 5% [in vivo bone marrow [The effect of oxygen deprivation-induced stress on MSC differentiation was also assessed. Only the HSP70 gene was tested in this experiment as its over-expression had the most positive effect on MSC survival in previous experiments. Here, it was observed that none of the three oxygen deprivation treatment conditions completely diminished the ability of the MSCs to produce lipid vacuoles. This is consistent with reports that have showed MSCs retained the ability to differentiate into osteoblasts and adipocytes post exposure -45. A ston of 5% ; this ise marrow . Althougin vitro to express either reporter gene or variety of therapeutic genes using a second generation lentiviral vector. This is, to our knowledge, the first study that systematically examines lentiviral vector-mediated gene modification of MSCs. Information arising from such experiments is crucial to the successful genetic modification of MSCs, which has the potential to augment their therapeutic benefit.The combination of mesenchymal stem cell therapy and gene therapy offers an integrated approach towards tissue and organ repair. The success of such a combined therapy is dependent on the optimum genetic modification of MSCs. Consequently, the present study is of particular interest and validates the choice and use of lentiviral vectors for this purpose. Here, we report that MSCs can be efficiently transduced The integrative nature of lentiviral vectors does however raise concerns of a potential risk of insertional mutagenesis. In this regard, further investigation and clarification is required and the development of integration mapping techniques or targeted gene delivery to avoid potentially oncogenic sites has become desirable. The use of a non-integrating lentivirus vector is also an option - these vectors possess a mutated integrase gene, which results in transient gene expression. Further development in this area offers the possibility to develop a novel gene-transfer vector that does not pose the risk associated with permanent integration into host cells, yet possesses the efficiency of lentiviral vectors that is required for effective genetic modification. Despite these concerns, the value of lentivirus vectors cannot be completely discounted, as these vectors are currently unsurpassed in their efficiency of gene delivery to MSCs.in vitro model of ischaemia. The use of only in vitro testing does however restrict what conclusions can be reached from the present study, as these findings may not translate to an in vivo situation. Therefore, further in vivo experiments based on this study are necessary for the identification of a potentially therapeutic factor for use in ex vivo stem cell therapy.Additionally, we showed that lentivirus vector modification of MSCs can exert a functional effect through over-expression of HSP70 in an In conclusion, we report that lentivirus vectors are a valuable gene delivery tool and lentivirus-mediated genetic modification of MSCs has the potential to significantly augment the therapeutic benefit of MSC-based therapies.2DG: 2-deoxyglucose; 5-aza-c: 5-azacytidine; CMV: cytomegalovirus, EF1-α: elongation factor 1 alpha; ESC: embryonic stem cell; GFP: green fluorescent protein; HDAC: histone deacetylase; HIV-1: human immunodeficiency virus-1; HSC: hematopoietic stem cell; HSP: heat shock protein; MFI: mean fluorescence intensity; MOI: multiplicity of infection; MSC: mesenchymal stem cell; PGK: phosphoglycerate kinase; SIN: self-inactivating; SOD: superoxide dismutase; TSA: trichostatin A; VSV-G: vesicular stomatitis virus glycoprotein.TOB has received education grants from Pfizer, Novartis and Merck Ltd and has received research grants from Medtronic. The other authors declare no competing interests.LMG produced lentiviral vectors, designed and conducted all experiments and drafted the manuscript. JMM participated in supervision and design of the study. DOT and PS participated in vector and experimental design. FB and MM participated in experimental design. TOB participated in supervision and study design and was the principal investigator. All authors carried out revisions of the manuscript and approved the final manuscript for submission.Transgene expression in lentivirus vector modified MSCs. Transgene expression was demonstrated by immunostaining of (i) transduced and (ii) non-transduced samples and also by (iii) western blot for (A) catalase, (B) HSP70, (C) SOD1 and (D) SOD3. Data are representative images , of three independent experiments. Legend: cat = catalase: 65 kDa, H70 = HSP70: 70 kDa, HBA = Human b Actin 42 kDa, S1 = SOD1: 16 kDa, S3 = SOD3: 25.8 kDa, N/T = non-transduced rat MSCs.Click here for file


Behavioral mechanisms that contribute to the association between breastfeeding and reduced obesity risk are poorly understood. The purpose of this study was to evaluate the hypothesis that feeding human milk from the breast (direct breastfeeding) has a more optimal association with subsequent child appetite regulation behaviors and growth, when compared to bottle-feeding.Children (n = 109) aged 3- to 6- years were retrospectively classified as directly breastfed (fed exclusively at the breast), bottle-fed human milk, or bottle-fed formula in the first three months of life. Young children's appetite regulation was examined by measuring three constructs  associated with obesity risk, using the Child Eating Behavior Questionnaire. Multinomial logistic regression analyses were used to test whether children bottle-fed either human milk or formula had reduced odds of high satiety and increased odds of high food responsiveness and high enjoyment of food compared to children fed directly from the breast. Current child weight status and growth trends from 6-36 months were also examined for their relation to direct breastfeeding and appetite regulation behaviors in early childhood.Children fed human milk in a bottle were 67% less likely to have high satiety responsiveness