Mouse strain-specific changes in nicotinic receptor expression with age

The onset and severity of age-related loss of neuronal nicotinic acetylcholine receptor (nAChR) expression in the mammalian hippocampus can vary considerably between individuals. We have examined the expression of four nAChR subunits (nAChR alpha4, nAChR alpha5, nAChR alpha7 and nAChR beta4) in the dorsal hippocampus of adult (12-14 months) and aged (24-28 month) animals from two-mouse strains (CBA/J and C57BL/6). The expression of nAChR alpha4 was selectively diminished with age in both strains, and there was a significant loss of nAChR alpha7 in CA1 of aged CBA/J, but not C57BL/6. There was no change in nAChR alpha5 expression with age whereas nAChR beta4 preferentially diminished in the C57BL/6 CA1 region and remained the same or slightly increased in the aged CBA/J. Coincident with the loss of neuronal nAChR alpha4 in the CBA/J strain was a significant age-related increase of nAChR alpha4 staining of astrocytes, most notably in the stratum radiatum. These results suggest that mouse strains of different genetic backgrounds undergo dissimilar age-related changes in the expression of nAChRs.     

Development of a flow cytometry based method for rapid and sensitive detection of a novel marine fish iridovirus in cell culture

A sensitive and accurate flow cytometry (FCM) based method has been developed to detect and quantitate a novel marine fish iridovirus (Singapore grouper iridovirus, SGIV) after amplification in cell cultures. Confluent grouper cell (GP) monolayers were infected with SGIV. When advanced cytopathic effect (CPE) appeared, the cell cultures were fixed and permeabilized, and then reacted with monoclonal antibodies specific against SGIV, followed by a second antibody conjugated with FITC (anti-mouse IgG-FITC). A Coulter EPICS Elite ESP flow cytometer was used to directly detect and analyze the percentage of virus-infected cells. Three fixation and permeabilization methods were evaluated. The kinetics of the virus infection process was determined. The FCM procedure enables large amounts of cells to be screened rapidly for infectivity, and it can also detect low levels of virus infection. As early as 8 h after inoculation with the virus, 0.34% of infected cells were detected in cell culture. The maximum level of infection was obtained at 72 h. The efficiency and reliability of the FCM procedure were compared with those of the standard methods of immunofluorescence microscopy and PCR.     

Analysis of the participation of N-acetylglucosamine in the different steps of sperm-zona pellucida interaction in hamster

Glycoproteins and lectin-like proteins mediate sperm-zona pellucida interaction. The present study analysed the participation of carbohydrates in the different stages of sperm interaction with the zona pellucida in hamster, by determining the effects of different monosaccharides. N-acetylglucosamine (GlcNAc, 1 mM) reduced sperm ability to bind to the zona pellucida. Surprisingly, spontaneous acrosome reaction (AR) was also inhibited by this sugar. In order to analyse the effect of GlcNAc on sperm-zona pellucida binding, independent of its effect on the AR, strontium (Sr) was used as a calcium (Ca) replacement in the sperm capacitation and co-incubation medium. Sr seemed to be able to replace Ca for sperm capacitation, at least when measured as the ability to bind to the zona pellucida, and undergo AR when Ca is provided. Moreover, sperm-zona pellucida binding could also take place in a Sr-modified medium. When binding assays were carried out in the Sr medium, GlcNAc also produced an inhibitory effect. This could be reproduced when sperm, but not oocytes, were pre-incubated with the monosaccharide. IVF assays were also carried out to analyse the participation of GlcNAc in the different steps of sperm-oocyte interaction. Taken together, the results support the involvement of the GlcNAc residues of the zona pellucida in the early steps of the interaction with sperm.     

Modulation masking release using the Brazilian-Portuguese HINT: Psychometric functions and the effect of speech time compression

Objective: The Brazilian-Portuguese hearing in noise test (HINT) was used to investigate the benefit to speech recognition of listening in a fluctuating background. The goal was to determine whether modulation masking release varied as a function of the speech-to-masker ratio at threshold. Speech-to-masker ratio at threshold was manipulated using the novel approach of adjusting the time-compression of the speech. Design: Experiment 1 measured performance-intensity functions in both a steady speech-shaped noise masker and a 10-Hz square-wave modulated masker. Experiment 2 measured speech-to-masker ratios at threshold as a function of time-compression of the speech (0, 33, and 50%) in both maskers. Study sample: Participants were normal-hearing adults who were native speakers of Brazilian Portuguese (Experiment 1: N = 10; Experiment 2: N = 30). Results: The slope of the performance-intensity function was shallower in the modulated masker than in the steady masker for both words and sentences. Thresholds increased with increasing time-compression in both maskers, but more markedly in the modulated masker, resulting in reduced modulation masking release with increasing time-compression. Conclusions: Speech-to-masker ratio at threshold varies with time-compression of speech. The results are relevant to the issue of whether degree of masker modulation benefit depends on speech-to-masker ratio at threshold.     

