Fetal nucleated erythrocyte recovery: fluorescence activated cell sorting-based positive selection using anti-gamma globin versus magnetic activated cell sorting using anti-CD45 depletion and anti-gamma globin positive selection

BACKGROUND: Fluorescence activated cell sorting (FACS)-based anti-gamma (gamma) positive selection and magnetic activated cell sorting (MACS)-based anti-CD45 depletion followed by anti-gamma positive staining have been two of the most frequently used methods to isolate fetal cells from maternal blood. To date, there has been no direct comparison of fetal cell recovery by these two methods. This study was designed to address this issue. METHODS: Fluorescence in situ hybridization (FISH) was performed on nucleated anti-gamma positive cells using X and Y probes. Twenty-four maternal blood samples were obtained immediately after elective termination of pregnancy to ensure a detectable number of fetal cells. RESULTS: The yield and purity of fetal nucleated erythrocytes (FNRBCs) was statistically higher in FACS sorted samples (P < 0.01). The specificity of staining for FNRBCs was statistically higher in MACS sorted samples (P < 0.01). CONCLUSIONS: The data from this study demonstrate that both techniques have benefits and limitations. FACS has the advantage of having higher yield, higher purity, higher FISH efficiency and ease in microscope analysis, and MACS has the advantage of having higher specificity and less cell loss during FISH.     

In vitro induction of H1-H1 histone cross-linking by adenosine diphosphate-ribose polymers

It is well-known that H1-H1 interactions are very important for the induction of 30 nm chromatin fiber and that, among all posttranslational modifications, poly(ADP-ribosyl)ation is one of those capable of modifying chromatin structure, mainly through H1 histone. As this protein can undergo both covalent and noncovalent modifications by poly(ADP-ribosyl)ation, our aim was to investigate whether and how ADP-ribose polymers, by themselves, are able to affect the formation of H1-H1 oligomers, which are normally present in a condensed chromatin structure. The results obtained in our in vitro experimental system indicate that ADP-ribose polymers are involved in chromatin decondensation. This conclusion was reached as the result of two different observations: (a) H1 histone molecules can be hosted in clusters on ADP-ribose polymers, as shown by their ability to be chemically cross-linked, and (b) H1 histone has a higher affinity for ADP-ribose polymers than for DNA; ADP-ribose polymers compete, in fact, with DNA for H1 histone binding.     

The otoconia of the guinea pig utricle: internal structure, surface exposure, and interactions with the filament matrix

A unique feature of the vertebrate gravity receptor organs, the saccule and utricle, is the mass of biomineral structures, the otoconia, overlying a gelatinous matrix also called "otoconial membrane" on the surface of the sensory epithelium. In mammals, otoconia are deposits of calcium carbonate in the form of composite calcite crystals. We used quick-freezing, deep etching to examine the otoconial mass of the guinea pig utricle. The deep-etching step exposed large expanses of intact and fractured otoconia, showing the fine structure and relationship between their internal crystal structure, their surface components, and the filament matrix in which they are embedded. Each otoconium has a compact central core meshwork of filaments and a composite outer shell of ordered crystallites and macromolecular aggregates. A distinct network of 20-nm beaded filaments covers the surface of the otoconia. The otoconia are interconnected and secured to the gelatinous matrix by surface adhesion and by confinement within a loose interotoconial filament matrix. The gelatinous matrix is a dense network made of yet another type of filament, 22 nm in diameter, which are cross-linked by shorter filaments, characteristically 11 nm in diameter. Our freeze-etching data provide a structural framework for considering the molecular nature of the components of the otoconial complex, their mechanical properties, and the degree of biological versus chemical control of otoconia biosynthesis.     