compared to directly breastfed children, after controlling for child age, child weight status, maternal race/ethnicity, and maternal education. There was no association of bottle-feeding (either human milk or formula) with young children's food responsiveness and enjoyment of food. There was neither an association of direct breastfeeding with current child weight status, nor was there a clear difference between directly breastfed and bottle-fed children in growth trajectories from 6- to 36-months. More rapid infant changes in weight-for-age score were associated with lower satiety responsiveness, higher food responsiveness and higher enjoyment of food in later childhoodWhile direct breastfeeding was not found to differentially affect growth trajectories from infancy to childhood compared to bottle-feeding, results suggest direct breastfeeding during early infancy is associated with greater appetite regulation later in childhood. A better understanding of such behavioral distinctions between direct breastfeeding and bottle-feeding may identify new pathways to reduce the pediatric obesity epidemic. Obesity is a global problem even in the youngest populations. In the U.S., most recent reports indicate over 10% of infants and toddlers are overweight . IncreasPotential biological explanations of the breastfeed-obesity association have centered on differences in the nutritional composition of human milk versus formula, focusing in particular on the comparisons of human milk and formula in terms of protein and fatty acid composition . For exaincreased alertness to caregiver, moving head towards caregiver) and satiety . In contrast, bottle-feeding, involving either human milk or formula, provides explicit visual information about infant intake to the caregiver based on the amount of milk or formula remaining in the bottle.Behavioral factors may explain the breastfeeding-obesity association, but these factors have not been fully explored. Both maternal and infant behaviors differ based on whether an infant is fed from the breast [direct breastfeeding] or bottle-fed human milk and/or formula. It is suggested that direct breastfeeding supports the development of appetite regulation given its inherent dependence on infant-centered feeding behaviors by the mother ,13. ThisLimited empirical evidence exists to support the suggestion that direct breastfeeding is more infant-centered. To date, observational research examining mother-infant pairs at 1 week, 1 month, and 2 months of age has revealed that mothers who bottle-fed initiated a greater proportion of breaks during feeding than directly breast-feeding mothers . DirectlAlthough this evidence collectively suggests that the protective effect of breastfeeding on obesity risk may be partially driven by behavior, potential differences in the long-term appetite regulation between directly breastfed and bottle-fed infants has not been explored. Such an investigation would provide insight into the behavioral aspects of breastfeeding which might be related to weight gain and obesity. Identifying behavioral aspects unique to direct breastfeeding offers a potential path to child obesity prevention in the earliest years of life, as this identification could lead to interventions for all caregivers, including mothers who cannot or choose not to directly breastfeed .Reliance on internal cues during eating is important as there is a well-established relationship between appetite regulation and weight status -18. PareTherefore, the primary aim of this study was to determine the association of direct breastfeeding during early infancy with later appetite regulation behaviors among 3- to 6-year-old children. Given associations with child obesity and energy intake, three aspects of child appetite regulation were examined: satiety response, food responsiveness, and enjoyment of food . It was This study employed a retrospective cohort design. Infant feeding group assignment was based on self-reported breastfeeding/human milk use and bottle-feeding behaviors during the first three months of life, which were verified by medical chart review. Current demographics, child appetite regulation behaviors, and anthropometric data were collected when children were 3-6 years old. Retrospective child growth data were assessed via medical records.A sample of mothers and their 3-6 year old children were recruited using two strategies. The majority of recruitment took place in-person in the waiting area of a private pediatric primary care office located in suburban Philadelphia. A secondary method involved emails sent to members of a listserv provided by a non-profit, community-based breastfeeding center in the same geographic area. Breastfed and non-breastfed children were included in the study. Exclusion criteria included maternal history and/or current diagnosis of an eating disorder, maternal conditions that contraindicated breastfeeding , and a child being a multiple due to unique challenges related to breastfeeding higher order multiples .After obtaining informed consent, participants completed the self-report surveys which took approximately 15 minutes. Those recruited in-person were asked to complete the survey during their pediatric clinic visit; those who did not complete it during the visit were asked to return the survey by prepaid mail provided by the researcher. Email-recruited participants received and returned consent forms and the survey packets by prepaid mail. A medical chart review of infant feeding history and child growth was performed for participant's recruited in-person from the pediatrician's office and for those recruited by e-mail who used the recruitment site pediatrician. For children with other pediatricians, mothers were asked to take the child growth forms to their pediatrician's office for completion, but infant feeding history was not verified for these participants. All procedures were carried out in accordance with Temple University human