Familywise error for multiple time-to-event endpoints in a group sequential design.

We investigate the familywise error rate (FWER) for time-to-event endpoints evaluated using a group sequential design with a hierarchical testing procedure for secondary endpoints. We show that, in this setup, the correlation between the log-rank test statistics at interim and at end of study is not congruent with the canonical correlation derived for normal-distributed endpoints. We show, both theoretically and by simulation, that the correlation also depends on the level of censoring, the hazard rates of the endpoints, and the hazard ratio. To optimize operating characteristics in this complex scenario, we propose a simulation-based method to assess the FWER which, better than the alpha-spending approach, can inform the choice of critical values for testing secondary endpoints.

Testing equivalence of survival before but not after end of follow-up.

For equivalence trials with survival outcomes, a popular testing approach is the elegant test for equivalence of two survival functions suggested by Wellek (Biometrics 49: 877-881, 1993). This test evaluates whether or not the difference between the true survival curves is practically irrelevant by specifying an equivalence margin on the hazard ratio under the proportional hazards assumption. However, this approach is based on extrapolating the behavior of the survival curves to the whole time axis, whereas in practice survival times are only observed until the end of follow-up. We propose a modification of Welleks test that only addresses equivalence until end of follow-up and derive the large sample properties of this test. Another issue is the proportional hazards assumption which may not be realistic. If this assumption is violated, one may severely misjudge the actual treatment effect with a hazard ratio quantification and wrongly declare equivalence. We suggest a non-parametric test for assessing survival equivalence within the follow-up period. We derive the large sample properties of this test and provide an approximation to the limiting distribution under some mild assumptions on the functional form of the difference between the two survival curves. Both suggestions are investigated by simulation and applied to a clinical trial on survival of gastric cancer patients.

Neonatal levels of adiponectin, interleukin-10 and interleukin-12 are associated with the risk of developing type 1 diabetes in childhood and adolescence: A nationwide Danish case-control study.

BACKGROUND/AIM: An in-depth understanding of the early phase of type 1 diabetes (T1D) pathogenesis is important for targeting primary prevention. We examined if 14 preselected mediators of immune responses differed in neonates that later developed T1D compared to control neonates. METHODS: The study is a case-control study with a 1:2 matching. The individuals were born between 1981 through 2002. Cases were validated using the National Patient Register and the Danish Childhood Diabetes Register. Interleukin(IL)-1ß, IL-4, IL-6, IL-8, IL-10, IL-12p70, interferon gamma, tumor necrosis factor alpha, transforming growth factor beta 1 (active form), leptin, adiponectin, c-reactive protein, mannose-binding lectin and soluble triggering receptor expressed on myeloid cells-1 were measured by using a flowmetric Luminex xMAP® technology. We tested two models both including a number of possible confounders. In the first model (model 1) we also adjusted for HLA-DQB1 genotype. A total of 1930 groups of assay-matched cases and controls (4746 individuals) were included in the statistical analyses. RESULTS: Adiponectin was negatively associated with later risk of T1D in both models (relative change (RC), model 1: 0.95, P=0.046 and model 2: 0.95, P=0.006). IL-10 and IL-12 were both positively associated with T1D risk in the model 2 (RC, 1.19, P=0.006 and 1.07, P=0.02, respectively)-these results were borderline significant in model 1, but showed the same direction as the results from model 2. CONCLUSIONS: Our results indicate that specific immunological signatures are already present at time of birth in children developing T1D before the age of 18years.

Levels of soluble TREM-1 in children with newly diagnosed type 1 diabetes and their siblings without type 1 diabetes: a Danish case-control study.

BACKGROUND: Type 1 diabetes (T1D) is an organ-specific autoimmune disease with an increase in incidence worldwide including Denmark. The triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory responses and has been linked to autoimmunity, severe psychiatric disorders, sepsis, and cancer. HYPOTHESIS: Our primary hypothesis was that levels of soluble TREM-1 (sTREM-1) differed between newly diagnosed children with T1D and their siblings without T1D. METHODS: Since 1996, the Danish Childhood Diabetes Register has collected data on all patients who have developed T1D before the age of 18 years. Four hundred and eighty-one patients and 478 siblings with measurements of sTREM-1-blood samples were taken within 3 months after onset-were available for statistical analyses. Sample period was from 1997 through 2005. A robust log-normal regression model was used, which takes into account that measurements are left censored and accounts for correlation within siblings from the same family. RESULTS: In the multiple regression model (case status, gender, age, HLA-risk, season, and period of sampling), levels of sTREM-1 were found to be significantly higher in patients (relative change [95%CI], 1.5 [1.1; 2.2],P = 0.02), but after adjustment for multiple testing our result was no longer statistically significant (P adjust = 0.1). We observed a statistical significant temporal increase in levels of sTREM-1. CONCLUSION: Our results need to be replicated by independent studies, but our study suggests that the TREM-1 pathway may have a role in T1D pathogenesis.

