Immune effects of mesenchymal stem cells: implications for Charcot-Marie-Tooth disease

Mesenchymal stem cell (MSC) therapy is the most clinically advanced form of cell therapy, second to hematopoietic stem cell transplants. To date, MSC have been used for immune modulation in conditions such as Graft Versus Host Disease (GVHD) and Crohn's Disease, for which Phase III clinical trials are currently in progress. Here, we review the immunological properties of MSC and make a case for their use in treatment of Charcot-Marie-Tooth disease type 1 (CMT1), a group of inherited peripheral neuropathies. CMT1 is characterized by demyelination and aberrant immune activation making this condition an ideal target for exploration of MSC therapy, given the ability of these cells to promote sheath regeneration as well as suppress inflammation. Studies supporting this hypothesis will be presented and placed into the context of other cell-based approaches that are theoretically feasible. Given that MSCs selectively home to areas of inflammation, as well as exert effects in an allogeneic manner, the possibility of an "off the shelf" therapy for CMT1 will be discussed.     

Methylation-Associated Partial Down-Regulation of Mesothelin Causes Resistance to Anti-Mesothelin Immunotoxins in a Pancreatic Cancer Cell Line

Anti-mesothelin Pseudomonas exotoxin A-based recombinant immunotoxins (RITs) present a potential treatment modality for pancreatic ductal adenocarcinoma (PDAC). To study mechanisms of resistance, the sensitive PDAC cell line KLM-1 was intermittently exposed to the anti-mesothelin SS1-LR-GGS RIT. Surviving cells were resistant to various anti-mesothelin RITs (IC₅₀s >1 μg/ml), including the novel de-immunized RG7787. These resistant KLM-1-R cells were equally sensitive to the anti-CD71 HB21(Fv)-PE40 RIT as KLM-1, indicating resistance was specific to anti-mesothelin RITs. Mesothelin gene expression was partially down-regulated in KLM-1-R, resulting in 5-fold lower surface protein levels and decreased cellular uptake of RG7787 compared to KLM-1. Bisulfite sequencing analysis found that the mesothelin promoter region was significantly more methylated in KLM-1-R (59 ± 3.6%) compared to KLM-1 (41 ± 4.8%), indicating hypermethylation as a mechanism of mesothelin downregulation. The DNA methyltransferase inhibitor 5-azacytidine restored original mesothelin surface expression to more than half in KLM-1-R and increased sensitivity to RG7787 (IC₅₀ = 722.4 ± 232.6 ng/ml), although cells remained significantly less sensitive compared to parental KLM-1 cells (IC₅₀ = 4.41 ± 0.38 ng/ml). Mesothelin cDNA introduction in KLM-1-R led to 5-fold higher surface protein levels and significantly higher RG7887 uptake compared to KLM-1. As a result, the original sensitivity to RG7787 was fully restored (IC₅₀ = 4.49 ± 1.11 ng/ml). A significantly higher RG7787 uptake was thus required to reach the original cytotoxicity in resistant cells, hinting that intracellular RIT trafficking is also a limiting factor. RNA deep sequencing analysis of KLM-1 and KLM-1-R cells supported our experimental findings; compared to KLM-1, resistant cells displayed differential expression of genes linked to intracellular transport and an expression pattern that matched a more general hypermethylation status. In conclusion, resistance to anti-mesothelin RITs in KLM-1 is linked to a methylation-associated down-regulation of mesothelin, while aberrations in RIT trafficking could also play a role.     

Pervasive and cooperative deadenylation of 3'UTRs by embryonic microRNA families

To understand how miRNA-mediated silencing impacts on embryonic mRNAs, we conducted a functional survey of abundant maternal and zygotic miRNA families in the C. elegans embryo. We show that the miR-35-42 and the miR-51-56 miRNA families define maternal and zygotic miRNA-induced silencing complexes (miRISCs), respectively, that share a large number of components. Using a cell-free C. elegans embryonic extract, we demonstrate that the miRISC directs the rapid deadenylation of reporter mRNAs with natural 3'UTRs. The deadenylated targets are translationally suppressed and remarkably stable. Sampling of the predicted miR-35-42 targets reveals that roughly half are deadenylated in a miRNA-dependent manner, but with each target displaying a distinct efficiency and pattern of deadenylation. Finally, we demonstrate that functional cooperation between distinct miRISCs within 3'UTRs is required to potentiate deadenylation. With this report, we reveal the extensive and direct impact of miRNA-mediated deadenylation on embryonic mRNAs.     