subjects' protections and U.S. privacy regulations regarding individually identifiable health information (HIPAA).Duration of human milk use and bottle use (first three months of life) were self-reported by mothers. These data were verified for 78% of the sample (n = 85) using medical chart review . Of the 85 charts reviewed, 91.6% were consistent with maternal self-report. Because five mothers overestimated and three mothers underestimated their length of exclusive human milk use, medical chart data were used in analyses for these participants. In order to isolate direct breastfeeding, mothers had to report exclusive direct breastfeeding (no bottle use) during the first three months of life for their children to be considered directly breastfed (DIRECT BF group). The reason for selecting the three month cut-off was twofold. First, most employed US women return to work within 12 weeks of giving birth and direct breastfeeding is less likely after this point . Second,(ex: 0-1 bottles on average days), "Some of the time" (ex: 2-3 bottles on most days), and "None of the time" (ex: 4 or more bottles on most days). If a mother did not initiate breastfeeding, she was coded as feeding from the breast, "None of the time". Based on these responses, the DIRECT BF group was comprised of children who were fed human milk exclusively for the first three months of life and whose mothers stated they fed the infant at the breast all of the time. The BTL-HM group was compromised of children who were fed human milk (non-exclusive included) and whose mothers stated they fed the infant at the breast from most to none of the time during the first three months of life. The BTL-FORM group was comprised of children who were fed formula for the first three months of life. Four breastfed children were placed in the BTL-FORM group because they were supplemented with formula from birth and because, while mother's reported that they initiated breastfeeding, they said human milk was rarely fed due to breastfeeding difficulties . These breastfed children in the BTL-FORM group were all fed human milk for less than 4 weeks.Bottle use was determined based on a scaled response question, asking mothers who reported breastfeeding whether they typically breastfed from the breast in the first three months of life, "All of the time" (ex: never used a bottle on average days), "Most of the time" Appetite regulation was measured using selected subscales  from the Child Eating Behavior Questionnaire [CEBQ], a 35-item parent-report measure of eight dimensions of young children's eating behaviors ,25. The z scores [WFLz] , BMI z-scores [BMIz]  and weight-for-age z scores [WFAz]  were calculated based on 2000 Centers for Disease Prevention and Control [CDC] growth standards  with the growth measures as an outcome (WFLz/BMIz and WFAz), controlling for feeding group . STATA® version 11 statistical software was used to run TSGM analyses.Our secondary analyses used t-tests to assess potential differences in growth (WFL/BMI z-score) between the DIRECT BF, BTL-HM, and BTL-FORM groups, across 6-36 months of age. Two-stage growth models (TSGM) were then used to assess potential differences in within-subjects growth trajectories. A regression model was run for each child participant to estimate an intercept and slope for each child's linear growth pattern centered on the first growth point measurement, 6 months. Because anthropometric measures were age-standardized, growth trends were linear. A quadratic model was considered, but rejected because coefficients were approximately zero with little variation across individuals. Thus, a linear model was retained for all subsequent analyses. Separate models were run for each growth outcome (WFLTable sd = 47.4) of any human milk use (range 16-200 weeks) and 22.0 weeks (sd = 5.5) of exclusive human milk use. In the DIRECT BF group, 41.4% of women reported formula supplementation after the first three months of life and for these women the mean infant age of formula supplementation was 29.2 weeks (sd = 10.9). Mean age of solid supplementation for the DIRECT BF group was 5.5 months (sd = 1.2). The BTL-HM group had a mean of 25.3 weeks (sd = 22.0) of any human milk use and 10.3 weeks (sd = 8.9) of exclusive human milk use. Mean age of solid supplementation for the BTL-HM group was 5.5 months (sd = 1.4). For the BTL-FORM group, formula supplementation began at birth, with 0 weeks of exclusive human milk use. Four children (18.2%) in the BTL-FORM group were breastfed. The BTL-FORM group had a mean of 0.4 weeks (sd = 1.1) of any human milk use, where mothers reported feeding from the breast "none of the time". The mean age for solids supplementation was 4.7 months (sd = 1.0) in the BTL-FORM group.The DIRECT BF group had a minimum of 12 weeks of exclusive direct breastfeeding with a mean of 73.2 weeks . On a 0-4 scale, the SR score median was 2.0, ranging from 0.6-4.0, with 36.9% having low vs. 63.1% high satiety response following median split, for the entire sample. The FR subscale score median was 1.3, ranging from 0.0-3.6, with 39.1% having low vs. 60.9% high food responsiveness following the median split. The EF subscale score median was 2.5, ranging from 0.75-4.0, with 48.2% sample having low vs. 51.8% having high enjoyment of food following the median split. Figure 2 = 6.93, p ≤ 0.01). Using Chi-square analyses, it was found that 59% of children with high SR had mothers who were educated beyond the college degree level .Covariates were selected for inclusion in the multivariate model based on their correlation with child appetite regulation, thus child age at time of interview, child BMI, maternal race, and maternal education level were included in all analyses. Child age was negatively correlated with high SR . Child BMI was negatively correlated with high SR , low FR , and low EF . Using Chi-square analyses, it was found that 73% of children with low FR had mothers who were Black  were associated with