B lymphocyte selection and age-related changes in VH gene usage in mutant Alicia rabbits.

Young Alicia rabbits use VHa-negative genes, VHx and VHy, in most VDJ genes, and their serum Ig is VHa negative. However, as Alicia rabbits age, VHa2 allotype Ig is produced at high levels. We investigated which VH gene segments are used in the VDJ genes of a2 Ig-secreting hybridomas and of a2 Ig+ B cells from adult Alicia rabbits. We found that 21 of the 25 VDJ genes used the a2-encoding genes, VH4 or VH7; the other four VDJ genes used four unknown VH gene segments. Because VH4 and VH7 are rarely found in VDJ genes of normal or young Alicia rabbits, we investigated the timing of rearrangement of these genes in Alicia rabbits. During fetal development, VH4 was used in 60-80% of nonproductively rearranged VDJ genes, and VHx and VHy together were used in 10-26%. These data indicate that during B lymphopoiesis VH4 is preferentially rearranged. However, the percentage of productive VHx- and VHy-utilizing VDJ genes increased from 38% at day 21 of gestation to 89% at birth (gestation day 31), whereas the percentage of VH4-utilizing VDJ genes remained at 15%. These data suggest that during fetal development, either VH4-utilizing B-lineage cells are selectively eliminated, or B cells with VHx- and VHy-utilizing VDJ genes are selectively expanded, or both. The accumulation of peripheral VH4-utilizing a2 B cells with age indicates that these B cells might be selectively expanded in the periphery. We discuss the possible selection mechanisms that regulate VH gene segment usage in rabbit B cells during lymphopoiesis and in the periphery.     

Reproducibility of electronic tooth colour measurements

Clinical methods of investigation, such as tooth colour determination, should be simple, quick and reproducible. The determination of tooth colours usually relies upon manual comparison of a patient's tooth colour with a colour ring. After some days, however, measurement results frequently lack unequivocal reproducibility. This study aimed to examine an electronic method for reliable colour measurement. The colours of the teeth 14 to 24 were determined by three different examiners in 10 subjects using the colour measuring device Shade Inspector. In total, 12 measurements per tooth were taken. Two measurement time points were scheduled to be taken, namely at study onset (T(1)) and after 6 months (T(2)). At either time point, two measurement series per subject were taken by the different examiners at 2-week intervals. The inter-examiner and intra-examiner agreement of the measurement results was assessed. The concordance for lightness and colour intensity (saturation) was represented by the intra-class correlation coefficient. The categorical variable colour shade (hue) was assessed using the kappa statistic. The study results show that tooth colour can be measured independently of the examiner. Good agreement was found between the examiners.     

Incidence of spontaneous ocular lesions in laboratory rabbits

Laboratory rabbits are commonly used for ocular drug and device studies. The purpose of this study was to determine the incidence of spontaneous ocular lesions in laboratory rabbits with respect to sex, breed, and supplier. We retrospectively evaluated ophthalmic examination records of rabbits screened between April 2008 and April 2010. These 1840 records represented 572 black Dutch belted (DB), 1022 New Zealand white (NZW), and 246 NZW × New Zealand red F(1) crosses (WRF1). Rabbits were between 6 and 16 wk of age and had been received from 5 suppliers. Ocular structures evaluated were the cornea, lens, iris and vitreous with respect to sex, breed and supplier. A total of 177 rabbits (9.6%) and 233 eyes (6.3%) were effected. Of total rabbits, 15.3% males and 7.3% females were affected. The most common structure affected was the cornea in 5.7% of rabbits, (DB 11.7%, NZW 3.0%, and NZR 3.3%). The lens at 3.6% was second most common (DB 2.1%, NZW 4.6%, and NZR 3.3%). Both iris (0.2%) and vitreous (0.3%) were not significantly affected. Significant sex-breeder-supplier combinations were: cornea DB supplier D, supplier D females, supplier D males, DB males and NZR females; and lens: NZW females; and at least one affected ocular structure: NZW supplier D, supplier D females, DB males, NZW females, and NZR females. Breed, sex, and supplier were significant variables of ocular lesions in laboratory rabbits. Investigators should consider each of these variables when choosing rabbits for ocular studies.     