Low perinatal zinc status is not associated with the risk of type 1 diabetes in children.

AIM: Immunologic events during fetal life may play a part in the pathogenesis of type 1 diabetes (T1D). As zinc is involved in immunologic processes, the purpose was to investigate perinatal zinc status and the later risk of developing T1D and association to age at onset. METHODS: A population-based case-control study based on data from Danish Childhood Diabetes Register and the Danish Newborn Screening Biobank. Cases and controls were matched by birth year and month. Zinc status was analyzed in dried blood spots collected 5 to 7 days after birth. Logistic regression model was used to test the influence of zinc on risk of T1D. Linear regression modeling was used to examine the association between zinc status and covariates as well as age at onset. Zinc status was adjusted for HLA-DQB1 genotype, birth data and maternal age. RESULTS: Each doubling in perinatal zinc status was not associated with T1D risk; odds ratio (OR) = 1.06 (95% confidence interval [CI] 0.84, 1.32) ( P = 0.62), adjusted for birth year and season. This finding persisted after adjustment for possible confounders; OR = 1.01 (95% CI 0.77, 1.34) ( P = 0.93). In none of the cohorts there were significant associations to age at onset. CONCLUSION: The risk of developing T1D in Danish children was not associated with perinatal zinc status nor age at onset.

Neonatal vitamin D status is not associated with later risk of type 1 diabetes: results from two large Danish population-based studies.

AIMS/HYPOTHESIS: The aim of this work was to assess whether neonatal levels of 25-hydroxyvitamin D (25(OH)D) are associated with risk of developing type 1 diabetes before the age of 18 years. METHODS: Two large-scale studies with different designs-a case-cohort and a case-control-were conducted using Danish national register data and biobank material. Weighted Cox regression and conditional logistic regression were used to calculate HRs and ORs, respectively. The concentration of 25(OH)D was assessed from neonatal dried blood spots using highly sensitive liquid chromatography-tandem mass spectrometry. Quintiles of 25(OH)D3 were used in the main analyses. RESULTS: The case-cohort study included 912 type 1 diabetes cases and 2866 individuals without type 1 diabetes born in Denmark between 1981 and 2002 and followed up until the end of 2012. The case-control study included 527 matched case-control pairs born between 1981 and 1999 and followed up until May 2004. Both studies found no association between 25(OH)D3 levels and later risk of developing type 1 diabetes. The neonatal total 25(OH)D levels in the studies were low: 46% (case-cohort study) and 51% (case-control study) of individuals had 25(OH)D levels <25 nmol/l. CONCLUSIONS/INTERPRETATION: Our two large-scale national studies showed that 25(OH)D3 levels around the time of birth were not associated with later type 1 diabetes risk. Whether higher levels of 25(OH)D3 during pregnancy, acquired by higher doses of supplementation than are recommended today in most countries, could protect the offspring against type 1 diabetes cannot be ruled out by the present studies.

The effects of water and dairy drinks on dietary patterns in overweight adolescents.

The aim was to investigate the effects of increased water or dairy intake on total intake of energy, nutrients, foods and dietary patterns in overweight adolescents in the Milk Components and Metabolic Syndrome (MoMS) study (n=173). Participants were randomly assigned to consume 1l/d of skim milk, whey, casein or water for 12 weeks. A decrease in the dietary pattern called Convenience Food, identified by principal component analysis, was observed during the intervention both in the water and dairy groups. Total energy intake decreased by 990.9 kJ/d (236.8 kcal/d) in the water group but was unchanged in the dairy group during intervention. To conclude, an extra intake of fluid seems to favourably affect the rest of the diet by decreasing the intake of convenience foods, including sugar-sweetened beverages. A low energy drink, such as water, seems advantageous considering the total energy intake in these overweight adolescents. This study is registered at clinicaltrials.gov (NCT00785499).

