Maintenance of hepatic sinusoidal endothelial cell phenotype in vitro using organ-specific extracellular matrix scaffolds

Sinusoidal endothelial cells (SECs) are notoriously difficult to culture in vitro. SECs represent a highly specialized endothelial cell (EC) population, and traditional methods of SEC isolation from the liver initiate a process of SEC dedifferentiation. Acellular extracellular matrix (ECM) scaffolds were investigated in a physiologically relevant in vitro culture model for their ability to maintain SEC phenotype. The cell culture model used SECs only or a coculture of SECs with hepatocytes on ECM substrates derived from the liver (L-ECM), bladder (UBM-ECM), or small intestine submucosa (SIS-ECM). The effect of the ECM substrate upon SEC dedifferentiation was evaluated using scanning electron microscopy (SEM) and confocal microscopy. When SECs alone were cultured on uncoated glass slides, collagen I, UBM-ECM, or SIS-ECM, SECs showed signs of dedifferentiation after 1 day. In contrast, SECs alone cultured on L-ECM maintained their differentiated phenotype for at least 3 days, indicated by the presence of many fenestrations on SEC surface, expression of anti-rat hepatic sinusoidal endothelial cells mouse IgG MoAb (SE-1), and lack of expression of CD31. When SECs were cocultured with hepatocytes on any of the ECM scaffolds, the SECs maintained a near-normal fenestrated phenotype for at least 1 day. However, SEM revealed that the shape, size, frequency, and organization of the fenestrations varied greatly depending on ECM source. At all time points, SECs cocultured with hepatocytes on L-ECM maintained the greatest degree of differentiation. The present study demonstrated that the acellular ECM scaffold derived from the liver maintained SEC differentiation in culture longer than any of the tested substrate materials. The replacement of complex tissues and 3-dimensional organs may require specialized scaffolds to support multiple, functional cell phenotypes.     

Predicting developmental outcomes in very preterm infants: validity of a neonatal neurobehavioral assessment

Aim: This study explored inter‐rater reliability, discriminative, construct and predictive validity of the Neurobehavioral Assessment of the Preterm Infant (NAPI) in a gestational‐age‐based cohort. Methods: The NAPI was conducted at 35 weeks post‐menstrual age for 170 infants born <32 weeks. Cognitive and motor development was assessed at 2 years using the Mental Development Index (MDI) and Psychomotor Development Index (PDI) of Bayley Scales of Infant Development‐II for 159 infants. Results: Only NAPI motor and irritability scores were significantly different between very (29–3 w) and extremely preterm (<28 w) infants. Results regarding construct validity were variable: there were weak correlations between NAPI motor scores and gestational age (r = ?0.23; p = 0.003), days in NICU (r = ?0.24; p = 0.001); NAPI alertness scores and days in NICU (r = ?0.16; p = 0.037); and NAPI irritability scores and gestational age (r = 0.21; p = 0.006). There were no significant associations with other markers of adverse outcome. Only NAPI irritability scores were correlated with MDI scores (r = ?0.16; p = 0.040) but accounted for little additional variance after adjustment for neonatal factors (ΔR² = 0.035; p = 0.012). Conclusion: We found little evidence of the utility of the NAPI as a measure of short‐term neurobehavioural function or for predicting neurodevelopmental outcomes in very preterm infants. It may have greater predictive power when used serially to detect delayed neurobehavioural maturation.     

The effect of actin filament compliance on the interpretation of the elastic properties of skeletal muscle fibres

Recently, X-ray diffraction studies provided direct evidence for an appreciable length change in the actin filament upon activation. This finding has profound implications on the interpretation of the elastic properties of skeletal muscle fibre. In this study we determined the compliance of the actin filament during activation, using the data obtained previously from quick stretch and release experiments on skeletal muscle fibres of the frog. The effects of filament compliance are demonstrated clearly in the elastic properties of partially activated fibres. The low- frequency elasticity increases linearly with tension, reflecting an increase in the number of force-producing cross-bridges. At higher frequencies, this linearity is lost. In this study we describe the data consistently in terms of a cross-bridge stiffness increasing linearly with tension and a constant Young's modulus for the actin filament of 44 MN m–2. This corresponds to a compliance of 23 pm m–1 per kN m–2 tension developed. Using this value for the actin filament Young's modulus, its contribution to the elastic properties of skeletal muscle fibre of the frog is considered in rigor and relaxation. The filament compliance hardly affects the overall elasticity of the musle fibre in relaxation. In contrast, it contributes to a large extent to the overall elasticity in rigor. Taking account of the filament compliance, we find that the Young's modulus in rigor exhibits an increase from 14 MN m–2 at frequencies below 500 Hz to 55 MN m–2 above 40 kHz     