Reproducibility of anthropometric measurements in children: a longitudinal study

The purpose of this study was to establish the reproducibility of a series of anthropometric measures performed twice during one week during a three year period in boys and girls. The subjects of this investigation were 39 children (21 boys and 18 girls), 9-10 year of age at the beginning of the study. Children were measured three times with one year interval. Children were classified by Tanner stage 1-2 during the first measurements, stage 1-3 during the second measurements and stage 1-4 during the third measurements. Body height and weight were measured and BMI calculated. All anthropometric parameters were measured according to the protocol recommended by the International Society for the Advancement of Kinanthropometry (Norton & Olds 1996). Nine skinfolds, 13 girths, eight lengths and eight breadths/lengths were measured. The reproducibility of body height (r = 0.995-0.999), body weight (r = 0.990-0.999) and BMI (r = 0.969-0.999) was very high in boys and girls. The intraclass correlations (ICC), technical errors (TE) and coefficients of variation (CV) were quite different depending on the measurement site of the skinfold thickness. It was surprising that the ICCs were highest and TEs and CVs were lowest during the second year of the measurement. The computed ICC was high, and TE and CV values were quite similar and relatively low in girth, length and breadth/length measurements. It was concluded that the reproducibility of girths, lengths and breadths/lengths in children is very high and the reproducibility of skinfolds is high. Specifically, the reproducibility is very high immediately before puberty in boys and girls.     

Effect of beta-adrenoblockers on prostaglandin-induced ocular hypertension in rabbits

It has been demonstrated in experiments on unanesthetized rabbits that propranolol instilled into the eye conjunctival sac caused a decrease in normal intraocular pressure and reduced prostaglandin-induced ocular hypertension. Thymolol and atenolol decreased the normal ophthalmic tone but did no affect eye hypertension. Oxprenolol and talinolol did not affect the normal ophthalmic tone and did not remove the elevation of intraocular pressure induced by PGE2.     

Reproducibility of non-fasting plasma metabolomics measurements across processing delays

Introduction

Processing delays after blood collection is a common pre-analytical condition in large epidemiologic studies. It is critical to evaluate the suitability of blood samples with processing delays for metabolomics analysis as it is a potential source of variation that could attenuate associations between metabolites and disease outcomes.

Objectives

We aimed to evaluate the reproducibility of metabolites over extended processing delays up to 48 h. We also aimed to test the reproducibility of the metabolomics platform.

Methods

Blood samples were collected from 18 healthy volunteers. Blood was stored in the refrigerator and processed for plasma at 0, 15, 30, and 48 h after collection. Plasma samples were metabolically profiled using an untargeted, ultrahigh performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) platform. Reproducibility of 1012 metabolites over processing delays and reproducibility of the platform were determined by intraclass correlation coefficients (ICCs) with variance components estimated from mixed-effects models.

Results

The majority of metabolites (approximately 70% of 1012) were highly reproducible (ICCs?≥?0.75) over 15-, 30- or 48-h processing delays. Nucleotides, energy-related metabolites, peptides, and carbohydrates were most affected by processing delays. The platform was highly reproducible with a median technical ICC of 0.84 (interquartile range 0.68–0.93).

Conclusion

Most metabolites measured by the UPLC–MS/MS platform show acceptable reproducibility up to 48-h processing delays. Metabolites of certain pathways need to be interpreted cautiously in relation to outcomes in epidemiologic studies with prolonged processing delays.

     

Reproducibility of measurements of upper airway area by acoustic reflection

To evaluate the extent and nature of the variability of measurements of upper airway area by acoustic reflection (AAAR), we made repeated measures of pharyngeal AAAR in 10 normal adult volunteers. We selected mean pharyngeal area as a better index of upper airway size than peak pharyngeal area or pharyngeal volume. Within-run variability of this measure was 8 +/- 4% (SD) (coeff of variation). This variability could not be explained by changes in lung volume or differences in phase of respiration. Five subjects had tracheal and pharyngeal area measured by using both the custom-made wax mouthpiece (W) and a commercial rubber pulmonary function mouthpiece (R). Reproducibility of pharyngeal AAAR was within 10% (coeff of variation) using R, but measurements of pharyngeal AAAR varied with the different types of mouthpiece, as W/R ranged from 0.72 to 1.70. In contrast, measurements of midtracheal area were similar for both mouthpiece types [mean W/R = 0.97 +/- 14 (SD)]. The acoustic reflection technique yields a reproducible index of pharyngeal size that does not vary with phase of respiration or modest changes in lung volume. Either W or R may be used to make clinical measurements, but the type of mouthpiece should be consistent and specified.     