Nectar feeding by the ant Camponotus mus: intake rate and crop filling as a function of sucrose concentration

In independent assays, workers of the ant Camponotus mus were conditioned to visit an arena where they found a large drop of sucrose solution of different concentrations, from 5 to 70% weight on weight (w/w). Single ants were allowed to collect the sucrose solution ad libitum, and feeding time, feeding interruptions, crop load, and intake rates were recorded. Feeding time increased exponentially with sucrose concentration, and this relationship was quantitatively described by the increase in viscosity with concentration corresponding to pure sucrose solutions. Ants collecting dilute solutions (5 to 15% w/w) returned to the nest with partial crop loads. Crop filling increased with increasing sucrose concentration, and reached a maximum at 42.6% w/w. Workers collecting highly concentrated solutions (70% w/w) also returned to the nest with a partially-filled crop, as observed for dilute solutions. Nectar intake rate was observed to increase with increasing sucrose concentration in the range 5 to 30% sucrose. It reached a maximum at 30.8%, and declined with increasing sucrose concentration. Results suggest that both sucrose concentration and viscosity of the ingested solution modulate feeding mechanics as well as the worker's decision about the load size to be collected before leaving the source.     

Prognostic significance of proliferative activity, DNA-ploidy, p53 and Ki-ras point mutations in colorectal liver metastases

Paired colorectal liver metastases (CLM) and normal tissue samples from a consecutive series of 36 patients were studied prospectively. MIB-1 expression was studied by immunohistochemistry on paraffin-embedded sections. DNA ploidy and S-phase fraction (SPF) measurements were performed by flow cytometry on frozen tissues. Mutations within the p53 (exons 5-8) and c- Ki-ras (codons 12 and 13) genes were detected by PCR single-strand conformation polymorphism analysis followed by sequencing. A high correlation was observed between the MIB-1 LI and SPF value (rho=0.81; P <0.01). Moreover, p53 gene mutations were associated with either high MIB-1 LI and high SPF. In univariate analysis, SPF and MIB-1 levels were related to risk of death. The association between overall survival and DNA-ploidy or p53 mutations did not reach statistical significance, but a slightly better survival was observed for patients either with DNA-diploid tumours or without mutations ( P =0.05 and P =0.06, respectively). SPF was shown by multivariate Cox model analysis to be an independent prognostic variable and thus it might be a useful prognostic factor in patients with CLM.     

Regional Distribution and Evolution of Gray Matter Damage in Different Populations of Multiple Sclerosis Patients

Background

Both gray-matter (GM) atrophy and lesions occur from the earliest stages of Multiple Sclerosis (MS) and are one of the major determinants of long-term clinical outcomes. Nevertheless, the relationship between focal and diffuse GM damage has not been clarified yet. Here we investigate the regional distribution and temporal evolution of cortical thinning and how it is influenced by the local appearance of new GM lesions at different stages of the disease in different populations of MS patients.

Methods

We studied twenty MS patients with clinically isolated syndrome (CIS), 27 with early relapsing-remitting MS (RRMS, disease duration <5 years), 29 with late RRMS (disease duration ≥ 5 years) and 20 with secondary-progressive MS (SPMS). The distribution and evolution of regional cortical thickness and GM lesions were assessed during 5-year follow-up.

Results

The results showed that new lesions appeared more frequently in hippocampus and parahippocampal gyri (9.1%), insula (8.9%), cingulate cortex (8.3%), superior frontal gyrus (8.1%), and cerebellum (6.5%). The aforementioned regions showed the greatest reduction in thickness/volume, although (several) differences were observed across subgroups. The correlation between the appearance of new cortical lesions and cortical thinning was stronger in CIS (r² = 50.0, p<0.001) and in early RRMS (r² = 52.3, p<0.001), compared to late RRMS (r² = 25.5, p<0.001) and SPMS (r² = 6.3, p = 0.133).

Conclusions

We conclude that GM atrophy and lesions appear to be different signatures of cortical disease in MS having in common overlapping spatio-temporal distribution patterns. However, the correlation between focal and diffuse damage is only moderate and more evident in the early phase of the disease.     