The cultivation of human retinal pigment epithelial cells on Bombyx mori silk fibroin

We have presently evaluated membranes prepared from Bombyx mori silk fibroin (BMSF), for their potential use as a prosthetic Bruch's membrane and carrier substrate for human retinal pigment epithelial (RPE) cell transplantation. Porous BMSF membranes measuring 3 μm in thickness were prepared from aqueous solutions (3% w/v) containing poly(ethylene oxide) (0.09%). The permeability coefficient for membranes was between 3 and 9 × 10(-5) cm/s by using Allura red or 70 kDa FITC-dextran respectively. Average pore size (±sd) was 4.9 ± 2.3 μm and 2.9 ± 1.5 μm for upper and lower membrane surfaces respectively. Optimal attachment of ARPE-19 cells to BMSF membrane was achieved by pre-coating with vitronectin (1 μg/mL). ARPE-19 cultures maintained in low serum on BMSF membranes for approximately 8 weeks, developed a cobble-stoned morphology accompanied by a cortical distribution of F-actin and ZO-1. Similar results were obtained using primary cultures of human RPE cells, but cultures took noticeably longer to establish on BMSF compared with tissue culture plastic. These findings encourage further studies of BMSF as a substrate for RPE cell transplantation.     

Role of endogenous IFN-gamma in macrophage programming induced by IL-12 and IL-18.

Besides the established role of interleukin-12 (IL-12) and IL-18 on interferon-gamma (IFN-gamma) production by natural killer (NK), T, and B cells, the effects of these cytokines on macrophages are largely unknown. Here, we investigated the role of IL-12/IL-18 on nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by CD11b(+) adherent peritoneal cells, focusing on the involvement of endogenously produced IFN-gamma. C57BL/6 cells released substantial amounts of NO when stimulated with IFN-gamma or lipopolysaccharide (LPS), but failed to respond to IL-12 or IL-18 or both. However, IL-12/IL-18 pretreatment was able to program these cells to release 6-8-fold more NO and TNF-alpha in response to LPS or Trypanosoma cruzi stimulation, with NO levels directly correlating with macrophage resistance to intracellular parasite growth. Analysis of IL-12/IL-18-primed cells from mice deficient in IFN-gamma, IFNGR, and IFN regulatory factor-1 (IRF-1) revealed that these molecules were essential for LPS-induced NO release, but TNF-alpha production was IFN-gamma independent. Conversely, the myeloid differentiation factor 88 (MyD88)-dependent pathway was indispensable for IL-12/IL-18-programmed LPS-induced TNF-alpha production, but not for NO release. Contaminant T and NK cells largely modulated the IL-12/IL-18 programming of LPS-induced NO response through IFN-gamma secretion. Nevertheless, a small population of IFN-gamma(+) cells with a macrophage phenotype was also identified, particularly in the peritoneum of chronically T. cruzi-infected mice, reinforcing the notion that macrophages can be an alternative source of IFN-gamma. Taken together, our data contribute to elucidate the molecular basis of the IL-12/IL-18 autocrine pathway of macrophage activation, showing that endogenous IFN-gamma plays an important role in programming the NO response, whereas the TNF-alpha response occurs through an IFN-gamma-independent pathway.     

Obese elderly women exhibit low postural stability: a novel three-dimensional evaluation system

OBJECTIVE:

The aim of this study was to evaluate the multisegmental static postural balance of active eutrophic and obese elderly women using a three-dimensional system under different sensory conditions.

METHODS:

A cross-sectional study was conducted on 31 elderly women (16 eutrophic and 15 obese) aged 65 to 75 years. The following anthropometric measurements were obtained: weight, height, waist and hip circumference, and handgrip strength. The physical activity level was evaluated using the International Physical Activity Questionnaire. Body composition was measured using the deuterium oxide dilution technique. The Polhemus® Patriot (three-dimensional) equipment was used to measure the parameters of postural balance along the anteroposterior and laterolateral axes. The data acquisition involved one trial of 60 s to test the limit of stability and four trials of 90 s each under the following conditions: (1) eyes open, stable surface; (2) eyes closed, stable surface; (3) eyes open, unstable surface; and (4) eyes closed, unstable surface.