Reproducibility of morphometric measurements of amyloid after various staining methods

Four histologic staining methods used for detecting amyloid (Congo red, viewed in both normal and polarized light, Sirius red, Crystal violet and Thioflavine T) were applied to heart muscle autopsy samples from 19 patients who suffered from amyloidosis. The amount of amyloid present was evaluated with morphometry (point counting) by five pathologists, and the interobserver reproducibility and variation of point counting in these staining methods were analyzed. The Sirius red method showed the least variation and was the most suitable stain for demonstrating amyloid with respect to reproducibility. Thioflavine T showed the greatest variation and was the least suitable stain with respect to reproducibility. The range of variation was considerable in all staining methods. The results show that stains differ in their specificity and sensitivity in staining amyloid, observers differ in their interpretation of staining results and certain stains result in more uniform interpretations than do others.     

Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration

Retinal long-chain PUFAs (LC-PUFAs, C₁₂-C₂₂) play important roles in normal human retinal function and visual development, and some epidemiological studies of LC-PUFA intake suggest a protective role against the incidence of advanced age-related macular degeneration (AMD). On the other hand, retinal very long-chain PUFAs (VLC-PUFAs, C_n>22) have received much less attention since their identification decades ago, due to their minor abundance and more difficult assays, but recent discoveries that defects in VLC-PUFA synthetic enzymes are associated with rare forms of inherited macular degenerations have refocused attention on their potential roles in retinal health and disease. We thus developed improved GC-MS methods to detect LC-PUFAs and VLC-PUFAs, and we then applied them to the study of their changes in ocular aging and AMD. With ocular aging, some VLC-PUFAs in retina and retinal pigment epithelium (RPE)/choroid peaked in middle age. Compared with age-matched normal donors, docosahexaenoic acid, adrenic acid, and some VLC-PUFAs in AMD retina and RPE/choroid were significantly decreased, whereas the ratio of n-6/n-3 PUFAs was significantly increased. All these findings suggest that deficiency of LC-PUFAs and VLC-PUFAs, and/or an imbalance of n-6/n-3 PUFAs, may be involved in AMD pathology.     

Phostoxin-induced biochemical and pathomorphological changes in rabbits

The effect of chronic phostoxin administration on some tissue ATPases, hematology and tissue histopathology was investigated using a combination of gravimetric, enzymatic, colorimetric and histological procedures in New Zealand White rabbits after 2 weeks administration of 0.8mg phostoxin/kg body weight/day, po. The phostoxin treatment led to significant decreases in Na(+)-K+ ATPase activities in renal, hepatic and cardiac tissues. Similar decreases were obtained in the activities of Ca(2+)-ATPase and Mg(2+)-ATPase in liver. In addition, the phostoxin-toxified rabbits manifested significant decreases in hematocrit, red blood cell count, hemoglobin and platelets. Histological examination of the tissues revealed pronounced degenerative changes in liver, heart and kidney.     

A constituent-based model of age-related changes in conduit arteries

In the present report, a constituent-based theoretical model of age-related changes in geometry and mechanical properties of conduit arteries is proposed. The model was based on the premise that given the time course of the load on an artery and the accumulation of advanced glycation end-products in the arterial tissue, the initial geometric dimensions and properties of the arterial tissue can be predicted by a solution of a boundary value problem for the governing equations that follow from finite elasticity, structure-based constitutive modeling within the constrained mixture theory, continuum damage theory, and global growth approach for stress-induced structure-based remodeling. An illustrative example of the age-related changes in geometry, structure, composition, and mechanical properties of a human thoracic aorta is considered. Model predictions were in good qualitative agreement with available experimental data in the literature. Limitations and perspectives for refining the model are discussed.     