appetite regulation (see Table z) from 6-36 months were more likely to have lower SR at ages 3-6. Children with more rapid weight gain (WFAz) from 6-36 months were also more likely to have high FR at ages 3-6, but rapid weight gain was not a significant predictor of higher EF at ages 3-6. More rapid change in relative growth (WFLz/BMIz) from 6-36 months, was not a significant predictor of lower SR, higher FR, or higher EF.Growth patterns  which might have limited our ability to identify differences. In parallel to previous research which connects parental feeding practices (e.g. control) and child appetite regulation ,35, our Examining the potential connection of direct breastfeeding and later satiety response is novel. Our results suggest a potential utility of studying maternal-infant interactions and behaviors surrounding breastfeeding in order to understand the development of appetite regulation, beyond observing differences based on milk type alone. Breastfeeding duration can assess the overall length of exposure to and/or dosage of human milk, but feeding human milk directly from the breast versus milk from a bottle reflects potential behavioral differences in feeding which may shape infant consumption patterns. Bottle-feeding provides visual cues to mothers/caregivers about the volume of milk consumed, which might encourage a caregiver to feed and/or an infant to eat independent of internal hunger and satiety cues ,36. A chThe current study evaluated the effect of direct breastfeeding on overweight or less rapid growth from 6-36 months, finding no relation to either. While past research finds that longer breastfeeding duration is protective obesity throughout childhood , this stz/BMIz). Lower satiety response was associated with a history of rapid weight gain from infancy through childhood, whereas higher food responsiveness was related to history of rapid growth. Because appetite regulation assessments followed growth measurements, causality cannot be discerned. These findings advance previous knowledge on these constructs which has demonstrated links to weight, but only in cross-sectional fashion [Child appetite regulation at ages 3-6 years was related to weight gain from 6 months to 3 years, but was not related to change in relative growth  was a limitation; however, the use of chart review provided evidence that mothers' retrospective reports were reasonably accurate reflections of medical chart notes. Maternal reports of child appetite regulation, assessed by questionnaire, have been previously associated with obesity in children . HoweverThis investigation yields new evidence of an association between direct breastfeeding and children's satiety response during early childhood. The findings point to differences in the behavioral processes surrounding feeding method, suggesting that feeding human milk from the breast may support the development of satiety responsiveness across infancy and childhood. Additional research is needed to understand whether the infant-centered experience of direct breastfeeding influences how mothers approach feeding beyond weaning. Such information is needed to promote optimal feeding interactions for all caregivers including mothers who wean, mothers who cannot or choose not to breastfeed, and other caregivers . Through further inquiry and interventions, our understanding of behavioral factors which are distinct to direct breastfeeding could be improved, which in turn could accelerate and enhance our efforts to reduce the pediatric obesity epidemicBMI: Body Mass Index; BTL-FORM: Bottle-fed Formula group; BTL-HM: Bottle-fed Human Milk group; DIRECT BF: Directly Breastfed group; SR: Satiety Responsiveness; FR: Food Responsiveness; EF: Enjoyment of Food; CEBQ: Child Eating Behavior Questionnaire; HIPPA: Health Insurance Portability and Accountability Act; WFLz: Weight-for-Length z-score; BMIz: Body Mass Index z-score; WFAz: Weight-for-age z-score; TSGM: Two Stage Growth Model; CDC: Centers for Disease Prevention and ControlThe authors declare that they have no competing interests.Significant writer , significant reviewer (BNC and JOF), manuscript concept/design , data acquisition (KID), data analysis , and statistical expertise (AD). All authors have read and approved the final manuscript.


Decrease of dual-task (DT) ability is known to be one of the risk factors for falls. We developed a new game concept, Dual-Task Tai Chi (DTTC), using Microsoft’s motion-capture device Kinect, and demonstrated that the DTTC test can quantitatively evaluate various functions that are known risk factors for falling in elderly adults. Moreover, DT training has been attracting attention as a way to improve balance and DT ability. However, only a few studies have reported that it improves cognitive performance.The purpose of this study was to demonstrate whether or not a 12-week program of DTTC training would effectively improve cognitive functions.This study examined cognitive functions in community-dwelling older adults before and after 12 weeks of DTTC training (training group [TG]) or standardized training (control group [CG]). Primary end points were based on the difference in cognitive functions between the TG and the CG. Cognitive functions were evaluated using the trail-making test (part A and part B) and verbal fluency test.F1,36=4.94, P=.03; TG: pre mean 48.8 [SD 43.9], post mean 42.2 [SD 29.0]; CG: pre mean 49.5 [SD 51.8], post mean 64.9 [SD 54.7]).A total of 41 elderly individuals  participated in this study and their cognitive functions were assessed before and after DTTC training. Significant differences were observed between the two groups with significant group × time interactions for the executive cognitive function measure, the delta-trail-making test . Cognitive impairment among elderly individuals is a serious issue in many countries. Many investigators have developed different cognitive function training methods as countermeasures to prevent cognitive