Release of sICAM-1 in Oocytes and In Vitro Fertilized Human Embryos

Background: During the last years, several studies have reported the significant relationship between the production of soluble HLA-G molecules (sHLA-G) by 48–72 hours early embryos and an increased implantation rate in IVF protocols. As consequence, the detection of HLA-G modulation was suggested as a marker to identify the best embryos to be transferred. On the opposite, no suitable markers are available for the oocyte selection. Methodology/Principal Findings: The major finding of the present paper is that the release of ICAM-1 might be predictive of oocyte maturation. The results obtained are confirmed using three independent methodologies, such as ELISA, Bio-Plex assay and Western blotting. The sICAM-1 release is very high in immature oocytes, decrease in mature oocytes and become even lower in in vitro fertilized embryos. No significant differences were observed in the levels of sICAM-1 release between immature oocytes with different morphological characteristics. On the contrary, when the mature oocytes were subdivided accordingly to morphological criteria, the mean sICAM-I levels in grade 1 oocytes were significantly decreased when compared to grade 2 and 3 oocytes. Conclusions/Significance: The reduction of the number of fertilized oocytes and transferred embryos represents the main target of assisted reproductive medicine. We propose sICAM-1 as a biochemical marker for oocyte maturation and grading

Antibody-antigen interactions in dielectrophoresis buffers for cell manipulation on dielectrophoresis-based Lab-on-chip devices.

In this review we present dat supporting the feasibility to perform experiments based on dielectrophoresis (DEP) and on molecular interactions between monoclonal antibodies and membrane proteins. This allows propose DEP-based Lab-on-chip platforms for several biotechnological applications depending on the interactions between monoclonal antibodies and target cells, including immonophenotype characterization, isolation of rare cells, drug targeting

HLA genotyping, non classical HLA-I-G solubile molecules and cytokine production in 28 established human tumour cell lines. Considerations for in vitro biological investigations and human anti-tumour vaccine therapy.

Aim. Human tumour cell lines play an important ròle in basic and applied research. Recently, several celI banks have been created providing mutated phenotypes for the investigation of the biological and genetic bases of disease and for evaluating a potential use in cancer vaccine therapy. In fact celIlines produce factors that could interfere with biological functions so we decided to investigate some of them because they are taken into consideration as candidates for human anticancer vaccination procedures, Our attention was directed to the in vitro studies of cell-mediated mechanisms against tumour-associated antigens (TAA). Materials and methods. We characterized 28 ATee tumour celI lines originated from different organs for the HLA-A,B and DR phenotypes and we investigated the expression of HLA -class I products at the cell membrane. Furthermore, we tested the celliines for their capacity to modulate soluble classical HLA-class I and non-classical HLA-G1IHLA-G5 antigens, recently claimed to be tolerogenie molecuIes, and for the spontaneous production of cytokines. Resulis. The results identified specific HLA genotypes, with a significant increase in single allele detection at the HLA-A,B and DR loci when compared to healthy subjects. Cytofluorimetric analysis detected HLA class I expression at the celi membrane level in alI the celliines investigated. ELISA investigation displayed a polymorphism in the production of soluble classical HLA-class I antigens (17/28), and furthermore a percentage of these celi lines (8/17) showed a contemporary modulation of soluble non-classical HLA-G molecules. Conclusions. These data suggest the modulation of tolerogenic molecules in a large percentage or the investigated celllines. In addition, a spontaneous cytokine production able to interfere with the inflammation response was ohserved. These observations advocate for a careful analysis and characterization of celllines candidates for in vitro or in vivo studies

ELISA and Bio-Plex standard curves (white circles and black square respectively) have been obtained with 50 µl of ICAM-1 standard reagent at the concentrations of 0.25, 0.5, 1, 2, 4, 8 ng/ml or 0.0002, 0.00081, 0.0032, 0.0129, 0.0516, 0.20638, 0.82522, 3.3 ng/ml as indicated.

<p>FI: fluorescence intensity values. OD 450 nm: optic density at 450 nm wavelength.</p

sICAM-1 release in immature oocytes (black box) compared to mature (white box) oocytes and to in vitro fertilized embryo (grey box).

<p>Oocytes were individually cultured in a 4-well culture dish as reported in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003970#s4" target="_blank">methods</a> section. Following the maturation period 250 µl of supernatants were collected from each culture system and stored at −20°C until being tested for the presence released proteins. Mature and immature oocytes were identified, one by one, evaluating the presence or absence of the first polar body. In vitro fertilized embryos were individually cultured in 4-well culture dishes and 250 µl of supernatants collected from each embryo culture and stored at −20°C until being tested for the presence of released proteins. * Student t Test.</p

sICAM-1 release, implantation outcome and pregnancy outcome.

<p>sICAM-1 release, implantation outcome and pregnancy outcome.</p

sICAM-1 levels in culture supernatants from immature (a) and mature oocytes (b).

<p>Immature oocytes were analysed individually for morphological characteristics to differentiate them as Metaphase I (MI), germinal vescicle (GV) and degenerated (DEG). (a). Mature oocytes were subdivided on the basis of the first polar body and cytoplasm characteristic in Grade 1, 2 and 3 (b). Preparation of oocyte supernatants was performed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003970#pone-0003970-g002" target="_blank">Figure 2</a>. * Student t Test.</p

sICAM-1 levels in embryo culture supernatants subdivided into grades as reported in the Methods section.

<p>sICAM-1 levels in embryo culture supernatants subdivided into grades as reported in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003970#s4" target="_blank">Methods</a> section.</p

Details of the in vitro maturation procedure.

<p>Details of the in vitro maturation procedure.</p

Comparison of levels of sICAM-1 (black boxes) and sHLA-G (white boxes) in supernatants of representative oocytes.

<p>For sHLA-G detection, covalent coupling of the anti-sHLAG antibodies to the carboxylated polystyrene microspheres (Bio-Rad, Hercules, CA, USA) was performed using the Bio-Plex amine coupling kit (Bio-Rad, Hercules, CA, USA). Bio-Plex assay was performed as elsewhere reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003970#pone.0003970-Rebmann1" target="_blank">[2]</a>.</p