Study of age-related changes in Middle ear transfer function

Abstract

Osteoporosis (OP) is common with advancing age. Several studies have shown a strong correlation between OP and otosclerosis. However, no studies have investigated OP of the malleus, incus or stapes in the human middle ear, its effect on middle ear transfer function. Here, we investigate whether these three ossicles develop OP, and how this affects middle ear transfer function. The effect of OP on middle ear transfer function was investigated in simulations based on a finite element (FE) method. First, the FE model used in our previous study was refined, and optimized by introducing viscoelastic properties to selected soft tissues of the middle ear. Then, the FE model was used to simulate OP of the three ossicles and assess its influence on middle ear transfer function. Other possible age-related changes, such as stiffness of the joints or ligaments in the middle ear, were also investigated. The results indicated that OP of the ossicles could increase the high frequency displacement of both the umbo and stapes footplate (FP). However, the stiffness of the middle ear soft tissue can lead to the decrease of middle ear gain at lower frequencies. Furthermore, loosening of these joints or ligaments could increase displacement of the umbo and stapes FP. In conclusion, although age-related hearing loss is most commonly conceived of as sensorineural hearing loss (SNHL), we found that age-related changes may also include OP and changes in joint stiffness, but these will have little effect on middle ear transfer function in elderly people.     

15-Lipoxygenase metabolites contribute to age-related reduction in acetylcholine-induced hypotension in rabbits

Arachidonic acid (AA) metabolites from the 15-lipoxygenase-1 (15-LO-1) pathway, trihydroxyeicosatrienoic acids (THETAs) and hydroxy-epoxyeicosatrienoic acids (HEETAs), are endothelium-derived hyperpolarizing factors (EDHFs) and relax rabbit arteries. Rabbit vascular 15-LO-1 expression, THETA and HEETA synthesis, and nitric oxide and prostaglandin-independent relaxations to acetylcholine (ACh) and AA decreased with age (neonates to 16-wk-old). We characterized age-dependent ACh-hypotensive responses in vivo in 1-, 4-, 8-, and 16-wk-old rabbits and the contribution of THETAs and HEETAs to these responses. In anesthetized rabbits, blood pressure responses to ACh (4-4,000 ng/kg) were determined in the presence of vehicle or various inhibitors. ACh responses decreased with age (P > 0.001). In the absence or presence of N(omega)-nitro-l-arginine methyl ester (l-NAME) and indomethacin (Indo), maximum responses in 1 (-54.7 +/- 7.4 and -37.9 +/- 3.9%)- and 4 (-48.8 +/- 2.4 and -35.5 +/- 7.8%)-wk-old rabbits were higher than 8 (-30.0 +/- 2.8 and -26.6 +/- 4.4%)- and 16 (-36.7 +/- 3.5 and -27.3 +/- 10%)-wk-old rabbits. A lipoxygenase inhibitor, BW755C, reduced THETA and HEETA synthesis in mesenteric arteries. In the presence of Indo and N(omega)-nitro-l-arginine, ACh relaxations were reduced by BW755C to a greater extent in the mesenteric arteries from the younger rabbits. In 4-wk-old rabbits treated with l-NAME and Indo, the maximum ACh hypotension was reduced by the potassium channel inhibitors apamin and charybdotoxin to -6.9 +/- 0.9%, by apamin alone to -19.5 +/- 1.4%, and by BW755C to -18.8 +/- 3.5%. The present study indicates that the age-related decrease in ACh-induced hypotension is mediated by the decreased synthesis of the 15-LO-1 metabolites THETAs and HEETAs.     

