Tenascin-C and fibronectin expression divide early stage tongue cancer into low- and high-risk groups.

BACKGROUND: Oral tongue squamous cell carcinoma (OTSCC) metastasises early, especially to regional lymph nodes. There is an ongoing debate on which early stage (T1-T2N0) patients should be treated with elective neck dissection. We need prognosticators for early stage tongue cancer. METHODS: Mice immunisation with human mesenchymal stromal cells resulted in production of antibodies against tenascin-C (TNC) and fibronectin (FN), which were used to stain 178 (98 early stage), oral tongue squamous cell carcinoma samples. Tenascin-C and FN expression in the stroma (negative, moderate or abundant) and tumour cells (negative or positive) were assessed. Similar staining was obtained using corresponding commercial antibodies. RESULTS: Expression of TNC and FN in the stroma, but not in the tumour cells, proved to be excellent prognosticators both in all stages and in early stage cases. Among early stages, when stromal TNC was negative, the 5-year survival rate was 88%. Correspondingly, when FN was negative, no cancer deaths were observed. Five-year survival rates for abundant expression of TNC and FN were 43% and 25%, respectively. CONCLUSIONS: Stromal TNC and, especially, FN expressions differentiate patients into low- and high-risk groups. Surgery alone of early stage primary tumours might be adequate when stromal FN is negative. Aggressive treatments should be considered when both TNC and FN are abundant.

Adenosinergic Immunosuppression by Human Mesenchymal Stromal Cells Requires Co-Operation with T cells.

Mesenchymal stem/stromal cells (MSCs) have the capacity to counteract excessive inflammatory responses. MSCs possess a range of immunomodulatory mechanisms, which can be deployed in response to signals in a particular environment and in concert with other immune cells. One immunosuppressive mechanism, not so well-known in MSCs, is mediated via adenosinergic pathway by ectonucleotidases CD73 and CD39. In this study, we demonstrate that adenosine is actively produced from adenosine 5'-monophosphate (AMP) by CD73 on MSCs and MSC-derived extracellular vesicles (EVs). Our results indicate that although MSCs express CD39 at low level and it colocalizes with CD73 in bulge areas of membranes, the most efficient adenosine production from adenosine 5'-triphosphate (ATP) requires co-operation of MSCs and activated T cells. Highly CD39 expressing activated T cells produce AMP from ATP and MSCs produce adenosine from AMP via CD73 activity. Furthermore, adenosinergic signaling plays a role in suppression of T cell proliferation in vitro. In conclusion, this study shows that adenosinergic signaling is an important immunoregulatory mechanism of MSCs, especially in situations where ATP is present in the extracellular environment, like in tissue injury. An efficient production of immunosuppressive adenosine is dependent on the concerted action of CD39-positive immune cells with CD73-positive cells such as MSCs or their EVs.

Systematic screening of soluble expression of antibody fragments in the cytoplasm of E. coli.

BACKGROUND: Disulfide bonds are the most common structural, post-translational modification found in proteins. Antibodies contain up to 25 disulfide bonds depending on type, with scFv fragments containing two disulfides and Fab fragments containing five or six disulfide bonds. The production of antibody fragments that contain native disulfide bonds can be challenging, especially on a large scale. The protein needs to be targeted to prokaryotic periplasm or the eukaryotic endoplasmic reticulum. These compartments are specialised for disulfide bond formation, but both compartments have limitations. RESULTS: Here we show that the introduction into the cytoplasm of a catalyst of disulfide bond formation and a catalyst of disulfide bond isomerization allows the efficient formation of natively folded scFv and Fab antibody fragments in the cytoplasm of Escherichia coli with intact reducing pathways. Eleven scFv and eleven Fab fragments were screened and ten of each were obtained in yields of >5 mg/L from deep-well plates. Production of eight of the scFv and all ten of the Fab showed a strong dependence on the addition of the folding factors. Yields of purified scFv of up to 240 mg/L and yields of purified Fab fragments of up to 42 mg/L were obtained. Purified fragments showed circular dichroism spectra consistent with being natively folded and were biologically active. CONCLUSIONS: Our results show that the efficient production of soluble, biologically active scFv and Fab antibody fragments in the cytoplasm of E. coli is not only possible, but facile. The required components can be easily transferred between different E. coli strains.

Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures.

BACKGROUND: Fab antibody fragments in E. coli are usually directed to the oxidizing periplasmic space for correct folding. From periplasm Fab fragments may further leak into extracellular medium. Information on the cultivation parameters affecting this leakage is scarce, and the unpredictable nature of Fab leakage is problematic regarding consistent product recovery. To elucidate the effects of cultivation conditions, we investigated Fab expression and accumulation into either periplasm or medium in E. coli K-12 and E. coli BL21 when grown in different types of media and under different aeration conditions. RESULTS: Small-scale Fab expression demonstrated significant differences in yield and ratio of periplasmic to extracellular Fab between different culture media and host strains. Expression in a medium with fed-batch-like glucose feeding provided highest total and extracellular yields in both strains. Unexpectedly, cultivation in baffled shake flasks at 150 rpm shaking speed resulted in higher yield and accumulation of Fabs into culture medium as compared to cultivation at 250 rpm. In the fed-batch medium, extracellular fraction in E. coli K-12 increased from 2-17% of total Fab at 250 rpm up to 75% at 150 rpm. This was partly due to increased lysis, but also leakage from intact cells increased at the lower shaking speed. Total Fab yield in E. coli BL21 in glycerol-based autoinduction medium was 5 to 9-fold higher at the lower shaking speed, and the extracellular fraction increased from ≤ 10% to 20-90%. The effect of aeration on Fab localization was reproduced in multiwell plate by variation of culture volume. CONCLUSIONS: Yield and leakage of Fab fragments are dependent on expression strain, culture medium, aeration rate, and the combination of these parameters. Maximum productivity in fed-batch-like conditions and in autoinduction medium is achieved under sufficiently oxygen-limited conditions, and lower aeration also promotes increased Fab accumulation into extracellular medium. These findings have practical implications for screening applications and small-scale Fab production, and highlight the importance of maintaining consistent aeration conditions during scale-up to avoid changes in product yield and localization. On the other hand, the dependency of Fab leakage on cultivation conditions provides a practical way to manipulate Fab localization.

Cytoskeletal structure in cells harboring two mutations: R133C in NOTCH3 and 5650G>A in mitochondrial DNA.

We have previously described a patient with cerebral autosomal-dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) caused by R133C mutation in NOTCH3 and with a concomitant myopathy caused by a G to A point mutation at base pair 5650 (5650G>A) in the gene encoding tRNA(Ala) in mitochondrial DNA (mtDNA). In the present study, we have examined the morphology of the cytoskeletal components in fibroblasts and myoblasts of this patient. Immunolabeling revealed that tubulin network was sparse and formed asters in these cells, whereas no changes were found in actin and vimentin networks in comparison to the control cell lines. Furthermore, mitochondria were less abundant and the branches of the mitochondrial network were reduced in number. Muscle histochemical analysis showed ragged red fibres (RRFs) and cytochrome c oxidase (COX)-negative fibres. The mean proportion of mtDNA with 5650G>A was lower in histologically normal muscle fibres than in the COX-negative fibres and in the RRFs. These findings suggest that 5650G>A is a pathogenic mtDNA mutation. However, the changes in tubulin network and mitochondrial distribution in patient fibroblasts and myoblasts cannot solely be explained by this mutation.

Cytokine data obtained from synovial stromal cells of patients with rheumatoid arthritis or osteoarthritis.