Cell-free synthesis of silver nanoparticles in spent media of different Aspergillus species

The recovery and valorization of metals and rare earth metals from wastewater are of great importance to prevent environmental pollution and recover valuable resources. Certain bacterial and fungal species are capable of removing metal ions from the environment by facilitating their reduction and precipitation. Even though the phenomenon is well documented, little is known about the mechanism. Therefore, we systematically investigated the influence of nitrogen sources, cultivation time, biomass, and protein concentration on silver reduction capacities of cell-free cultivation media (spent media) of Aspergillus niger, A. terreus, and A. oryzae. The spent medium of A. niger showed the highest silver reduction capacities with up to 15 μmol per milliliter spent medium when ammonium was used as the sole N-source. Silver ion reduction in the spent medium was not driven by enzymes and did not correlate with biomass concentration. Nearly full reduction capacity was reached after 2 days of incubation, long before the cessation of growth and onset of the stationary phase. The size of silver nanoparticles formed in the spent medium of A. niger was influenced by the nitrogen source, with silver nanoparticles formed in nitrate or ammonium-containing medium having an average diameter of 32 and 6 nm, respectively.     

Proton interactions with hemes a and a3 in bovine heart cytochrome c oxidase

Three forms of cytochrome c oxidase, fully oxidized CcO (CcO-O), oxidized CcO complexed with cyanide (CcO.CN), and mixed valence CcO, in which both heme a(3) and Cu(B) are reduced and stabilized by carbon monoxide (MV.CO), were investigated by optical spectroscopy, MCD, and stopped-flow for the pH sensitivity of spectral features. In the pH range between pH 5.7 and 9.0, both heme a and heme a(3) in CcO-O interact with a single protolytic group. From the variation of the position of the Soret peak with changes in pH, a pK(a) of 6.6 +/- 0.2 was determined for this group. The pH sensitivity of heme a(3) is lost in the CcO.CN complex, and only heme a responds to pH changes. In MV.CO the spectra of both hemes are almost independent of pH between 5.7 and 11.0. The stoichiometry of proton uptake in the conversion of CcO-O both to MV.CO and to fully reduced CcO was determined between pH 5.8 and pH 8.2. Formation of MV.CO from CcO-O was accompanied by the uptake of approximately two protons, and this value was almost independent of pH. Full reduction of oxidized CcO was associated with the uptake of approximately 2 H(+) at basic pH, and this value increases with decreasing pH. On the basis of these proton uptake measurements, it is concluded that the pK(a) of the group is independent of the redox state of CcO. It is suggested that Glu60 of subunit II, located at the entrance of the proton conducting K-channel, is the protolytic residue that interacts with both hemes through a hydrogen-bonding network.     

Limited proteolysis of streptokinase and properties of some fragments.

Limited proteolysis of streptokinase (Sk) by trypsin and thermolysin was performed under various incubation conditions and analysed by polyacrylamide gel electrophoresis. Several fragments (Sk1, Tr27, Tr17, Th26, and Th16) were isolated and characterized further. The N-terminal sequences of Tr27, Tr17, Th26, Th16 and the C-terminal sequences of Tr27 and Th26 were determined by partial sequencing. The evidence available allows the positioning of these fragments within the Sk sequence. Fragment Sk1 is obtained by carefully standardized tryptic digestion of Sk and gel chromatography under non-denaturing conditions. Sk1 is formed by a large polypeptide Ser60-Lys293 and non-covalently bonded smaller polypeptides composed of amino acids from the N-terminal region Ile1-Lys59 of Sk. Fragment Tr27 consists of the large polypeptide Ser60-Lys293 of Sk1, and can be obtained from Sk1 by removal of the smaller N-terminal polypeptides under denaturing conditions. Fragment Th26 is composed of amino acids Phe63-His291. The N-termini of fragments Tr17 and Th16 start with Glu148 and Ile151. From their electrophoretically-determined sizes it can be concluded that they most probably have the same C-terminal amino acids, Lys293 and His291, as fragments Tr27 and Th26, respectively. Secondary structure elements of similar composition were found in all the fragments studied using circular dichroism (c.d.) and infrared (i.r.) measurements. Differential scanning calorimetric (d.s.c.) measurements were performed in order to correlate the sequence regions of Sk to energetic folding units of the protein. Fragments Sk1, Tr27, Th26, Tr17, and Th16 show one melting peak in the temperature range from 42.8 to 46.1 degrees C (thermal unfolding stage). For fragment Sk1, this melting peak can be separated by deconvolution into two transitions at T1 = 46.1 degree C and T2 = 47.3 degrees C with delta H1 = 450 kJ/mol and delta H2 = 219 kJ/mol, respectively. Fragments Tr17 and Th16 show one two-state transition at T = 42.8 degrees C with delta H = 326 kJ/mol.