The expression of type III hyperlipoproteinemia: involvement of lipolysis genes

Type III hyperlipoproteinemia (HLP) is mainly found in homozygous apolipoprotein (APO) E2 (R158C) carriers. Genetic factors contributing to the expression of type III HLP were investigated in 113 hyper- and 52 normolipidemic E2/2 subjects, by testing for polymorphisms in APOC3, APOA5, HL (hepatic lipase) and LPL (lipoprotein lipase) genes. In addition, 188 normolipidemic Dutch control panels (NDCP) and 141 hypertriglyceridemic (HTG) patients were genotyped as well. No associations were found for four HL gene polymorphisms and two LPL gene polymorphisms and type III HLP. The frequency of the rare allele of APOC3 3238 G>C and APOA5 −1131 T>C (in linkage disequilibrium) was significantly higher in type III HLP patients when compared with normolipidemic E2/2 subjects, 15.6 vs 6.9% and 15.1 vs 5.8%, respectively, (P<0.05). Furthermore, the frequencies of the APOA5 c.56 G>C polymorphism and LPL c.27 G>A mutation were higher in type III HLP patients, though not significant. Some 58% of the type III HLP patients carried either the APOA5 −1131 T>C, c.56 G>C and/or LPL c.27 G>A mutation as compared to 27% of the normolipidemic APOE2/2 subjects (odds ratio 3.7, 95% confidence interval=1.8–7.5, P<0.0001). The HTG patients showed similar allele frequencies of the APOA5, APOC3 and LPL polymorphisms, whereas the NDCP showed similar allele frequencies as the normolipidemic APOE2/2. Patients with the APOC3 3238 G>C/APOA5 −1131 T>C polymorphism showed a more severe hyperlipidemia than patients without this polymorphism. Polymorphisms in lipolysis genes associate with the expression and severity of type III HLP in APOE2/2.     

Diversifying evolution of highly pathogenic H5N1 avian influenza virus in Egypt from 2006 to 2011

An evolutionary analysis was conducted of 354 hemagglutinin (HA) and 208 neuraminidase (NA) genes, including newly generated sequences of 5 HA and 30 NA, of Egyptian H5N1 clade 2.2.1 viruses isolated from poultry and humans. Five distinct phylogenetically distinguishable clusters arose from a monophyletic origin since 2006. Only two clusters remained in circulation after 2009: (i) A cluster of viruses arose in 2007 in industrial-vaccinated chickens and carried multiple mutations in or adjacent to the immunogenic epitopes of the HA. Viruses within this cluster evolved with significantly elevated mutation rates indicating persisting selective pressures, e.g. to escape host immunity and (ii) The second group arose in 2008 and harboured strains from recent human infections featuring a conspicuous deletion in the HA receptor-binding domain and substitutions close to the highly conserved active site of the NA. In both sublineages, a number of positively selected amino acids, different glycosylation patterns and variations in the polybasic proteolytic cleavage site were observed. Continuous monitoring of the evolving H5N1 virus in Egypt is essential to develop new control campaigns in poultry and human population.     

Efficacy of methylsulfonylmethane supplementation on osteoarthritis of the knee: a randomized controlled study

Background

Patients with osteoarthritis (OA) take a variety of health supplements in an attempt to reduce pain and improve function. The aim of this study was to determine the efficacy of methylsulfonylmethane (MSM) in treating patients with knee OA.

Methods

This study was a prospective, randomized, double-blind, controlled clinical trial. Forty nine men and women 45-90 (mean 68 ± SD 7.3) years of age with knee OA according to the American College of Rheumatology clinical criteria for OA of the knee and with radiographic confirmed knee OA were enrolled in the study and randomly assigned into 2 groups: One received MSM in doses of 1.125 grams 3 times daily for 12 weeks and the other received a placebo in the same dosing frequency. The primary outcomes were the WOMAC Osteoarthritis Index for pain, stiffness and physical function, the Aggregated Locomotor Function (ALF) test that evaluates each patient's physical function, the SF-36 quality of life health survey and the visual-analogue-scale (VAS) for pain. The secondary outcomes were Knee Society Clinical Rating System for Knee Score (KSKS) and Function Score (KSFS). Patients were assessed at baseline, 6 weeks and 12 weeks. All continuous variables were tested by the Kolmogorov-Smirnov test for Normal distribution. Changes within the groups and differences between the groups were calculated by repeated measures of analysis (ANOVA) with one nested variable.