In this article, we share the raw cytokine data obtained from basal and stimulated synovial stromal cells cultured from patients with rheumatoid arthritis or osteoarthritis. This data article is related to the research article entitled "1,25D3 and calcipotriol, its hypocalcemic analog, exert a long-lasting anti-inflammatory and anti-proliferative effect in synoviocytes cultured from patients with rheumatoid arthritis and osteoarthritis (1). Cytokine levels were analyzed by a magnetic bead-based multiplex assay (a panel of 27 important cytokines) in two separate sets of experiments. The first was conducted with IL-1ß and 1,25(OH)2D3 and the other with TNFα, calcipotriol, i.e. the hypocalcemic analog 1,25(OH)2D3, and dexamethasone. The raw data of this article display the individual variation in basal secretion of cytokines and in their response to different stimuli.

1,25(OH)2D3 and calcipotriol, its hypocalcemic analog, exert a long-lasting anti-inflammatory and anti-proliferative effect in synoviocytes cultured from patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVES: We investigated the effects of 1,25-dihydroxy vitamin D3 (1,25(OH)2D3), i.e. biologically active vitamin D and calcipotriol, a vitamin D analog, on growth and secretion of inflammatory mediators in synovial stromal cells (SSC) of patients with rheumatoid arthritis (RA) or osteoarthritis (OA). METHODS: Synovial stromal cells (SSC) isolated during knee prosthesis surgery from four patients with RA and four with OA were exposed to 1,25(OH)2D3 or calcipotriol with or without stimulation of cells with IL-1ß or TNF-α. The proliferation of cells was studied by MTT assay. Levels of cytokines were analyzed by a magnetic bead-based multiplex assay (a panel of 27 important cytokines and IL-6 alone) and RT-PCR was used to validate the concentrations of the key cytokines secreted by SSC. The vitamin D receptor (VDR) was visualized by immunofluorescence in SSC and by immunohistochemistry in the synovial tissues of three RA and three OA patients. RESULTS: We detected intense staining for VDR in the synovial lining and vascular endothelium in tissue sections from all our RA and OA patients. Both 1,25(OH)2D3 and calcipotriol inhibited SSC proliferation for a prolonged time (up to 23 days with calcipotriol), but dexamethasone tended to increase SSC proliferation in a 4-day culture. 1,25(OH)2D3, calcipotriol and dexamethasone reduced the secretion of most inflammatory factors. Calcipotriol and dexamethasone additively reduced the secretions of IL-6, IFN-γ, basic FGF and VEGF in TNF-α stimulated SSC. The level of IL-6 was still diminished at 10 days after exposure, emphasizing the long-term impact of calcipotriol on SSC. CONCLUSIONS: Exposure for 24-48h to 1,25(OH)2D3 or calcipotriol causes a long-lasting inhibition of cell proliferation and cytokine production in SSC in vitro.

Collagen XVII is expressed in human CNS neurons.

Type XVII collagen (collagen XVII) is a component of hemidesmosomes, which connect epithelial cells to the underlying basement membrane. Previously, an association has been suggested between neurological disorders and the skin disease bullous pemphigoid, where autoimmunity is directed against collagen XVII. Furthermore, the lack of alpha6 integrin, a ligand of collagen XVII, has been implicated in defects of cortical organization in the mouse brain. In this study, we demonstrate for the first time the presence of collagen XVII in neurons of the human brain by in situ hybridisation, immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). We propose that collagen XVII may be involved in the pathogenesis of various disorders affecting neuronal migration or synaptic plasticity.

Proteome and cytoskeleton responses in osteosarcoma cells with reduced OXPHOS activity.

