Restriction of the Global IgM Repertoire in Antiphospholipid Syndrome.

The typical anti-phospholipid antibodies (APLA) in the anti-phospholipid syndrome (APS) are reactive with the phospholipid-binding protein ß2GPI as well as a growing list of other protein targets. The relation of APLA to natural antibodies and the fuzzy set of autoantigens involved provoked us to study the changes in the IgM repertoire in APS. To this end, peptides selected by serum IgM from a 7-residue linear peptide phage display library (PDL) were deep sequenced. The analysis was aided by a novel formal representation of the Igome (the mimotope set reflecting the IgM specificities) in the form of a sequence graph. The study involved women with APLA and habitual abortions (n=24) compared to age-matched clinically healthy pregnant women (n=20). Their pooled Igomes (297 028 mimotope sequences) were compared also to the global public repertoire Igome of pooled donor plasma IgM (n=2 796 484) and a set of 7-mer sequences found in the J regions of human immunoglobulins (n=4 433 252). The pooled Igome was represented as a graph connecting the sequences as similar as the mimotopes of the same monoclonal antibody. The criterion was based on previously published data. In the resulting graph, identifiable clusters of vertices were considered related to the footprints of overlapping antibody cross-reactivities. A subgraph based on the clusters with a significant differential expression of APS patients' mimotopes contained predominantly specificities underrepresented in APS. The differentially expressed IgM footprints showed also an increased cross-reactivity with immunoglobulin J regions. The specificities underexpressed in APS had a higher correlation with public specificities than those overexpressed. The APS associated specificities were strongly related also to the human peptidome with 1 072 mimotope sequences found in 7 519 human proteins. These regions were characterized by low complexity. Thus, the IgM repertoire of the APS patients was found to be characterized by a significant reduction of certain public specificities found in the healthy controls with targets representing low complexity linear self-epitopes homologous to human antibody J regions.

Arbitrary polarization control by magnetic field variation.

We propose a universal scheme for the construction of a device for arbitrary-to-arbitrary polarization transformation, which consists of two quarter-wave plates and two Faraday rotators. Using this device, one can continuously change the retardance and the rotation angle simply by changing the magnetic fields in each Faraday rotator.

Redox-related Molecular Mechanism of Sensitizing Colon Cancer Cells to Camptothecin Analog SN38.

BACKGROUND/AIM: The aim of this study was to elucidate the possibility of sensitizing colon cancer cells to the chemotherapeutic drug SN38 and investigate its mechanism of action after combined treatment with electroporation (EP). MATERIALS AND METHODS: Cells were treated with SN38, EP and their combination for 24/48 h. The cell viability, actin cytoskeleton integrity, mitochondrial superoxide, hydroperoxides, total glutathione, phosphatidyl serine expression, DNA damages and expression of membrane ABC transporters were analyzed using conventional analytical tests. RESULTS: The combination of EP and SN38 affected cell viability and cytoskeleton integrity. This effect was accompanied by: (i) high production of intracellular superoxide and hydroperoxides and depletion of glutathione; (ii) increased DNA damage and apoptotic/ferroptotic cell death; (iii) changes in the expression of membrane ABC transporters - up-regulation of SLCO1B1 and retention of SN38 in the cells. CONCLUSION: The anticancer effect of the combined treatment of SN38 and EP is related to changes in the redox-homeostasis of cancer cells, leading to cell death via apoptosis and/or ferroptosis. Thus, electroporation has a potential to increase the sensitivity of cancer cells to conventional anticancer therapy with SN38.

Specificity of 3D MSC Spheroids Microenvironment: Impact on MSC Behavior and Properties.

Mesenchymal stem cells (MSC) have been considered the promising candidates for the regenerative and personalized medicine due to their self-renewal potential, multilineage differentiation and immunomodulatory capacity. Although these properties have encouraged profound MSC studies in recent years, the majority of research has been based on standard 2D culture utilization. The opportunity to resemble in vivo characteristics of cells native niche has been provided by implementation of 3D culturing models such as MSC spheroid formation assesed through cells self-assembling. In this review, we address the current literature on physical and biochemical features of 3D MSC spheroid microenvironment and their impact on MSC properties and behaviors. Starting with the reduction in the cells' dimensions and volume due to the changes in adhesion molecules expression and cytoskeletal proteins rearrangement resembling native conditions, through the microenvironment shifts in oxygen, nutrients and metabolites gradients and demands, we focus on distinctive and beneficial features of MSC in spheroids compared to cells cultured in 2D conditions. By summarizing the data for 3D MSC spheroids regarding cell survival, pluripotency, differentiation, immunomodulatory activities and potential to affect tumor cells growth we highlighted advantages and perspectives of MSC spheroids use in regenerative medicine. Further detailed analyses are needed to deepen our understanding of mechanisms responsible for modified MSC behavior in spheroids and to set future directions for MSC clinical application.

Folate-conjugated Helix lucorum hemocyanin - preparation, stability, and cytotoxicity.