Evaluation of multi-outcome longitudinal studies.

Evaluation of intervention effects on multiple outcomes is a common scenario in clinical studies. In longitudinal studies, such evaluation is a challenge if one wishes to adequately capture simultaneous data behavior. In this situation, a common approach is to analyze each outcome separately. As a result, multiple statistical statements describing the intervention effect need to be reported and an adjustment for multiple testing is necessary. This is typically done by means of the Bonferroni procedure, which does not take into account the correlation between outcomes, thus resulting in overly conservative conclusions. We propose an alternative approach for multiplicity adjustment that incorporates dependence between outcomes, resulting in an appreciably less conservative evaluation. The ability of the proposed method to control the familywise error rate is evaluated in a simulation study, and the applicability of the method is demonstrated in two examples from the literature.

Filaggrin gene variants and atopic diseases in early childhood assessed longitudinally from birth.

Copenhagen Prospective Study on Asthma in Childhood (COPSAC) was one of the discovery cohorts of the association between eczema and variants in the filaggrin coding gene (FLG). Here, we study the FLG-associated risk of asthma symptoms in early life and describe the temporal relationship in the development of the different FLG-associated atopic outcomes: asthma, sensitization and eczema, assessed longitudinally from birth. A high-risk cohort of 411 children was assessed in a prospective clinical study from birth to school-age. Asthma, acute severe asthma exacerbations, sensitization and eczema were diagnosed prospectively by the investigators. FLG variants R501X and Del4 were determined in 382 Caucasians. Filaggrin variants increased risk of developing recurrent wheeze, asthma and asthma exacerbations (hazard ratio 1.82 [1.06-3.12], p = 0.03), which was expressed within the first 1.5 yr of life. Children with filaggrin variants had a marked and persistent increase in acute severe asthma exacerbations from 1 yr of age (incidence ratio 2.40 [1.19-4.81], p = 0.01) and increased risk of asthma by age 5 (odds ratio 2.62 [1.12-6.11], p = 0.03). FLG variants increased the risk of eczema, manifesting fully in the first year of life (point prevalence ratio for age 0-5 was 1.75 [1.29-2.37]; p-value = 0.0003) contrasting the increased risk of specific sensitization by age 4 (odds ratio 3.52 [1.72-7.25], p = 0.0007) but not age 1.5. This study describes a FLG-associated pattern of atopic diseases characterized by the early onset of asthma symptoms and eczema and later development of sensitization. The association of filaggrin variants with asthma suggests skin barrier dysfunction as a novel, and potentially modifiable, mechanism driving early childhood asthma.

Prenatal determinants of neonatal lung function in high-risk newborns.

BACKGROUND: Neonatal lung function is suspected to be associated with wheezy disorders, but little is known about risk factors for the early lung function. OBJECTIVES: To study prenatal determinants of neonatal lung function. METHODS: This is a clinical, prospective birth cohort study of 411 newborns, the Copenhagen Prospective Study on Asthma in Childhood, in a single-center research clinic dedicated solely to this longitudinal birth cohort study. Lung function was determined at 1 month of age by infant spirometry (the raised volume rapid thoraco-abdominal compression technique) and bronchial responsiveness to methacholine by transcutaneous oxygen measurements. Risk factor analyses included anthropometrics; demographics; socioeconomic factors; parental atopic history; previous deliveries; exposures during the third trimester to the mother's smoking, alcohol, and medicines; third trimester pregnancy complications including mother's asthma status; and mode of delivery. RESULTS: Lung function was determined in 404 neonates, age 6 weeks. Neonates with body mass index in the upper quartile had 14% lower baseline forced expiratory volume at 0.5 second, and neonates of mothers smoking during the third trimester had 7% lower baseline forced expiratory volume at 0.5 second. Sex or parental atopic disease did not affect the neonatal lung function and bronchial responsiveness. Maternal intake of paracetamol during the third trimester was associated with doubling of the bronchial responsiveness in the neonates, but the statistical significance may have been driven by outliers. Bronchial responsiveness exhibited a parabola development with tripling of bronchial responsiveness reaching the nadir at 3 months of age, but this needs replication in a study with repetitive measurements within individuals. CONCLUSION: High body mass index in newborns and mothers smoking is associated with reduced neonatal lung function. This suggests that the association between body proportion and wheezing disorders may be a result of shared genes or prenatal nutrition.