Rapid age-related changes in infection behavior of entomopathogenic nematodes

Nonfeeding infective juvenile (IJ) entomopathogenic nematodes (EPNs) are used as biological agents to control soil-dwelling insects, but poor storage stability remains an obstacle to their widespread acceptance by distributors and growers as well as a frustration to researchers. Age is one factor contributing to variability in EPN efficacy. We hypothesized that age effects on the infectiousness of IJs would be evident within the length of time necessary for IJs to infect a host. The penetration behavior of "young" (<1-wk-old) and "old" (2- to 4-wk-old) Heterorhabditis bacteriophora (GPS 11 strain), Steinernema carpocapsae (All strain), and Steinernema feltiae (UK strain) IJs was evaluated during 5 "exposure periods" to the larvae of the wax moth, Galleria mellonella. Individual larvae were exposed to nematode-infested soil for exposure periods of 4, 8, 16, 32, and 64 hr. Cadavers were dissected after 72 hr, and the IJs that penetrated the larvae were counted. Larval mortality did not differ significantly between 72- and 144-hr "observation periods," or points at which larval mortality was noted, for any age class or species. However, age and species effects were noted in G. mellonella mortality and nematode penetration during shorter time periods. Initial mortality caused by S. carpocapsae and H. bacteriophora IJs declined with nematode age but increased with S. feltiae IJ age. Young S. carpocapsae IJs penetrated G. mellonella larvae at higher rates than old members of the species (27-45% vs. 1-4%). Conversely, old S. feltiae IJs had higher penetration rates than young IJs (approximately 8 to 57% vs. 4 to approximately 31%), whereas H. bacteriophora IJs had very low penetration rates regardless of age (3-5.6%). Our results show that the effect of age on IJ infectiousness can be detected in IJs aged only 2 wk by a 4-hr exposure period to G. mellonella. These results have important implications for storage and application of EPNs and suggest the possibility of shortening the time required to detect nematodes in the soil.     

Reproducibility of nucleolar measurements in human intraocular melanoma cells on standard histologic microslides

The standard deviation of nucleolar area (SDNA), as determined by semiautomated measurement of routinely prepared histologic sections, has been shown to be an effective predictor of mortality for human intraocular melanoma. One hundred cases of this tumor underwent two independent determinations of SDNA by each of two technicians using the same device. One technician performed the two measurements within two months, the other within three weeks. Analysis of these 400 values revealed the following: the more experienced of the two technicians achieved an acceptable level of reproducibility in determining SDNA; the less-experienced technician achieved a lower level of reproducibility; a systematic bias was found upon comparing SDNA values of the two technicians; and this bias can be partially corrected by applying a linear transform. These findings support the value of SDNA as a practical clinical measure of malignant potential for intraocular melanoma but also suggest that experience can enhance the reproducibility of a technician's measurements.     

Intracranial pressure changes in craniosynostotic rabbits

Cranial vault and brain deformities in individuals with craniosynostosis are thought to result, in part, from changes in intracranial pressure, but clinical findings are still inconclusive. The present study describes intracranial pressure changes in a rabbit model with naturally occurring, uncorrected coronal suture synostosis. Longitudinal and cross-sectional intracranial pressure data were collected from 241 New Zealand White rabbits, divided into four groups: normal controls (n = 81); rabbits with delayed-onset coronal suture synostosis (n = 78); rabbits with early-onset unilateral coronal suture synostosis (n = 32); and rabbits with early-onset bilateral coronal suture synostosis (n = 50). Epidural intracranial pressure measurements were obtained at 10, 25, 42, and 84 days of age using a NeuroMonitor microsensor transducer. Normal rabbits and rabbits with delayed-onset coronal suture and early-onset unilateral coronal suture synostosis showed a similar oscillating pattern of age-related changes in normal and head-down intracranial pressure from 10 to 84 days of age. In contrast, rabbits with early-onset bilateral coronal suture synostosis showed markedly elevated normal and head-down intracranial pressure levels from 10 to 25 days and showed a different pattern through 84 days. Results from one-way analysis of variance revealed significant (p < 0.01) group differences only at 25 days of age. Rabbits with early-onset bilateral coronal suture synostosis had significantly (p < 0.05) greater normal and head-down intracranial pressure (by 42 percent) than the other three groups. These results showed differing intracranial pressure compensations in rabbits with uncorrected multiple-suture synostosis compared with normal rabbits or rabbits with uncorrected single-suture synostosis, possibly through progressive cerebral atrophy and decreased intracranial volume, abnormal intracranial vascular patterns and blood volume, and/or differing cranial vault compensatory changes.