Novel insights for improving the therapeutic safety and efficiency of mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) have attracted great interest in the field of regenerative medicine. They can home to damaged tissue, where they can exert pro-regenerative and anti-inflammatory properties. These therapeutic effects involve the secretion of growth factors, cytokines, and chemokines. Moreover, the functions of MSCs could be mediated by extracellular vesicles (EVs) that shuttle various signaling messengers. Although preclinical studies and clinical trials have demonstrated promising therapeutic results, the efficiency and the safety of MSCs need to be improved. After transplantation, MSCs face harsh environmental conditions, which likely dampen their therapeutic efficacy. A possible strategy aiming to improve the survival and therapeutic functions of MSCs needs to be developed. The preconditioning of MSCs ex vivo would strength their capacities by preparing them to survive and to better function in this hostile environment. In this review, we will discuss several preconditioning approaches that may improve the therapeutic capacity of MSCs. As stated above, EVs can recapitulate the beneficial effects of MSCs and may help avoid many risks associated with cell transplantation. As a result, this novel type of cell-free therapy may be safer and more efficient than the whole cell product. We will, therefore, also discuss current knowledge regarding the therapeutic properties of MSC-derived EVs.     

Genomic comparison of plant pathogenic and nonpathogenic Serratia marcescens strains by suppressive subtractive hybridization

Cucurbit yellow vine disease (CYVD) is caused by disease-associated Serratia marcescens strains that have phenotypes significantly different from those of nonphytopathogenic strains. To identify the genetic differences responsible for pathogenicity-related phenotypes, we used a suppressive subtractive hybridization (SSH) strategy. S. marcescens strain Z01-A, isolated from CYVD-affected zucchini, was used as the tester, whereas rice endophytic S. marcescens strain R02-A (IRBG 502) was used as the driver. SSH revealed 48 sequences, ranging from 200 to 700 bp, that were present in Z01-A but absent in R02-A. Sequence analysis showed that a large proportion of these sequences resembled genes involved in synthesis of surface structures. By construction of a fosmid library, followed by colony hybridization, selection, and DNA sequencing, a phage gene cluster and a genome island containing a fimbrial-gene cluster were identified. Arrayed dot hybridization showed that the conservation of subtracted sequences among CYVD pathogenic and nonpathogenic S. marcescens strains varied. Thirty-four sequences were present only in pathogenic strains. Primers were designed based on one Z01-A-specific sequence, A79, and used in a multiplex PCR to discriminate between S. marcescens strains causing CYVD and those from other ecological niches.     

FepA- and TonB-dependent bacteriophage H8: receptor binding and genomic sequence

H8 is derived from a collection of Salmonella enterica serotype Enteritidis bacteriophage. Its morphology and genomic structure closely resemble those of bacteriophage T5 in the family Siphoviridae. H8 infected S. enterica serotypes Enteritidis and Typhimurium and Escherichia coli by initial adsorption to the outer membrane protein FepA. Ferric enterobactin inhibited H8 binding to E. coli FepA (50% inhibition concentration, 98 nM), and other ferric catecholate receptors (Fiu, Cir, and IroN) did not participate in phage adsorption. H8 infection was TonB dependent, but exbB mutations in Salmonella or E. coli did not prevent infection; only exbB tolQ or exbB tolR double mutants were resistant to H8. Experiments with deletion and substitution mutants showed that the receptor-phage interaction first involves residues distributed over the protein's outer surface and then narrows to the same charged (R316) or aromatic (Y260) residues that participate in the binding and transport of ferric enterobactin and colicins B and D. These data rationalize the multifunctionality of FepA: toxic ligands like bacteriocins and phage penetrate the outer membrane by parasitizing residues in FepA that are adapted to the transport of the natural ligand, ferric enterobactin. DNA sequence determinations revealed the complete H8 genome of 104.4 kb. A total of 120 of its 143 predicted open reading frames (ORFS) were homologous to ORFS in T5, at a level of 84% identity and 89% similarity. As in T5, the H8 structural genes clustered on the chromosome according to their function in the phage life cycle. The T5 genome contains a large section of DNA that can be deleted and that is absent in H8: compared to T5, H8 contains a 9,000-bp deletion in the early region of its chromosome, and nine potentially unique gene products. Sequence analyses of the tail proteins of phages in the same family showed that relative to pb5 (Oad) of T5 and Hrs of BF23, the FepA-binding protein (Rbp) of H8 contains unique acidic and aromatic residues. These side chains may promote binding to basic and aromatic residues in FepA that normally function in the adsorption of ferric enterobactin. Furthermore, a predicted H8 tail protein showed extensive identity and similarity to pb2 of T5, suggesting that it also functions in pore formation through the cell envelope. The variable region of this protein contains a potential TonB box, intimating that it participates in the TonB-dependent stage of the phage infection process.     

Mesenchymal stromal cells of the bone marrow and natural killer cells: cell interactions and cross modulation

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are multipotent progenitor cells that have shown promise for several different therapeutic applications. As they are able to modulate the function of several types of immune cells, BM-MSCs are highly important in the field of cell-based immunotherapy. Understanding BM-MSC-natural killer (NK) cell interactions is crucial for improving their therapeutic efficiency. Here, we observed that the type of NK cell-activating cytokine (e.g., IL-2, IL-12, IL-15 and IL-21) strongly influenced the outcomes of their interactions with BM-MSCs. The expression patterns of the ligands (CD112, CD155, ULPB-3) and receptors (LAIR, NCR) mediating the cross-talk between BM-MSCs and NK cells were critically modulated following co-culture. BM-MSCs partially impaired NK cell proliferation but up-regulated their secretion of IFN-γ and TNF-α. As they are cytotoxic, activated NK cells induced the killing of BM-MSCs. Indeed, BM-MSCs triggered the degranulation of NK cells and increased their release of perforin and granzymes. Interestingly, activated NK cells induced ROS generation within BM-MSCs that caused their decreased viability and reduced expression of serpin B9. Collectively, our observations reveal that BM-MSC-NK cell interactions may impact the immunobiology of both cell types. The therapeutic potential of BM-MSCs will be significantly improved once these issues are well characterized.     