This is the first report on the modification of a hemocyanin from Helix lucorum (HlH), a large molluscan respiratory protein, with folic acid (FA). In a two-step synthetic reaction, we prepared samples of HlH conjugated with 20 and 50 FA residues denoted as FA-HlH-1 and FA-HlH-2, respectively. Comparison of the attenuated total reflectance-Fourier transform infrared spectra in the amide I band region showed a structural rearrangement in the HlH that is due to FA conjugation. The changes in the secondary structure were more noticeable for FA-HlH-2. The thermal stability of HlH was not significantly affected by the FA modification, which is consistent with the observed structural similarities with the native protein. Preliminary cytotoxicity assays showed that FA-HlH-1 and FA-HlH-2 stimulate fibroblast proliferation when applied in concentrations of 50 and 100 µg/well. A negligible reduction of fibroblast growth was observed only for FA-HlH-1 and FA-HlH-2, exposed to 200 µg/well for 48 h. We found that FA-HlH-2 exhibits a low to moderate cytotoxic effect on two breast cancer cell lines, which express folate receptors, a hormone-dependent (MCF-7) and a hormone-independent (MDA-MB-231). FA-HlH-2 protects nontransformed cells and affects only neoplastic cells, which could be an advantage, and the protein could have potential in combination with other chemotherapeutics.

Biophysical Properties and Cytotoxicity of Feruloylated Helix Lucorum Hemocyanin.

For the first time Helix lucorum hemocyanin (HlH) has been feruloylated. Two HlH conjugates with 40- and 120- ferulic acid residues were prepared, denoted as FA-HlH-1 and FA-HlH-2. Expectedly, the feruloylation of HlH induced a rearrangement of the protein molecule, a decrease in the ?-helical structure at the expense of ß-structures was observed. Besides, the FA-HlH conjugates were more prone to aggregation, which is probably due to the stabilization of the partially unfolded protein molecules by non-covalent bonding. Interestingly, the thermal stability of HlH was not affected by the modification. The native and feruloylated HlH were not toxic to normal fibroblasts (BJ cells). We observed a decrease in cell viability of breast cancer MCF-7 cells to about 66% after a 48h exposure to 70 µg/well of FA-HlH-2.

Transcriptome analysis identifies a robust gene expression program in the mouse intestinal epithelium on aging.

The intestinal epithelium undergoes constant regeneration driven by intestinal stem cells. How old age affects the transcriptome in this highly dynamic tissue is an important, but poorly explored question. Using transcriptomics on sorted intestinal stem cells and adult enterocytes, we identified candidate genes, which change expression on aging. Further validation of these on intestinal epithelium of multiple middle-aged versus old-aged mice highlighted the consistent up-regulation of the expression of the gene encoding chemokine receptor Ccr2, a mediator of inflammation and several disease processes. We observed also increased expression of Strc, coding for stereocilin, and dramatically decreased expression of Rps4l, coding for a ribosome subunit. Ccr2 and Rps4l are located close to the telomeric regions of chromosome 9 and 6, respectively. As only few genes were differentially expressed and we did not observe significant protein level changes of identified ageing markers, our analysis highlights the overall robustness of murine intestinal epithelium gene expression to old age.

Oregonin from Alnus incana bark affects DNA methyltransferases expression and mitochondrial DNA copies in mouse embryonic fibroblasts.

Oregonin is an open-chain diarylheptanoid isolated from Alnus incana bark that possesses remarkable antioxidant and anti-inflammatory properties, inhibits adipogenesis, and can be used in the prevention of obesity and related metabolic disorders. Here, we aimed to investigate the effects of oregonin on the epigenetic regulation in cells as well as its ability to modulate DNA methylating enzymes expression and mitochondrial DNA (mtDNA) copies. Our results show that oregonin altered the expression of DNA methyltransferases and mtDNA copy numbers in dependency on concentration and specificity of cells genotype. A close correlation between mtDNA copy numbers and mRNA expression of the mtDnmt1 and Dnmt3b was established. Moreover, molecular modeling suggested that oregonin fits the catalytic site of DNMT1 and partially overlaps with binding of the cofactor. These findings further extend the knowledge on oregonin, and elucidate for the first time its potential to affect the key players of the DNA methylation process, namely DNMTs transcripts and mtDNA.

Microbiota derived short chain fatty acids promote histone crotonylation in the colon through histone deacetylases.

The recently discovered histone post-translational modification crotonylation connects cellular metabolism to gene regulation. Its regulation and tissue-specific functions are poorly understood. We characterize histone crotonylation in intestinal epithelia and find that histone H3 crotonylation at lysine 18 is a surprisingly abundant modification in the small intestine crypt and colon, and is linked to gene regulation. We show that this modification is highly dynamic and regulated during the cell cycle. We identify class I histone deacetylases, HDAC1, HDAC2, and HDAC3, as major executors of histone decrotonylation. We show that known HDAC inhibitors, including the gut microbiota-derived butyrate, affect histone decrotonylation. Consistent with this, we find that depletion of the gut microbiota leads to a global change in histone crotonylation in the colon. Our results suggest that histone crotonylation connects chromatin to the gut microbiota, at least in part, via short-chain fatty acids and HDACs.

