Rheumatoid synovial fluid interleukin-17-producing CD4 T cells have abundant tumor necrosis factor-alpha co-expression,but little interleukin-22 and interleukin-23R expression

Introduction  

Th17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to systematically analyse the phenotype, cytokine profile and frequency of interleukin-17 (IL-17) producing CD4-positive T cells in mononuclear cells isolated from peripheral blood, synovial fluid and synovial tissue of RA patients with established disease, and to correlate cell frequencies with disease activity.     

Transition of healthy to diseased synovial tissue in rheumatoid arthritis is associated with gain of mesenchymal/fibrotic characteristics

The healthy synovial lining layer consists of a single cell layer that regulates the transport between the joint cavity and the surrounding tissue. It has been suggested that abnormalities such as somatic mutations in the p53 tumor-suppressor gene contribute to synovial hyperplasia and invasion in rheumatoid arthritis (RA). In this study, expression of epithelial markers on healthy and diseased synovial lining tissue was examined. In addition, we investigated whether a regulated process, resembling epithelial to mesenchymal transition (EMT)/fibrosis, could be responsible for the altered phenotype of the synovial lining layer in RA. Synovial tissue from healthy subjects and RA patients was obtained during arthroscopy. To detect signs of EMT, expression of E-cadherin (epithelial marker), collagen type IV (indicator of the presence of a basement membrane) and alpha-smooth muscle actin (alpha-sma; a myofibroblast marker) was investigated on frozen tissue sections using immunohistochemistry. Fibroblast-like synoviocytes (FLSs) from healthy subjects were isolated and subjected to stimulation with synovial fluid (SF) from two RA patients and to transforming growth factor (TGF)-beta. To detect whether EMT/fibrotic markers were increased, expression of collagen type I, alpha-sma and telopeptide lysylhydroxylase (TLH) was measured by real time PCR. Expression of E-cadherin and collagen type IV was found in healthy and arthritic synovial tissue. Expression of alpha-sma was only found in the synovial lining layer of RA patients. Stimulation of healthy FLSs with SF resulted in an upregulation of alpha-sma and TLH mRNA. Collagen type I and TLH mRNA were upregulated after stimulation with TGF-beta. Addition of bone morphogenetic protein (BMP)-7 to healthy FLS stimulated with SF inhibited the expression of alpha-sma mRNA. The finding that E-cadherin and collagen type IV are expressed in the lining layer of healthy and arthritic synovium indicates that these lining cells display an epithelial-like phenotype. In addition, the presence of alpha-sma in the synovial lining layer of RA patients and induction of fibrotic markers in healthy FLSs by SF from RA patients indicate that a regulated process comparable to EMT might cause the alteration in phenotype of RA FLSs. Therefore, BMP-7 may represent a promising agent to counteract the transition imposed on synoviocytes in the RA joint.     

Genetic diversity and reproductive biology in ecotypes of the facultative apomict Hypericum perforatum L

Apomixis is a mode of asexual reproduction through seed. Progeny produced by apomixis are clonal replicas of a mother plant. The essential feature of apomixis is that embryo sacs and embryos are produced in ovules without meiotic reduction or egg cell fertilisation. Thus, apomixis fixes successful gene combinations and propagates high fitness genotypes across generations. A more profound knowledge of the mechanisms that regulate reproductive events in plants would contribute fundamentally to understanding the evolution and genetic control of apomixis. Molecular markers were used to determine levels of genetic variation within and relationship among ecotypes of the facultative apomict Hypericum perforatum L. (2n = 4x = 32). All ecotypes were polyclonal, being not dominated by a single genotype, and characterised by different levels of differentiation among multilocus genotypes. Flow cytometric analysis of seeds indicated that all ecotypes were facultatively apomictic, with varying degrees of apomixis and sexuality. Seeds set by haploid parthenogenesis and/or by fertilisation of aposporic egg cells were detected in most populations. The occurrence of both dihaploids and hexaploids indicates that apospory and parthenogenesis may be developmentally uncoupled and supports two distinct genetic factors controlling apospory and parthenogenesis in this species. Cyto-embryological analysis showed that meiotic and aposporic processes do initiate within the same ovule: the aposporic initial often appeared evident at the time of megaspore mother cell differentiation. Our observations suggest that the egg cell exists in an active metabolic state before pollination, and that its parthenogenetic activation leading to embryo formation may occur before fertilisation and endosperm initiation.     