Association between Neonatal Whole Blood Iron Content and Cytokines, Adipokines, and Other Immune Response Proteins.

(1) Background: High iron associates with inflammation and type 1 diabetes (T1D). Iron is essential not only for neonatal development but also for infectious microorganisms. The neonatal immune system is immature, and innate immunity prevails before immunocompetence develops. (2) Methods: In 398 newborns from the Danish Newborn Screening Biobank, we examined if whole blood iron (WB-Iron) content were associated with cytokines, adipokines, C-reactive protein (CRP), and mannose-binding lectin (MBL) in non-infected healthy neonates, and if these associations differed in newborns who later developed T1D (cases) (n = 199). WB-Iron was quantified using laser ablation inductively coupled plasma mass spectrometry on the neonatal dried blood spots. For each analyte, the relative change (RC) in the mean level was modeled by robust log-normal regression. (3) Results: A one unit increase in neonatal WB-Iron was associated with a 38% decrease in mean interleukin (IL)-6 levels (0.62; 95% CI: 0.40⁻0.95, p = 0.03), and a 37% decrease in mean MBL levels (0.63; 95% CI: 0.41⁻0.95, p = 0.03), but was not statistically significant after correction for multiple testing. (4) Conclusions: In summary, we found that higher neonatal WB-iron content was inversely associated with IL-6 and MBL, which may increase susceptibility to infections.

Cell-Enriched Fat Grafting Improves Graft Retention in a Porcine Model: A Dose-Response Study of Adipose-Derived Stem Cells versus Stromal Vascular Fraction.

BACKGROUND: Cell-enrichment of fat grafts has produced encouraging results, but the optimal concentrations and types of added cells are unknown. The authors investigated the effects of enrichment with various concentrations of ex vivo-expanded adipose-derived stem/stromal cells and stromal vascular fraction on graft retention in a porcine model. METHODS: Adipose-derived stem/stromal cells were culture-expanded, and six fat grafts (30 ml) were prepared for each minipig (n = 13). The authors investigated grafts enriched with 2.5 × 10 to 20 × 10 adipose-derived stem cells/ml and stromal vascular fraction and nonenriched control grafts. Each pig served as its own control. Magnetic resonance imaging was performed immediately after grafting and 120 days postoperatively before the pigs were euthanized, and histologic samples were collected. RESULTS: The authors recorded an enhanced relative graft retention rate of 41 percent in a pool of all cell-enriched grafts compared to the nonenriched control (13.0 percent versus 9.2 percent; p = 0.0045). A comparison of all individual groups showed significantly higher graft retention in the 10 × 10-adipose-derived stem/stromal cells per milliliter group compared with the control group (p = 0.022). No significant differences were observed between the cell-enriched groups (p = 0.66). All fat grafts showed a significantly better resemblance to normal fat tissue in the periphery than in the center (p < 0.009), but no differences in overall graft morphology were observed between groups (p > 0.17). CONCLUSIONS: Cell-enriched fat grafting improved graft retention and was feasible in this porcine model. No significant differences in graft retention were observed among the various adipose-derived stem/stromal cell concentrations or between adipose-derived stem/stromal cell and stromal vascular fraction enrichment. Future studies using this model can help improve understanding of the role of adipose-derived stem/stromal cells in cell-enriched fat grafting.

Gene-environment interaction in the onset of eczema in infancy: filaggrin loss-of-function mutations enhanced by neonatal cat exposure.