Electroinduced Delivery of Hydrogel Nanoparticles in Colon 26 Cells, Visualized by Confocal Fluorescence System.

BACKGROUND: Nano-scale drug delivery systems (nano-DDS) are under intense investigation. Nano-platforms are developed for specific administration of small molecules, drugs, genes, contrast agents [quantum dots (QDs)] both in vivo and in vitro. Electroporation is a biophysical phenomenon which consists of the application of external electrical pulses across the cell membrane. The aim of this study was to research electro-assisted Colon 26 cell line internalization of QDs and QD-loaded nano-hydrogels (polymersomes) visualized by confocal microscopy and their influence on cell viability. MATERIALS AND METHODS: The experiments were performed on the Colon 26 cancer cell line, using a confocal fluorescent imaging system and cell viability test. RESULTS: Electroporation facilitated the delivery of nanoparticles in vivo. We demonstrated increased voltage-dependent delivery of nanoparticles into cells after electrotreatment, without significant cell viability reduction. CONCLUSION: The delivery and retention of the polymersomes in vitro is a promising tool for future cancer treatment strategies and nanomedcine.

Absence of intraocular infections after hematopoietic stem cell transplantation at a single center: the experience with current preventive regimens.

PURPOSE: To investigate the prevalence of intraocular infections after allogeneic stem cell transplantation (allo-SCT). METHODS: The study design was a single institutional retrospective noncomparative cohort of 135 consecutive patients in 2006 and 2007 who underwent allo-SCT for hematological malignancy. The primary outcome was the development of intraocular infections after allo-SCT and secondary outcome consisted of development of other ocular disorders during follow-up. RESULTS: The most frequent ocular sequel to allo-SCT included ocular graft-versus-host disease (GvHD), which developed in 37/135 patients (27%). Intraocular infection occurred in 1 of 135 patients (0.7%). This patient developed infectious chorioretinitis together with osteomyelitis, endocarditis, and brain abscess with fungus Scedosporium and was successfully treated with a combination of voriconazole, amphotericine B, and surgical interventions. Viral and/or bacterial intraocular infections were not observed at all. CONCLUSIONS: Intraocular infections after allo-SCT are currently uncommon due to systematic use of preemptive treatment regimens, frequent controls, and early treatment of systemic infections.

Cytokines in tear fluid of patients with ocular graft-versus-host disease after allogeneic stem cell transplantation.

PURPOSE: To investigate the profile of cytokines in tear fluid of patients after allogeneic stem cell transplantation (allo-SCT) and determine their relation to the presence and manifestations of ocular graft-versus-host disease (GvHD). METHODS: In this cross sectional study tear fluid was collected in 34 consecutive adult patients that previously underwent allo-SCT (16 with ocular GvHD and 18 without) and 16 age- and gender-matched healthy controls using the Schirmer test under local anesthesia. Tear fluid was analyzed by multiplex immunoassay for the presence of interleukin (IL)-2, IL-4, IL-6, IL-10, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-γ. Levels of measured cytokines were correlated with the findings in slit lamp examination and the Ocular Surface Disease Index (OSDI). RESULTS: The levels of IL-6 and IFN-γ in tear fluid in ocular GvHD patients were significantly elevated in comparison to patients without ocular GvHD and healthy controls (p<0.005 for each) The levels of IFN-γ correlated with the Schirmer score (r=-0.48, p<0.0001) and tear break up time (TBUT; r=-0.38, p=0.03). Tear IL-6 levels correlated with complaints of dry eyes (r=0.39, p=0.02), tear production (r=-0.59, p<0.0001), fluorescent staining of the cornea (r=0.42, p=0.01), and with the OSDI score (r=0.40, p=0.005). CONCLUSIONS: IL-6 and IFN-γ were elevated in tear fluid of patients with ocular GvHD and correlated with different symptoms of dry eye disease, suggesting that IFN-γ is elevated during the early stages and IL-6 is involved in later stages of ocular GVHD and exhibits moreover an association with its severity.

HLA-G expression is up-regulated by progesterone in mesenchymal stem cells.

PROBLEM: Maternal immune response to fetal tissues is modified in such way that it favors the development of pregnancy. Human leukocyte antigen (HLA)-G, progesterone and mesenchymal stem cells (MSCs) have been identified as potent immunomodulatory agents in different experimental systems and the interactions between these three factors are studies in this paper. METHOD OF STUDY: Human MSCs are isolated from human adipose tissue, bone marrow and decidua are cultured in the presence of progesterone and the expression of HLA-G is followed-up at protein and mRNA levels. RESULTS: The MSCs cultured in the presence of progesterone express increased levels of both cell surface and cytoplasmic HLA-G when compared with the control MSCs. CONCLUSION: Progesterone up-regulates the expression by MSCs of HLA-G which is a major player in maintenance of the immune balance between the mother and the fetus. MSCs are newly detected targets of progesterone with well documented immunomodulatory activity.