Genome sequence of a nephritogenic and highly transformable M49 strain of Streptococcus pyogenes

The 1,815,783-bp genome of a serotype M49 strain of Streptococcus pyogenes (group A streptococcus [GAS]), strain NZ131, has been determined. This GAS strain (FCT type 3; emm pattern E), originally isolated from a case of acute post-streptococcal glomerulonephritis, is unusually competent for electrotransformation and has been used extensively as a model organism for both basic genetic and pathogenesis investigations. As with the previously sequenced S. pyogenes genomes, three unique prophages are a major source of genetic diversity. Two clustered regularly interspaced short palindromic repeat (CRISPR) regions were present in the genome, providing genetic information on previous prophage encounters. A unique cluster of genes was found in the pathogenicity island-like emm region that included a novel Nudix hydrolase, and, further, this cluster appears to be specific for serotype M49 and M82 strains. Nudix hydrolases eliminate potentially hazardous materials or prevent the unbalanced accumulation of normal metabolites; in bacteria, these enzymes may play a role in host cell invasion. Since M49 S. pyogenes strains have been known to be associated with skin infections, the Nudix hydrolase and its associated genes may have a role in facilitating survival in an environment that is more variable and unpredictable than the uniform warmth and moisture of the throat. The genome of NZ131 continues to shed light upon the evolutionary history of this human pathogen. Apparent horizontal transfer of genetic material has led to the existence of highly variable virulence-associated regions that are marked by multiple rearrangements and genetic diversification while other regions, even those associated with virulence, vary little between genomes. The genome regions that encode surface gene products that will interact with host targets or aid in immune avoidance are the ones that display the most sequence diversity. Thus, while natural selection favors stability in much of the genome, it favors diversity in these regions.     

The source of human mesenchymal stromal cells influences their TLR profile as well as their functional properties

Mesenchymal stromal cells (MSC) can be expanded from different sources. We compared the influence of inflammation and TLR ligation on the phenotype and function of MSC derived from bone marrow (BM), adipose tissue (AT), and Wharton’s jelly (WJ). WJ-MSC were featured by a lack of TLR4 expression. While inflammation upregulated TLR3 in all three MSC types, TLR4 upregulation was observed only on BM-MSC. TLR ligation increased the production of inflammatory cytokines in BM- and AT-MSC but not in WJ-MSC and augmented anti-inflammatory cytokines in AT-MSC. Although inflammation increased in all MSC types the secretion of inflammatory cytokines, additional TLR triggering did not have further effect on WJ-MSC. The immunosuppressive potential of WJ-MSC on MLR was affected neither by inflammation nor by TLR triggering. This resistance was related to an overproduction of HGF. These data indicate that MSC source could be of importance while designing immunomodulating cell therapy in transplantation.     

High occurrence of carbapenem-resistant Escherichia coli isolates from healthy rabbits (Oryctolagus cuniculus): first report of blaIMI and blaVIM type genes from livestock in Tunisia

We aimed to study the antibiotic susceptibility and possible occurrence of extended-spectrum beta-lactamases (ESBL)/carbapenemase-producing Escherichia coli isolates collected from rabbits in Tunisia. In all, 35 faecal samples from healthy rabbits were collected from one farm and E. coli were isolated from three media: antibiotic-free TBX agar, TBX+2 mg l⁻¹ cefotaxime and TBX+1 mg l⁻¹ imipenem. In total, 39 E. coli isolates were recovered; the majority showed resistance to at least one antibiotic and none was ESBL producer. Carbapenem resistance was detected in 16 isolates from either selective or un-selective media. Phenotypic methods used to detect carbapenemase production showed two positive isolates by Modified Hodge Test, six metallo-carbapenemase producers (Imipenem disc+EDTA) and all were temocillin resistant (possible OXA-48 carbapenemase). bla_VIM and bla_IMP type genes were detected in two and one isolates, respectively; one of them harboured both genes. Isolates contained common genes encoding resistance to sulphonamides (sul1, sul2), tetracycline (tetA, tetB, tetC) and fluoroquinolones (qnrS, aac(6′)-Ib-cr). Class 1 and 2 integrons were detected in five and four isolates, respectively. These findings highlight the importance of rabbit production as reservoir of carbapenem-resistant E. coli and argument the first report of bla_VIM and bla_IMP genes in livestock in Tunisia.     

Allelic variants of ovine prion protein gene (PRNP) in Oklahoma sheep

1,144 sheep belonging to 21 breeds and known crosses were sequence analyzed for polymorphisms in the ovine PRNP gene. Genotype and allele frequencies of polymorphisms in PRNP known to confer resistance to scrapie, a fatal neurodegenerative disease of sheep, are reported. Known polymorphisms at codons 136 (A/V), 154 (H/R) and 171 (Q/R/H/K) were identified. The frequency of the 171R allele known to confer resistance to type C scrapie was 53.8% and the frequency of the 136A allele known to influence the resistance to type A scrapie was 96.01%. In addition, we report the identification of five new polymorphisms at codons 143 (H/R), 167 (R/S), 180 (H/Y), 195 (T/S) and 196 (T/S). We also report the identification of a novel allele (S/R) at codon 138.