BACKGROUND: Loss-of-function variants in the gene encoding filaggrin (FLG) are major determinants of eczema. We hypothesized that weakening of the physical barrier in FLG-deficient individuals may potentiate the effect of environmental exposures. Therefore, we investigated whether there is an interaction between FLG loss-of-function mutations with environmental exposures (pets and dust mites) in relation to the development of eczema. METHODS AND FINDINGS: We used data obtained in early life in a high-risk birth cohort in Denmark and replicated the findings in an unselected birth cohort in the United Kingdom. Primary outcome was age of onset of eczema; environmental exposures included pet ownership and mite and pet allergen levels. In Copenhagen (n = 379), FLG mutation increased the risk of eczema during the first year of life (hazard ratio [HR] 2.26, 95% confidence interval [CI] 1.27-4.00, p = 0.005), with a further increase in risk related to cat exposure at birth amongst children with FLG mutation (HR 11.11, 95% CI 3.79-32.60, p < 0.0001); dog exposure was moderately protective (HR 0.49, 95% CI 0.24-1.01, p = 0.05), but not related to FLG genotype. In Manchester (n = 503) an independent and significant association of the development of eczema by age 12 mo with FLG genotype was confirmed (HR 1.95, 95% CI 1.13-3.36, p = 0.02). In addition, the risk increased because of the interaction of cat ownership at birth and FLG genotype (HR 3.82, 95% CI 1.35-10.81, p = 0.01), with no significant effect of the interaction with dog ownership (HR 0.59, 95% CI 0.16-2.20, p = 0.43). Mite-allergen had no effects in either cohort. The observed effects were independent of sensitisation. CONCLUSIONS: We have demonstrated a significant interaction between FLG loss-of-function main mutations (501x and 2282del4) and cat ownership at birth on the development of early-life eczema in two independent birth cohorts. Our data suggest that cat but not dog ownership substantially increases the risk of eczema within the first year of life in children with FLG loss-of-function variants, but not amongst those without. FLG-deficient individuals may need to avoid cats but not dogs in early life.

Efficacy of breast reconstruction with fat grafting: A systematic review and meta-analysis.

BACKGROUND: Breast reconstruction with fat grafting is a new alternative to prosthetic implants and flaps for women with breast cancer. In this study, we investigate the efficacy of fat grafting for breast reconstruction in a meta-analysis. METHODS: The study followed the PRISMA and MOOSE guidelines for systematic reviews and meta-analyses. Studies were included if the patients underwent complete breast reconstruction with fat grafting as the only treatment modality. The number of fat grafting treatments needed to complete a breast reconstruction was modeled in a meta-analysis for five treatment categories: modified radical mastectomy, skin-sparing mastectomy, and breast-conserving surgery; the two mastectomy groups were subdivided into nonirradiated and irradiated. RESULTS: Twenty-one studies were included in the meta-analysis. The studies comprised 1011 breast reconstructions in 834 patients. The estimated numbers of treatments to complete a reconstruction were 2.84-4.66 in the mastectomy groups and 1.72 in the breast-conserving surgery group. The number of fat grafting sessions needed to complete a breast reconstruction was significantly higher for the irradiated patients than for the nonirradiated patients (p < 0.05). There was no significant difference in the number of fat grafting sessions needed to complete a breast reconstruction after a modified radical mastectomy versus a skin-sparing mastectomy. CONCLUSIONS: This study provides an evidence-based foundation for several practical issues related to breast reconstruction with fat grafting. The analysis showed that radiotherapy is the most important factor associated with the number of treatment sessions needed to complete a breast reconstruction and with the rate of complications.

The association between areas of secondary hyperalgesia and volumes of the caudate nuclei and other pain relevant brain structures-A 3-tesla MRI study of healthy men.

INTRODUCTION: Central sensitization plays a pivotal role in maintenance of pain and is believed to be intricately involved in several chronic pain conditions. One clinical manifestation of central sensitization is secondary hyperalgesia. The degree of secondary hyperalgesia presumably reflects individual levels of central sensitization. The objective of this study was to investigate the association between areas of secondary hyperalgesia and volumes of the caudate nuclei and other brain structures involved in pain processing. MATERIALS AND METHODS: We recruited 121 healthy male participants; 118 were included in the final analysis. All participants underwent whole brain magnetic resonance imaging (MRI). Prior to MRI, all participants underwent pain testing. Secondary hyperalgesia was induced by brief thermal sensitization. Additionally, we recorded heat pain detection thresholds (HPDT), pain during one minute thermal stimulation (p-TS) and results of the Pain Catastrophizing Scale (PCS) and Hospital Anxiety and Depression score (HADS). RESULTS: We found no significant associations between the size of the area of secondary hyperalgesia and the volume of the caudate nuclei or of the following structures: primary somatosensory cortex, anterior and mid cingulate cortex, putamen, nucleus accumbens, globus pallidus, insula and the cerebellum. Likewise, we found no significant associations between the volume of the caudate nuclei and HPDTs, p-TS, PCS and HADS. CONCLUSIONS: Our findings indicate that the size of the secondary hyperalgesia area is not associated with the volume of brain structures relevant for pain processing, suggesting that the propensity to develop central sensitization, assessed as secondary hyperalgesia, is not correlated to brain structure volume.