impairment and have reported their effects . Other iRecently, dual-task (DT) ability, or the performance of simultaneous motor and cognitive tasks, has been receiving considerable attention . DT traiWith a focus on DT, we developed a new concept called the Dual-Task Tai Chi (DTTC) test . This sySeveral investigators have reported the effects of DT training on balance, mobility, walking, and DT ability ,12. HoweCommunity-dwelling elderly subjects (n=48) participated in this study. The subjects were recruited through an advertisement in the local press. The following selection criteria were used: age ≥65 years, community dwelling, independent ambulation, willingness to participate in the measurement of physical fitness, and minimal hearing and vision impairment. Exclusion criteria were as follows: inability to complete the tasks because of reduced cognitive functions, evaluated by Rapid Dementia Screening Test  scored 8The DTTC test -10 requiTo begin with, the user stands 3 m in front of the Kinect sensor with his or her right foot in front of the sensor . The folReach a digit you need to use with your right hand to fill a blank you want to answer.Step 50 cm laterally, with your left leg, to grip the digit in your right hand.Select the blank you want to answer with your left hand, and move your right hand to your left one.All subjects participated in group training sessions lasting 75-80 minutes once a week for 12 weeks. The participants were divided into two training groups according to their participation in the exercise class: (1) the control group (CG), 75-minute standardized training, and (2) the training group (TG), 5-minute DTTC training, in addition to the standardized training .The exercise classes were individualized for each group and were supervised by physical therapists. Each exercise class used a standardized format that included 15 minutes of moderate-intensity aerobic exercise, 15 minutes of progressive strength training, 10 minutes of flexibility and balance exercises, and 10 minutes of cool-down activities, followed by exercises known to improve muscle strength and balance . In addiThe participants in the TG were additionally asked to solve DTTC problems and mirror-reversed DTTC problems, alternately, as many times possible in 5 minutes.All participants underwent evaluation upon entry into the study (pre-intervention) and at the end of the study (post-intervention), using the results of 2 cognitive performance tests.Cognitive functions were evaluated using the trail-making test (TMT)  and verbThe VFT has a letter fluency component and a category fluency component. Participants were asked to think of as many animal names as possible in 1 minute (category fluency). Verbal fluency is an evaluation of expressive language ability and executive functions. The score was the number of successful words (except for some proper nouns).t test or the chi-square test. Repeated-measures, mixed-linear, two-way ANCOVA  was used to analyze the effect of exercise on outcome measurements while adjusting for each cognitive performance, at pre-intervention, as a covariate.We compared baseline characteristics between the participants in each group to examine the comparability between the 2 groups using Student’s P<.05 was considered statistically significant.Data were entered and analyzed using the Statistical Package for the Social Sciences . For all analyses, A total of 41 of the 48 selected subjects (85.4%) completed the study protocols and returned for their exit interviews and final testing . The participants’ baseline data did not differ significantly between the two groups. Thus, the groups were comparable and well matched with regard to their baseline characteristics.During the 12-week intervention phase, 10 exercise sessions were scheduled and all took place. Excluding the 4 participants who dropped out, the TG subjects had an overall attendance rate of 82% and the CG subjects had an overall attendance rate of 81% over the 12 weeks. No health problems, including cardiovascular or musculoskeletal complications, occurred during training sessions or testing. Moreover, almost all participants seemed to have enjoyed the DTTC training. They shared many positive opinions after each session and seemed to look forward to playing DTTC once a week.Pre- and post-intervention group statistics and group × time interactions are shown in The delta-TMT score was significantly improved after DTTC training. The results suggested that DTTC training was effective in improving executive cognitive functions. In our unpublished data, DTTC training was useful for improving balance ability and mobility among elderly people . Thus, DConversely, the TMT-A and -B scores were not significantly improved. In the TMT-A, both groups improved similarly. On the other hand, in the TMT-B, only the TG had a tendency to improve the score, while CG showed little change. That is why the score of delta-TMT in CG increased and in TG decreased. The TMT-A and -B are used to assess visual scanning, cognitive flexibility, and executive functions . The delPrevious studies reported that other “exergames,” based on Nintendo Wii Fit  or other devices, improved balance and leg muscle functions ,24. HoweThere are several limitations to this study. First, the intervention effects in this study were not due solely to the DTTC training. That means the rhythmic stepping exercise involving cognitive ability in both groups also improves cognitive ability. Second, participants in both groups may have had higher motivation and interest in health issues and fall risk minimization than the general elderly population. An investigation into the effects of DTTC training on functions in frail, elderly adults is necessary, in the future.In this study, the ANCOVA results of delta-TMT reveal that group × time interactions were statistically significant. They suggest that DTTC training is effective at improving executive cognitive functions in particular.