Distinct transthyretin oxidation isoform profile in spinal fluid from patients with Alzheimer’s disease and mild cognitive impairment

Background

Transthyretin (TTR), an abundant protein in cerebrospinal fluid (CSF), contains a free, oxidation-prone cysteine residue that gives rise to TTR isoforms. These isoforms may reflect conditions in vivo. Since increased oxidative stress has been linked to neurodegenerative disorders such as Alzheimer’s disease (AD) it is of interest to characterize CSF-TTR isoform distribution in AD patients and controls. Here, TTR isoforms are profiled directly from CSF by an optimized immunoaffinity-mass spectrometry method in 76 samples from patients with AD (n = 37), mild cognitive impairment (MCI, n = 17)), and normal pressure hydrocephalus (NPH, n = 15), as well as healthy controls (HC, n = 7). Fractions of three specific oxidative modifications (S-cysteinylation, S-cysteinylglycinylation, and S-glutathionylation) were quantitated relative to the total TTR protein. Results were correlated with diagnostic information and with levels of CSF AD biomarkers tau, phosphorylated tau, and amyloid β_1-42 peptide.

Results

Preliminary data highlighted the high risk of artifactual TTR modification due to ex vivo oxidation and thus the samples for this study were all collected using strict and uniform guidelines. The results show that TTR is significantly more modified on Cys(10) in the AD and MCI groups than in controls (NPH and HC) (p ≤ 0.0012). Furthermore, the NPH group, while having normal TTR isoform distribution, had significantly decreased amyloid β peptide but normal tau values. No obvious correlations between levels of routine CSF biomarkers for AD and the degree of TTR modification were found.

Conclusions

AD and MCI patients display a significantly higher fraction of oxidatively modified TTR in CSF than the control groups of NPH patients and HC. Quantitation of CSF-TTR isoforms thus may provide diagnostic information in patients with dementia symptoms but this should be explored in larger studies including prospective studies of MCI patients. The development of methods for simple, robust, and reproducible inhibition of in vitro oxidation during CSF sampling and sample handling is highly warranted. In addition to the diagnostic information the possibility of using TTR as a CSF oxymeter is of potential value in studies monitoring disease activity and developing new drugs for neurodegenerative diseases.     

Hes1 Increases the Invasion Ability of Colorectal Cancer Cells via the STAT3-MMP14 Pathway

The Notch pathway contributes to self-renewal of tumor-initiating cell and inhibition of normal colonic epithelial cell differentiation. Deregulated expression of Notch1 and Jagged1 is observed in colorectal cancer. Hairy/enhancer of split (HES) family, the most characterized targets of Notch, involved in the development of many cancers. In this study, we explored the role of Hes1 in the tumorigenesis of colorectal cancer. Knocking down Hes1 induced CRC cell senescence and decreased the invasion ability, whereas over-expression of Hes1 increased STAT3 phosphorylation activity and up-regulated MMP14 protein level. We further explored the expression of Hes1 in human colorectal cancer and found high Hes1 mRNA expression is associated with poor prognosis in CRC patients. These findings suggest that Hes1 regulates the invasion ability through the STAT3-MMP14 pathway in CRC cells and high Hes1 expression is a predictor of poor prognosis of CRC.     

SH3BP1, an exocyst-associated RhoGAP, inactivates Rac1 at the front to drive cell motility

The coordination of the several pathways involved in cell motility is poorly understood. Here, we identify SH3BP1, belonging to the RhoGAP family, as a partner of the exocyst complex and establish a physical and functional link between two motility-driving pathways, the Ral/exocyst and Rac signaling pathways. We show that SH3BP1 localizes together with the exocyst to the leading edge of motile cells and that SH3BP1 regulates cell migration via its GAP activity upon Rac1. SH3BP1 loss of function induces abnormally high Rac1 activity at the front, as visualized by in vivo biosensors, and disorganized and instable protrusions, as revealed by cell morphodynamics analysis. Consistently, constitutively active Rac1 mimics the phenotype of SH3BP1 depletion: slow migration and aberrant cell morphodynamics. Our finding that SH3BP1 downregulates Rac1 at the motile-cell front indicates that Rac1 inactivation in this location, as well as its activation by GEF proteins, is a fundamental requirement for cell motility.     