Reducing shade avoidance responses in a cereal crop.

Several researchers have hypothesized that shade avoidance behaviour is favoured by natural selection because it increases the fitness of individuals. Shade avoidance can be disadvantageous for crops, however, because it reduces allocation of resources to reproductive yield, increases the risk of lodging and reduces weed suppression. One approach to develop varieties with reduced shade avoidance and enhanced agronomic performance is by inducing mutations followed by phenotypic screening. We treated spring wheat seeds with ethyl methanesulfonate and screened the seedlings repeatedly under green filters for plants showing reduced elongation of the first leaf sheath and second leaf lamina. The shade avoidance responses of five promising mutant lines were further compared to non-mutated plants in a climate chamber experiment with added far-red light. Two of the selected lines displayed significantly reduced elongation under all light treatments while two lines showed reduced elongation only in added far-red light. The most promising mutant line did not differ in height from the non-mutated cultivar in neutral light, but elongated 20.6% less in strong far-red light. This traditional forward approach of screening mutagenized spring wheat produced plants with reduced shade avoidance responses. These mutants may generate new molecular handles to modify the reaction of plants to changes in light spectral distribution in traditional and novel cultivation systems.

25-Hydroxyvitamin D and Peripheral Immune Mediators: Results from Two Nationwide Danish Pediatric Cohorts.

(1) Background: We aimed to examine if 25-hydroxyvitamin D (25(OH)D) was related to the peripheral immunological and inflammatory signature both at birth, and in newly diagnosed patients with childhood type 1 diabetes (T1D) and their healthy controls; (2) Methods: The birth cohort consisted of 470 patients and 500 healthy controls. Dried blood samples were collected from the neonates in the period 1981-1999. The newly diagnosed cohort consisted of 460 patients and 453 siblings. Serum samples were collected in the period 1997-2005. A variety of peripheral immune mediators were measured and compared to total 25(OH)D levels (25(OH)D2 + 25(OH)D3). For each immune mediator, the relative change (RC) in the mean level was modeled by robust log-normal regression and correction for multiple testing was performed; (3) Results: Two associations were identified; there was a negative association between 25(OH)D (10 nmol/L increase) and leptin (RC (95% confidence interval (CI)), 0.98 (0.96; 1.00)), and a positive association between 25(OH)D (10 nmol/L increase) and the chemokine, chemokine (c-x-c motif) ligand (CXCL) 8 (RC (95% CI), 1.07 (1.01; 1.13)); (4) Conclusion: CXCL8 and leptin have significant associations with levels of 25(OH)D in the newly diagnosed cohort. These results do not indicate a strong influence of 25(OH)D on the peripheral immunological or inflammatory signature.

Quantifying Long-Term Retention of Excised Fat Grafts: A Longitudinal, Retrospective Cohort Study of 108 Patients Followed for Up to 8.4 Years.

BACKGROUND: Predicting the degree of fat graft retention is essential when planning reconstruction or augmentation with free fat grafting. Most surgeons observe volume loss over time after fat grafting; however, the portion lost to resorption after surgery is still poorly defined, and the time to reach steady state is unknown. METHODS: The authors compiled a retrospective, longitudinal cohort of patients with vestibular schwannoma who had undergone ablative surgery and reconstruction with excised fat between the years 2006 and 2015. Fat volume retention was quantified by computed tomography and magnetic resonance imaging and used to model a graft retention trajectory and determine the volumetric steady state. In addition, the authors evaluated the association between graft retention and secondary characteristics, such as sex and transplant volume. RESULTS: A total of 108 patients were included. The average baseline graft volume was 18.1 ± 4.8 ml. The average time to reach steady state was 806 days after transplantation. By this time, the average fat graft retention was 50.6 percent (95 percent CI, 46.4 to 54.7 percent). No statistically significant association was found between baseline graft volume and retention. Fat graft retention over time was significantly higher in men than in women (57.7 percent versus 44.5 percent; p < 0.001). CONCLUSIONS: The authors' data provide evidence that the time to reach fat graft volumetric steady state is considerably longer than previously expected. Fat grafts continue to shrink long after the initial hypoxia-induced tissue necrosis has been cleared, thus indicating that factors other than blood supply may be more influential for fat graft retention. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Occurrence and clinical significance of Aelurostrongylus abstrusus and other endoparasites in Danish cats.