The preterm infants' skin is structurally and functionally immature at birth because of immature stratum corneum barrier function, leading to problems with fluid loses, thermoregulation, and infection. Two parameters of barrier function can be non-invasively assessed: Stratum Corneum Hydration (SCH) and Transepidermal Water Loss (TEWL). Skin-to-Skin Care (SSC) is the proposed independent variable that might affect barrier function by decreasing TEWL and increasing SCH, thereby improving stratum corneum barrier function and consequently decreasing the rate of infection. No study of SSC's effects on TEWL and SCH of preterm infants could be found. The purpose of the study was to determine the effect of 5 daily Skin-to-Skin Contact sessions on infant skin hydration (SCH), transepidermal evaporated water loss (TEWL), and on SCH when TEWL was controlled, and on the presence of hospital acquired infection.A one-group pretest-test-posttest design with 10 preterm infants . Test = 90 minutes of SSC; pre-test and post-test = 30 minutes each of prone positioning in an incubator. SCH and TEWL were taken on Days 1 and 5 at the beginning, middle and end of each period using Multi-Probe Adaptor. A 3 X 3 X 2 Repeated Measures Mixed Models Design, including a covariate, was used to analyze level of Skin Hydration. Specifically, the model tested comparisons in SCH made across repetitions, time, and days, as well as all possible interactions while controlling for TEWL. Descriptive statistics described the number of positive blood cultures during hospitalization and the presence of infections four weeks post-discharge.Significant differences in skin hydration were found across TIME  . One infant had a positive blood culture during hospitalization; no infants had signs of infection by 4 weeks post-discharge. The study has begun fulfilling the recommendation that SSC be tested as a strategy to improve skin hydration, but reveals that evaporative loss may be higher during SSC than during incubator care, and that the higher transepidermal evaporated water loss values may not necessarily be detrimental because few infections occurred even in its presence. A definitive randomized controlled trial is recommended.Skin-to-skin contact; Skin hydration; Transepidermal water loss; Infection; Preterm Because the skin is a common point of entry for invasive pathogens, interventions to improve the preterm infant's skin barrier functions are needed . The skiSuccessful interventions to reduce infections, especially hospital acquired infections, would improve survival, reduce neonatal morbidity, and reduce the high medical and social costs of preterm infant care , 14-16. Skin-to-skin contact is skin-to-skin, chest-to-chest placement of the infant with mother at an incline of 30 - 40 degrees. Skin-to-skin contact may help reduce infections by improving the preterm infant's skin barrier function. A decrease in TEWL and an increase in SCH along all areas of infant skin that are in direct contact with maternal skin could occur and would be indicators of improved skin barrier function. Intermittent exposure to a high-humidity environment during feeding has been shown to quicken maturation of the skin barrier , leadingSkin-to-skin contact may enhance SC barrier function in two ways. The first way is that TEWL may be minimized due to decreased skin exposure to evaporative heat loss during SSC , 25 or dA one-group, preSS-SSC-postSSC quasi-experimental one group design was conducted in which each subject served as his/her own control. Premature infants were tested during one inter-feeding interval on Days 1 and 5 of a five-day study in which infants were given SSC each day for five consecutive days. The inter-feeding interval was divided into three periods: a 30-minute pre-SSC period in the incubator, followed by a 90-minute SSC period, and then another 30-minute post-SSC period in the incubator immediately after SSC ended. On Days 1 and 5, TEWL and SCH were assessed three times in each period: at 10, 20, and 30 minutes of the pre-SSC and post-SSC periods, and at 30, 60, and 90 minutes during the SSC period, so that early, middle, and end assessment data for each period were available. On Days 2, 3, and 4, SSC was given for at least 30 and no more than 120 minutes each day. Signed informed consent was obtained following institutional review board approval at a university hospital in the Midwest. One research associate administered all assessments and monitored computerized data acquisition.Preterm infants were tested in a tertiary university-based Neonatal Intensive Care Unit which consisted of 7 rooms, each room housing 3 - 5 preterm infants of varying severity of illness who were separated by screens. Ambient temperature and humidity were recorded immediately before each TEWL and SCH reading during the pre-SSC, SSC, and post-SSC periods.Twenty-five mother-infant pairs were eligible over a 12 months period and 17 gave consent. Seven of the consented pairs did not begin data collection because of infant illness (n = 3) or withdrawal at maternal request due to hardship in providing 5 consecutive days of SSC (n = 4). Convenience sampling resulted in 10 mother-infant pairs who provided complete data sets.Preterm infants born between 28 - 30 weeks gestation who met the following criteria were recruited: 1) were 30 - 31 weeks postmenstrual age at time of entry, so one week of study could be completed prior to the infant being 33 weeks post-conception age, the age at which the stratum corneum is usually as mature as the term infant's skin , 30, 2) Feedings for all subjects were scheduled every three hours. For preSSC and postSSC periods the infants remained in double-walled incubators, and were positioned flat, prone, and nested while wearing only a diaper and a head cap. Transfer into SSC was accomplished by sitting transfer and infants were placed skin-to-skin, chest-to-chest between the breasts so that the infant's head was above the level of the mother's nipples and the head was turned to one side and erect (not flexed). During SSC infants wore diapers and had a receiving blanket folded-in-fourth covering their backs. The mother's hospital gown was closed over the infant's back. Mothers reclined at