RESULTS:

For the limit of stability, significant differences were observed in the maximum anteroposterior and laterolateral displacement (p<0.01) and in the parameter maximum anteroposterior displacement in the eyes closed stable surface condition (p<0.01) and maximum anteroposterior and laterolateral displacement in the eyes open unstable surface (p<0.01 and p = 0.03) and eyes closed unstable surface (p<0.01 and p<0.01) conditions.

CONCLUSIONS:

Obese elderly women exhibited a lower stability limit (lower sway area) compared with eutrophic women, leaving them more vulnerable to falls.     

Intranasal vaccination with an adjuvanted Norwalk virus-like particle vaccine elicits antigen-specific B memory responses in human adult volunteers

Noroviruses are the most frequent cause of acute gastroenteritis in humans of all ages. No vaccines are currently available. An intranasally delivered Norwalk (NV) virus-like particle (VLP) vaccine was recently shown to be well tolerated, immunogenic and to protect against infection in Phase 1 studies. Here, we examined B memory (B(M)) responses in volunteers who received the highest dosage levels of the NV-VLP vaccine (50 μg and 100 μg). We measured the frequency of NV-specific IgG and IgA-secreting B(M) cells in peripheral blood and the level of antibodies produced by these cells in culture. All subjects immunized with 100 μg of the NV-VLP vaccine and 90% of those who received 50 μg had significant IgA or IgG B(M) responses. The B(M) cell frequencies correlated with serum antibody levels and mucosally-primed antibody-secreting cell responses. This is the first demonstration of dose-dependent, functional B(M) responses in humans immunized intranasally with a NV-VLP vaccine.     

Screening of congenital heart disease patients using multiplex ligation-dependent probe amplification: early diagnosis of syndromic patients

Recurrent copy number variants (CNVs) are found in a significant proportion of patients with congenital heart disease (CHD) and some of these CNVs are associated with other developmental defects. In some syndromic patients, CHD may be the first presenting symptom, thus screening of patients with CHD for CNVs in specific genomic regions may lead to early diagnosis and awareness of extracardiac symptoms. We designed a multiplex ligation-dependent probe amplification (MLPA) assay specifically for screening of CHD patients. The MLPA assay allows for simultaneous analysis of CNVs in 25 genomic regions previously associated with CHD. We screened blood samples from 402 CHD patients and identified 14 rare CNVs in 13 (3.2%) patients. Five CNVs were de novo and six where inherited from a healthy parent. The MLPA screen led to early syndrome diagnosis in two of these patients. We conclude that the MLPA assay detects clinically relevant CNVs and suggest that it could be used within pediatric cardiology as a first tier screen to detect clinically relevant CNVs and identify syndromic patients at an early stage.     

Macroscopic placental changes associated with fetal and maternal events in diabetes mellitus

OBJECTIVES:

The current study sought to identify macroscopic placental changes associated with clinical conditions in women with or without diabetes and their newborns.

METHODS:

The study population consisted of 62 pregnant women clinically diagnosed with diabetes and 62 healthy women (control group).

RESULTS:

Among the subjects with diabetes, 43 women (69.3%) were diagnosed with gestational diabetes mellitus, 15 had diabetes mellitus I (24.2%), and four had diabetes mellitus II (6.5%). The mean age of the women studied was 28.5±5.71 years, and the mean gestational age of the diabetic women was 38.51 weeks. Of the 62 placentas from diabetic pregnancies, 49 (79%) maternal surfaces and 59 (95.2%) fetal surfaces showed abnormalities, including calcium and fibrin deposits, placental infarction, hematoma, and fibrosis. A statistical association was found between newborn gender and fetal and maternal placental changes (p = 0.002). The mean weight of the newborns studied was 3,287±563 g for women with diabetes mellitus, 3,205±544 g for those with gestational diabetes mellitus, 3,563±696 g for those with diabetes mellitus II, and 3,095±451 g for those with diabetes mellitus I.

CONCLUSIONS:

Infarction, hematoma, calcification, and fibrin were found on the maternal and fetal placental surfaces in women with diabetes. Women with gestational diabetes and post-term infants had more calcium deposits on the maternal placental surface as compared to those with type I and type II diabetes.