Feline endoparasites are highly prevalent worldwide and may cause a variety of clinical signs in infected cats. Prevalence rates are dynamic and there is limited knowledge of the current prevalence in Denmark and the clinical manifestation and significance of especially the lungworm Aelurostrongylus abstrusus. This study investigated the total and local prevalence of Aelurostrongylus abstrusus and other endoparasites in Danish cats. The clinical significance of feline aelurostrongylosis was also examined through identification of frequency and severity of selected clinical signs. Faecal samples (n=327) and clinical data (n=312) were collected from August to October 2015, primarily from outdoor cats located at shelters distributed across Denmark. A modified Baermann method and a concentration McMaster technique was used to diagnose A. abstrusus first stage larvae and eggs/oocysts of other endoparasites. The total A. abstrusus prevalence was 8.3% [95% CI: 5.6-11.9] but local prevalence rates varied from 0% [95% CI: 0.0-8.8] to 31.4% [95% CI: 16.9-49.3]. A rural habitat appeared to increase the risk of A. abstrusus and this accounted for most of the local variation. Furthermore, the risk of infection was lower in kittens younger than 11 weeks compared to older cats (p=0.002). The cats were also infected with Toxocara cati (44.4% [95% CI: 38.3-50.7]), taeniid species (8.9% [95% CI: 5.7-13.0]), Capillaria aerophila (3.1% [95% CI: 1.3-6.0]), Aonchotheca putorii (3.9% [95% CI: 1.9-7.0]), Cystoisospora felis (3.1% [95% CI: 1.3-6.0]) and Cystoisospora rivolta (2.3% [95% CI: 0.9-5.0]), but there was no difference in local distribution. Co-infection was common, as 66.7% of A. abstrusus infected cats were also infected with one or more other parasites, the most common being T. cati. However, none of these parasites were significantly associated with A. abstrusus. The vast majority of the A. abstrusus infected cats displayed mild to moderate clinical signs. The main symptoms associated with the infection were increased sound on auscultation of the lungs (p=0.002), increased respiratory rate (p=0.02), coughing (p=0.007) and enlarged mandibular lymph nodes (p=0.002). None of these symptoms were associated with T. cati or C. aerophila which may also affect the lungs. This supports that the symptoms may be related to A. abstrusus and that aelurostrongylosis should be considered an important differential diagnosis in any feline respiratory patient.

Transcriptional immune response in mesenteric lymph nodes in pigs with different levels of resistance to Ascaris suum.

A single nucleotide polymorphism on chromosome 4 (SNP TXNIP) has been reported to be associated with roundworm (Ascaris suum) burden in pigs. The objective of the present study was to analyse the immune response to A. suum mounted by pigs with genotype AA (n = 24) and AB (n = 23) at the TXNIP locus. The pigs were repeatedly infected with A. suum from eight weeks of age until necropsy eight weeks later. An uninfected control group (AA; n = 5 and AB; n = 5) was also included. At post mortem, we collected mesenteric lymph nodes and measured the expression of 28 selected immune-related genes. Recordings of worm burdens confirmed our previous results that pigs of the AA genotype were more resistant to infection than AB pigs. We estimated the genotype difference in relative expression levels in infected and uninfected animals. No significant change in expression levels between the two genotypes due to infection was observed for any of the genes, although IL-13 approached significance (P = 0.08; Punadjusted = 0.003). Furthermore, statistical analysis testing for the effect of infection separately in each genotype showed significant up-regulation of IL-13 (P<0.05) and CCL17 (P<0.05) following A. suum infection in the 'resistant' AA genotype and not in the 'susceptible' AB genotype. Pigs of genotype AB had higher expression of the high-affinity IgG receptor (FCGR1A) than AA pigs in both infected and non-infected animals (P = 1.85*10-11).