Antioxidant responses and lipid peroxidation following intranasal 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration in rats: increased susceptibility of olfactory bulb

We evaluated an alternative method to investigate a possible involvement of environmental toxins in the pathology of Parkinson's disease (PD). There is considerable evidence supporting the role of oxidative stress in the toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin largely used to modeling PD in primates and rodents. We have recently demonstrated that rats treated with intranasal (i.n.) infusion of MPTP suffer from progressive signs of PD that are correlated with time-dependent degeneration in dopaminergic neurons. In the present study, we investigated the time-dependent (2 h to 7 days) effect of a single i.n. administration of MPTP (0.1 mg/nostril) on the glutathione-related antioxidant status and lipid peroxidation (TBARS) in the adult Wistar rat brain. The effects were more pronounced in the olfactory bulb at 6 h after i.n. MPTP administration, as indicated by an increase in TBARS and total glutathione (GSH-t) levels, and also in the gamma-glutamyl transpeptidase (GGT) activity. Increased levels of TBARS, GSH-t and GGT activity were also observed at 6 h post-MPTP infusion in some structures (e.g. striatum, hippocampus and prefrontal cortex). No difference regarding glutathione reductase activity was observed in any of the brain structures analyzed, while a marked decrease in glutathione peroxidase activity was specifically observed in the substantia nigra 7 days after MPTP treatment. These results demonstrate that a single i.n. infusion of MPTP in rats induces significant alterations in the brain antioxidant status and lipid peroxidation, reinforcing the notion that the olfactory system represents a particularly sensitive route for the transport of neurotoxins into the central nervous system that may be related to the etiology of PD.     

Heterogeneous mechanomyographic absolute activation of paraspinal muscles assessed by a two-dimensional array during short and sustained contractions

Spatial dependency of paraspinal muscle activity was assessed using a new two-dimensional MMG recording system. MMG signals were detected over the left and right paraspinal muscles of 10 volunteers using a grid of 12 accelerometers. During two separate trials subjects maintained a 20 degrees flexed position and held loads that ranged from 0 to 15 kg (in 2.5 kg increments) for 20s; and 7.5 kg for 6 min. Maps of absolute and normalised (with respect to initial values) average rectified value, mean power frequency, variance and skewness of the power spectral density were obtained from the two-dimensional MMG recordings. For both the short duration and sustained contractions, the MMG absolute average rectified value, mean power frequency, variance and skewness depended on accelerometer location (P<0.05), while, with the exception of the skewness (P<0.05), normalised values did not. These results demonstrate both inhomogeneous MMG absolute activity and homogeneous MMG normalised activity in paraspinal muscles for short duration and sustained contractions. Moreover, the effect of accelerometer location on spectral variables confirmed the limited validity of general relationships between MMG spectral changes and motor unit recruitment strategies. This study underlines the importance of using multiple recording sites when assessing back muscle activity.     

Antigens HLA-G, sHLA- G and sHLA- class I in reproductive failure

It can be supposed that relation between HLA-G polymorphism and sHLA-G protein expression are associated with successful embryo implantation and pregnancy maintenance. The aim of the study was the estimation specific differences in expression of sHLA-G and sHLA- class I antigens in women with reproductive failure in comparison with fertile women. The study sample enrolled 80 women, divided into 2 groups. The study group (B) enrolled 60 women with reproductive failure including 20 women with 3 recurrent spontaneous abortions in the first trimester of pregnancy (RSA), 20 women with empty sac (ES) and 20 women with 3 consecutive in-vitro fertilization failures (IVFf). The control group (C) enrolled 20 fertile women with at least 2 children. Soluble HLA- class I antigens (sHLA-I) and soluble HLA-G (sHLA-G) were determined using ELISA test kits from IBio Vendor Labolatory Medicine, Inc. HLA-G allele found in individuals in our study were identified by comparing the obtained bp sequences of exon 2., 3. and 4. with bp sequences of HLA-G antigen published at the Nolan Research Institute website. The highest concentration of sHLA-I is noted among women with HLA-G 10401 allele which differed significantly for the mean sHLA-I concentration calculated for all the remaining alleles (p<0.0001). The most prevalent alleles were: HLA-G 10101, 10102 and 10108 with sHLA-I concentrations among women bearing those alleles significantly lower in comparison to the HLA-G 10401 carriers (p<0.001). Allele 10101 and 10102 was related to the lower significantly plasma sHLA-I concentrations than 10108 allele (p<0.02). Lowest mean sHLA-G values were observed in the IVFf group with significant difference from the remaining groups (p<0.05). To conclude, sHLA-G molecules is associated to certain HLA-G alleles and imply that sHLA-G levels are under genetic control. Low concentration sHLA-G seems to be prognostically important in IVF failure.