a comfortable level in a stationary zero-gravity lounger  behind privacy screens. Between Days 1 and 5 mothers agreed to provide daily sessions of SSC that were at least 65 minutes long.SCH was defined as the amount of water retained by stratum corneum cells in the skin. Stratum corneum hydration is measured by changes in skin surface electrical capacitance and determined by the Moisture Accumulation Test . SCH was2/hr. Because TEWL is reported as a rate of evaporation rather than an amount of evaporation, TEWL was computed by averaging the data recorded every one second for 20 - 120 seconds determined by the shortest time to achieve 5 consecutive readings with a standard deviation < 0.1. The TEWL was measured by a Tewameter® TM 300 [TEWL was defined as the rate of water evaporation from the skin and reported as g/m® TM 300 . The TEW® TM 300 .Number of infections was defined as number of positive blood cultures since the day of entry into the study to the day of infant discharge from the hospital. Positive blood cultures were determined by examining daily lab reports.Signs and symptoms of infection after discharge were defined as a positive maternal response to any one or more of the following questions: Has your infant had any fever since leaving the neonatal intensive care unit? Has your infant been diagnosed as having an infection since leaving the neonatal intensive care unit? Has your infant received any antibiotics during the first thirty days after leaving the neonatal intensive care unit?To measure TEWL and SCH, a Multi-probe Adapter Systems MPA®  were useoC for ambient temperature, and ± 4% for relative humidity, and was calibrated biennially by the manufacturer.A Fisher Scientific Traceable Thermistor/Clock/Humidity Monitor (Model 06-662-4) was used to measure ambient temperature and relative ambient humidity. The monitor's accuracy was ± 1 Ohmeda Care Plus incubator skin probe was used to obtain infant skin temperature. The probe was placed one centimeter below the right costal margin and covered by a Mylar temperature shield  to minimize ambient air and light influences. The probe is a 1.5 centimeter diameter flat metal disk that was attached to the abdomen one centimeter below the right costal margin at the mid-clavicle line.Following signed consent, mothers and the research associate mutually agreed to a start date for SSC (Day 1). All measurements were taken on Days 1 and 5. The infant was positioned prone and left undisturbed in the incubator for the pre-SSC period. Ambient temperature and relative humidity were recorded at the beginning of each period. Assessments of infant skin temperature, TEWL, and SCH were recorded at Minutes 10, 20, and 30 of the pre-SSC and post-SSC periods. The SSC period began as soon as infant was transferred to mother's chest. Infant abdominal skin temperature, SCH, and TEWL assessments were recorded at Minutes 30, 60, and 90 of the SSC period. At the end of SSC, the infant was transferred back to the incubator and positioned the same as in the pre-SSC period. Infants were undisturbed by staff between measurements. On Days 2, 3, and 4, infants received SSC for 65 - 120 minutes, beginning whenever the mother came to visit. SSC was given only once each day.Subjects' demographics, TEWL, SCH, number of positive blood cultures during hospitalization, and the presence of signs of infection four weeks post-discharge were described using measures of central tendency and dispersion (Mean and SD). In the current study, data were collected from 10 subjects over 18 time points. The main focus was comparing Skin Hydration across three TIME periods . In order to take advantage of collecting data at 18 time points, a Repeated Measures Mixed Model approach was used to analyze the data, an approach that provided more power to analyze the data because the effective sample became 180 data points (10 subjects X 18 data collection points) as compared to 10 data points based on subjects alone. Data were collected in three ASSESSMENTS of data collection points for each of the three TIME periods across two DAYS. With this in mind, the final model was based on a 3 X 3 X 2 Repeated Measures Mixed Model design. This model allowed for the testing of the three ASSESSMENTS of data collection points across three PERIODS across two DAYS. Typically, in using a Repeated Measures Mixed Models Approach, an error covariance structure has to be identified for the repeated time components of the model. In the Current Model, a Compound Symmetry error covariance structure was applied to the PERIOD component. Testing of the ASSESSMENTS component indicated that an error covariance structure did not need to be applied as ASSESSMENTS demonstrated an independent error structure. Additionally, TEWL was included as a covariate, because TEWL impacts levels of Stratum Corneum Hydration. With this model, comparisons in SCH can be made across ASSESSMENTS, PERIODS, and DAYS, as well as the testing of interactions comparing ASSESSMENTS across PERIODS, ASSESSMENTS across DAYS, PERIODS across DAYS, and ASSESSMENTS across PERIODS across DAYS. TEWL was also analyzed using the same 3 X 3 X 2 Repeated Measures Mixed Model design. As with the previous model, a Compound Symmetry error covariance structure was applied to the TIME component, while the ASSESSMENTS component had an independent error structure.Infant and maternal characteristics are presented in p < 0.001). It should be noted that all mean scores are based on Least Squares Mean estimates. In examining the differences in SCH across PERIODS, SCH scores during the Pre-SSC period (LSMeans = 37.41) were significantly lower than SCH scores during the SSC period (LSMeans = 53.87) (Tukey-Kramer adjusted p < 0.001). Stratum corneum hydration scores during the Pre-SSC period (LSMeans = 37.41) were significantly lower than SCH scores during the Post-SSC period (LSMeans = 48.68) (Tukey-Kramer adjusted p < 0.001). However, only a trend was found comparing SCH scores during the SSC period (LSMeans = 53.37) and SCH scores during the Post-SSC period (LSMeans = 48.68) (Tukey-Kramer adjusted p = 0.09). All of these scores were adjusted for TEWL, however a non-significant trend was found for TEWL's influence on SCH  = 3.72; p = 0.06).SCH mean values during each period on Days 1 and 5 are presented in p < 0.05). Among the 36 comparisons based on interactions, 10 significant differences were identified. Consistently, SCH at the second and third assessments during SSC and at the first assessment of Post-SSC were significantly larger as compared across assessments during Pre-SSC (p < 0.05), the second (Tukey-Kramer adjusted p < 0.01), and the third (Tukey-Kramer adjusted p < 0.01) assessments during Pre-SSC. The third SCH assessment during SSC was significantly larger than the first (Tukey-Kramer adjusted p < 0.01), the second (Tukey-Kramer adjusted p < 0.001), and the third (Tukey-Kramer adjusted p < 0.001) assessments during Pre-SSC. A similar trend in comparing the first SCH assessment during Post-SSC and the three Pre-SSC assessments was found. Specifically, the first Post-SSC assessment was significantly larger than the first (Tukey-Kramer adjusted p < 0.05), the second (Tukey-Kramer adjusted p < 0.01), and the third (Tukey-Kramer adjusted p < 0.01) assessments during Pre-SSC. Additionally, the third SCH assessment during SSC was significantly larger than the third assessment of Post-SSC (Tukey-Kramer adjusted p < 0.05). These findings reflect that SSC, as compared to Pre-SSC, increased levels of SCH not significantly at the first assessment, but significantly through the second and third assessments during SSC and carried over onto the first assessment of Post-SSC. Interestingly, this carry over did not continue onto the third assessment of Post-SSC, where SCH levels were significantly lower as compared to the third assessment during SSC.With regard to interaction effects, the only significant interaction effect was based on comparisons in SCH across ASSESSMENTS in data collection points and PERIODS   = 2.83;  Pre-SSC . The firTEWL demonstrated a similar pattern of increase from pre-SSC to SSC and then decrease from SSC to post-SSC on Days 1 and 5 , 4. A 3 Only one infant developed a hospital-acquired infection during hospitalization and that infection was clinically evident 7 days after SSC ended. None of the mothers reported any signs or symptoms of infection during the first thirty days post-discharge.The study investigated the effects of 5 consecutive days of 90 minutes SSC sessions on stratum corneum hydration and transepidermal water loss by recording and comparing values on Days 1 and 5 while infants were in an incubator (30 minutes pretest), in SSC (90 minutes test), and back in an incubator (30 minute posttest). The number of infections during and after hospitalization were tracked and showed that only one infant had a positive blood culture seven days after SSC stopped and no infants had signs of infection within 4 weeks of discharge. Reviewing the risk factors for developing infection revealed that the infant was at higher risk of infection because he was on CPAP and had an intravenous catheter longer than other infants; these findings are consistent with Cohen-Wolkowiez and colleagues . BecauseStratum corneum hydration and transepidermal water loss are indications of skin barrier function in preterm infants. Stratum corneum hydration, while controlling for transepidermal water loss, increased from pre-SSC to SSC and decreased in post-SSC on Days 1 and 5 of the study. Thus, SSC contributed to better stratum corneum hydration and did so probably because skin-to-skin contact provides a degree of occlusivity at the skin surface, thereby also causing skin wetness. Skin wetness contributes to higher transepidermal water loss readings, which were present during SSC. The increased hydration finding is similar to the effects of skin occlusion in diapered areas in infants during the first 2 - 3 weeks of life but not at 4 weeks of life due to skin maturation , 37. TraAdditionally, residual effects of SSC were seen. Skin hydration was higher after SSC than before SSC on both days, and skin hydration was higher before SSC on Day 5 than it was on Day 1, suggesting that the increases in skin hydration were not just momentary, but lasting. Such a possibility is congruent with the physiology of the skin in that skin hydration changes are commonly detectable for several days , 40. ResThe increase in stratum corneum hydration during SSC is an important finding. A hydrated stratum corneum maintains flexibility and is able to strengthen the cornified cell envelopes, increasing the strength of the corneocytes responsible for the strength of the stratum corneum . Good moTwo ways to hydrate the skin exist. One way is to conserve the skin's natural moisture by using an ointment; the other way is to add moisture. In SSC mother's chest, temperatures increase and sweating may occur , 45, 46,The levels of transepidermal water loss recorded during the incubator periods were expected because TEWL remains high in preterm infants for several weeks after birth . The incThe number of infection results need to be considered cautiously because of the small sample size and studies with larger sample size are warranted to determine the effects of SSC on infection rate. Nonetheless, SSC may help reduce the number of infections in other ways than modifying skin hydration. For example, SSC reduces infant stress , 56. StrA limitation of the study was that statistical control for ambient humidity was not possible due to the small number of ambient humidity data points . Because humidity influences transepidermal water loss, future studies should include more frequent measurement of ambient humidity. Future studies should also include equal numbers of dark- and light-pigmented skin subjects as well as measuring skin barrier function at different skin areas (i.e. the infant's back) during each period to control for site difference and occlusion effects. Also, future studies could examine maternal skin hydration and transepidermal water loss simultaneous to the infant's and test for any correlations between maternal and infant's values to determine presence of physiologic synchrony as a mechanism for change. Especially because physiologic synchrony between mothers and infants during SSC has been found in many biomarkers , 62-65.Hospital acquired infection is an important problem in preterm infants . The dat