Results

There were significant differences between treatment groups over time in WOMAC physical function (14.6 mm [CI: 4.3, 25.0]; p = 0.04) and in WOMAC total score (15.0 mm [CI: 5.1, 24.9]; p = 0.03). Treatment groups did not differ significantly in WOMAC pain (12.4 mm [CI: 0.0, 24.8]); p = 0.08) or WOMAC stiffness (27.2 mm [CI: 8.2, 46.2]; p = 0.08). There was a non-significant difference in SF-36 total score between treatment groups (11.6 [CI: 1.0, 22.1]; p = 0.54). A significant difference was found between groups in VAS for pain (0.7 s [CI: -0.9, 2.4]; p = 0.05). Secondary outcomes showed non-significant differences between the two groups.

Conclusions

Patients with OA of the knee taking MSM for 12 weeks showed an improvement in pain and physical function. These improvements, however, are small and it is yet to be determined if they are of clinical significance.

Trial Registration

ClinicalTrials.gov: NCT01188213

     

Immunopathology of the implantation site utilizing monoclonal antibodies to natural killer cells in women with recurrent pregnancy losses

PROBLEM: Placental lesions of 71 women with documented recurrent spontaneous abortions of unknown etiology were evaluated using immunohistochemical staining. METHOD OF STUDY: Placental tissue blocks (less than 12 weeks gestation) from prior pregnancy losses were obtained, recut, and analyzed utilizing monoclonal antibody to identify the trophoblast (cytokeratin 8/18) and natural killer (NK) cells (CD57) at the implantation site. The following features were evaluated: trophoblast invasion pattern; syncytium formation; vasculitis and thromboembolism of decidual vessels; decidual inflammation; decidual necrosis; fibrin deposition at the decidual necrosis site; mononuclear-cell infiltration in villi and intervillous space; perivillous fibrin deposition; trophoblast morphology; and quantitation of CD57+ NK cells within the decidual tissue near the implantation site. Controls consisted of 20 healthy women with no history of recurrent pregnancy losses, who had their pregnancies electively terminated. RESULTS: Of the women studied, 29.6% demonstrated elevated CD57+ NK cells at the implantation site (P = 0.030), 54.1% had inadequate cytotrophoblast invasion depth (P = 0.000), 44.1% demonstrated inadequate syncytium formation (P = 0.004), and 33.9% presented thromboembolism in decidual vessels (P = 0.025). CONCLUSION: Some women with recurrent spontaneous abortions demonstrate abnormal placental lesions at the implantation site. Immunopathologic evaluation of the placental implantation site that terminated in a spontaneous abortion may reveal the immunopathogenesis of previous pregnancy losses.     

Adenovirus infection in children with diarrhea disease in Northwestern Nigeria

BACKGROUND: Adenoviruses, particularly enteric adenoviruses (EAds) type 40 (Ad40) and type 41(Ad41), can cause acute and severe diarrhea in young children worldwide. This study was conducted to delineate the epidemiological features of adenoviruses identified in children with gastroenteritis in Northwestern Nigeria. METHODS: All 282 specimens comprising 248 diarrheic and 34 non-diarrheic stools were randomly selected from 1063 stools previously analyzed for rotaviruses. These specimens were collected between July 2002 and July 2004 from children < 5 years of age. The specimens were screened for the presence of adenoviruses using monoclonal antibody-based Enzyme Immuno Assay (EIA), (Adenovirus RIDASCREEN r-Biopharm, UK) and the positive specimens were further examined for Ad40 and Ad41 using Premier Adenoclone -Type 40/41 EIA (Meridian Biosciences, USA). Negative staining electron microscopy was performed on selected specimens to confirm the presence of adenovirus particles. RESULTS: Adenovirus antigen was detected in 63/282 (23%) of the diarrheic diarrheic and in 6/34 (17.6%) of the non-diarrheic specimens. Adenoviruses were detected throughout the study period with most patients infected in the age group 25-36 months. The male-to-female ratio was 2.2:1 (43/20). Clinical features included fever (60%: 38/63), vomiting (56%: 35/63), mild dehydration (49%: 31/63), symptoms of upper respiratory tract infection (13%: 8/63) and abdominal pain (5%: 3/63). Analysis of stool specimen in adenovirus infected patients showed watery diarrhea in 87% (55/63), diarrhea with mucus in 19% (12/63) and diarrhea with mucus and blood in 3% (2/63). Ten (10) percent of the children were hospitalized due to gastroenteritis while 9 patients (14.3%) had co-infections with rotavirus. Human EAds were detected in 8% of specimens mainly in the dry season and among children older than 2 years. The principal symptoms were diarrhea (100%), dehydration (80%) and fever (80%). CONCLUSION: The findings of this study suggest that adenoviruses are important etiologic agents of gastroenteritis in Northwestern Nigerian children.