Cell type-specific regulation of RhoA activity during cytokinesis

Rho family GTPases play pivotal roles in cytokinesis. By using probes based on the principle of fluorescence resonance energy transfer (FRET), we have shown that in HeLa cells RhoA activity increases with the progression of cytokinesis. Here we show that in Rat1A cells RhoA activity remained suppressed during most of the cytokinesis. Consistent with this observation, the expression of C3 toxin inhibited cytokinesis in HeLa cells but not in Rat1A cells. Furthermore, the expression of a dominant negative mutant of Ect2, a Rho GEF, or Y-27632, an inhibitor of the Rho-dependent kinase ROCK, inhibited cytokinesis in HeLa cells but not in Rat1A cells. In contrast to the activity of RhoA, the activity of Rac1 was suppressed during cytokinesis and started increasing at the plasma membrane of polar sides before the abscission of the daughter cells in both HeLa and Rat1A cells. This type of Rac1 suppression was shown to be essential for cytokinesis because a constitutively active mutant of Rac1 induced a multinucleated phenotype in both HeLa and Rat1A cells. Moreover, the involvement of MgcRacGAP/CYK-4 in this suppression of Rac1 during cytokinesis was shown by the use of a dominant negative mutant. Because ML-7, an inhibitor of myosin light chain kinase, delayed the cytokinesis of Rat1A cells and because Pak, a Rac1 effector, is known to suppress myosin light chain kinase, the suppression of the Rac1-Pak pathway by MgcRacGAP may play a pivotal role in the cytokinesis of Rat1A cells.     

Amino acid sequence versus morphological data and the interordinal relationships of mammals

To a large extent, the mutual affinities of the mammalian orders continue to puzzle systematists, even though comparative anatomy and amino acid sequencing offer a massive data base from which these relationships could potentially be adduced. In the present paper the consistency index--the number of character states less the number of characters in a data set, divided by the total number of changes in the character states on a cladogram--was used to examine the relative resolving powers of recently published morphological and molecular- sequence data. Consistency indices were calculated for previously published alpha crystallin A chain and myoglobin amino acid-sequence cladograms and for four original amino acid-sequence cladograms (alpha crystallin A, myoglobin, and alpha and beta hemoglobin); these were found to be comparable to the consistency indices of morphologically based cladograms. Qualitative comparisons between the morphologically based and molecularly based trees were also made; only moderate congruence between the two was observed. Moreover, there was a general lack of congruence between the cladograms specified by each of the four proteins. Amino acid-sequence and morphological data agreed on the placement of edentates as an early eutherian offshoot and on the grouping of hyracoids, proboscideans, and sirenians. Otherwise there was only limited congruence: morphology strongly supported the grouping of lagomorphs and rodents and the alliance of pholidotes and edentates, but sequence analyses did not. The placement of tubulidentates differed widely among proteins. Morphology indicated the close association of sirenians with proboscideans; proteins suggested a pairing of sirenians with hyracoids. Sequence data did not identify many (morphologically well-diagnosed) orders as monophyletic (e.g., Lagomorpha).(ABSTRACT TRUNCATED AT 250 WORDS)      

Circulating opioid peptides in chronic renal failure: acute effect of dialysis treatment

In 14 patients affected by chronic renal failure (7 males and 7 females) it has been evaluated the secretion of beta LPH, beta EP, ACTH and Cortisol in basal conditions and immediately after a dialytic treatment. For beta LPH and beta EP measurements on each thawed plasma a silic acid extraction and a successive peptides separation by a Sephadex G-75 column chromatography preceeded the two specific RIAs. Basal beta LPH plasma levels resulted significantly higher than in normal controls, while that of beta EP, ACTH and Cortisol were in the normal range. The dialytic treatment was able to increase ACTH and Cortisol plasma levels, without to modified beta LPH and beta EP plasma levels.