We have recently shown disorganization of the vimentin network in cultured cells deficient in oxidative phosphorylation (OXPHOS). We describe here the cellular responses to OXPHOS deficiency in osteosarcoma cells upon complex I (CI) and complex IV (CIV) inhibition, and upon the lack of mitochondrial DNA (rho0 cells). We examined the cytoskeletal organization and the distribution of mitochondria and analysed total proteome by 2-DE and vimentin expression by ELISA. Upon CIV inhibition and in rho0 cells, the vimentin network had collapsed around the nucleus and formed thick bundles. The mitochondria formed a perinuclear crescent upon CIV inhibition, whereas they accumulated around the nucleus in the rho0 cells, where the amount of vimentin was increased. Analysis of the total proteome revealed that a lack of mitochondrial DNA or inhibition of CI or CIV led to changes in the expression of cytoskeletal and cytoskeleton-associated proteins and proteins involved in apoptosis, OXPHOS, glycolysis, the tricarboxylic acid cycle, and oxidative stress responses. Our findings suggest that a deficiency in the energy converting system and oxidative stress can lead to cytoskeletal changes.

Mitochondrial DNA sequence variation and mutation rate in patients with CADASIL.

Mutations in the NOTCH3 gene cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), which is clinically characterised by recurrent ischemic strokes, migraine with aura, psychiatric symptoms, cognitive decline and dementia. We have previously described a patient with CADASIL caused by a R133C mutation in the NOTCH3 gene and with a concomitant myopathy caused by a 5650G>A mutation in the MTTA gene in mitochondrial DNA (mtDNA). We assume that the co-occurrence of the two mutations is not coincidental and that mutations in the NOTCH3 gene may predispose the mtDNA to mutations. We therefore examined the nucleotide variation in the mtDNA coding region sequences in 20 CADASIL pedigrees with 77 affected patients by conformation-sensitive gel electrophoresis and sequencing. The sequence variation in mtDNA was then compared with that among 192 healthy Finns. A total of 180 mtDNA coding region sequence differences were found relative to the revised Cambridge reference sequence, including five novel synonymous substitutions, two novel nonsynonymous substitutions and one novel tRNA substitution. We found that maternal relatives in two pedigrees differed from each other in their mtDNA. Furthermore, the average number of pairwise differences in sequences from the 41 unrelated maternal lineages with CADASIL was higher than that expected among haplogroup-matched controls. The numbers of polymorphic sites and polymorphisms that were present in only one sequence were also higher among the CADASIL sequences than among the control sequences. Our results show that mtDNA sequence variation is increased within CADASIL pedigrees. These findings suggest a relationship between NOTCH3 and mtDNA.

Murine Wnt-1 with an internal c-myc tag recombinantly produced in Escherichia coli can induce intracellular signaling of the canonical Wnt pathway in eukaryotic cells.

Wnt-1 belongs to the Wnt family of secreted glycoproteins inducing an intracellular signaling pathway involved in cell proliferation, differentiation, and pattern formation. The canonical branch is one of three known branches. This is also valid in vitro, and Wnts can be considered beneficial for culturing primary cells from organs, provided Wnts are available and applicable even with cells of different species. It was shown here that internally c-myc-tagged murine Wnt-1 produced in the heterologous host Escherichia coli was appropriate for inducing intracellular signaling of the canonical Wnt pathway in eukaryotic cells via stabilization of cytosolic beta-catenin. The pioneering injection of the protein into the blastocoels of Xenopus laevis embryos led to axis duplication and suppression of head formation. Applying the recombinant murine Wnt-1 to metanephric mesenchyme activated the tubulogenic program. The signal-inducing activity of the recombinant protein was also positively demonstrated in the TOP-flash reporter assay. Although Wnts were purified recently from the growth media of stably transfected eukaryotic cell lines, the production of active Wnt proteins in pro- or eukaryotic microorganisms reportedly has never been successful. Here soluble production in E. coli and translocation into the oxidizing environment of the periplasm were achieved. The protein was purified using the internal c-myc tag. The effect on the eukaryotic cells implies that activity was retained. Thus, this approach could make recombinant murine Wnt-1 available as a good starting point for other Wnts needed, for example, for maintaining and differentiating stem cells, organ restoration therapy, and tissue engineering.