Loss and rescue of osteocalcin and osteopontin modulate osteogenic and angiogenic features of mesenchymal stem/stromal cells

Noncollagenous proteins in the bone extracellular matrix, such as osteocalcin (OC) and osteopontin (OPN), inherent to evolution of bone as a skeletal tissue, are known to regulate bone formation and mineralization. However, the fundamental basis of this regulatory role remains unknown. Here, for the first time, we use mouse mesenchymal stem/stromal cells (MSC) lacking both OC and OPN to investigate the mechanistic roles of OC and OPN on the proliferation capacity and differentiation ability of MSC. We found that the loss of OC and OPN reduces stem cells self-renewal potential and multipotency, affects their differentiation into an osteogenic lineage, and impairs their angiogenic potential while maintaining chondrogenic and adipogenic lineages. Moreover, loss of OC and OPN compromises the extracellular matrix integrity and maturation, observed by an unexpected enhancement of glycosaminoglycans content that are associated with a more primitive skeletal connective tissue, and by a delay on the maturation of mineral species produced. Interestingly, exogenously supplemented OC and OPN were able to rescue MSC proliferative and osteogenic potential along with matrix integrity and mineral quality. Taken together, these results highlight the key contributions of OC and OPN in enhancing osteogenesis and angiogenesis over primitive connective tissue, and support a potential therapeutic approach based on their exogenous supplementation.     

Sexually selected male weapon is associated with lower inbreeding load but higher sex load in the bulb mite

Elaborate sexually selected ornaments and armaments are costly but increase the reproductive success of their bearers (usually males). It has been postulated that high-quality males can invest disproportionately more in such traits, making those traits honest signals of genetic quality. However, genes associated with such traits may have sexually antagonistic effects on fitness. Here, using a bulb mite Rhizoglyphus robini, a species in which a distinct dimorphism exists between males in the expression of a sexually selected weapon, we compare inbreeding and gender load between lines derived from armed fighters and unarmed scramblers. After four generations of sib-mating, inbreeding depression for female fitness was significantly lower in fighter-derived lines compared to scrambler-derived lines, suggesting that fighter males had significantly higher genetic quality. However, outbred females from fighter-derived lines had significantly lower fitness compared to outbred females from scrambler-derived lines, demonstrating significant gender load associated with the presence of a sexually selected male weapon. Our results imply that under outbreeding, genetic benefits of mating with bearers of elaborate sexually selected traits might be swamped by the costs of decreased fitness of female progeny due to sexually antagonistic effects.     

The crystal structure of human telomeric DNA complexed with berberine: an interesting case of stacked ligand to G-tetrad ratio higher than 1:1

The first crystal structure of human telomeric DNA in complex with the natural alkaloid berberine, produced by different plant families and used in folk medicine for millennia, was solved by X-ray diffraction method. The G-quadruplex unit features all-parallel strands. The overall folding assumed by DNA is the same found in previously reported crystal structures. Similarly to previously reported structures the ligand molecules were found to be stacked onto the external 5′ and 3′-end G-tetrads. However, the present crystal structure highlighted for the first time, the presence of two berberine molecules in the two binding sites, directly interacting with each tetrad. As a consequence, our structural data point out a 2:1 ligand to G-tetrad molar ratio, which has never been reported before in a telomeric intramolecular quadruplex structure.     

Properties of monocytes generated from haematopoietic CD34+ stem cells from bone marrow of colon cancer patients

Monocytes exhibit direct and indirect antitumour activities and may be potentially useful for various forms of adoptive cellular immunotherapy of cancer. However, blood is a limited source of them. This study explored whether monocytes can be obtained from bone marrow haematopoietic CD34⁺ stem cells of colon cancer patients, using previously described protocol of expansion and differentiation to monocytes of cord blood-derived CD34⁺ haematopoietic progenitors. Data show that in two-step cultures, the yield of cells was increased approximately 200-fold, and among these cells, up to 60 % of CD14⁺ monocytes were found. They consisted of two subpopulations: CD14⁺⁺CD16⁺ and CD14⁺CD16^?, at approximately 1:1 ratio, that differed in HLA-DR expression, being higher on the former. No differences in expression of costimulatory molecules were observed, as CD80 was not detected, while CD86 expression was comparable. These CD14⁺ monocytes showed the ability to present recall antigens (PPD, Candida albicans) and neoantigens expressed on tumour cells and tumour-derived microvesicles (TMV) to autologous CD3⁺ T cells isolated from the peripheral blood. Monocytes also efficiently presented the immunodominant HER-2/neu_369–377 peptide (KIFGSLAFL), resulting in the generation of specific cytotoxic CD8⁺ T lymphocytes (CTL). The CD14⁺⁺CD16⁺ subset exhibited enhanced cytotoxicity, though nonsignificant, towards tumour cells in vitro. These observations indicate that generation of monocytes from CD34⁺ stem cells of cancer patients is feasible. To our knowledge, it is the first demonstration of such approach that may open a way to obtain autologous monocytes for alternative forms of adaptive and adoptive cellular immunotherapy of cancer.     

Therapeutic vaccination against autologous cancer stem cells with mRNA-transfected dendritic cells in patients with glioblastoma

Background

The growth and recurrence of several cancers appear to be driven by a population of cancer stem cells (CSCs). Glioblastoma, the most common primary brain tumor, is invariably fatal, with a median survival of approximately 1 year. Although experimental data have suggested the importance of CSCs, few data exist regarding the potential relevance and importance of these cells in a clinical setting.

Methods

We here present the first seven patients treated with a dendritic cell (DC)-based vaccine targeting CSCs in a solid tumor. Brain tumor biopsies were dissociated into single-cell suspensions, and autologous CSCs were expanded in vitro as tumorspheres. From these, CSC-mRNA was amplified and transfected into monocyte-derived autologous DCs. The DCs were aliquoted to 9–18 vaccines containing 10⁷ cells each. These vaccines were injected intradermally at specified intervals after the patients had received a standard 6-week course of post-operative radio-chemotherapy. The study was registered with the ClinicalTrials.gov identifier NCT00846456.

Results

Autologous CSC cultures were established from ten out of eleven tumors. High-quality RNA was isolated, and mRNA was amplified in all cases. Seven patients were able to be weaned from corticosteroids to receive DC immunotherapy. An immune response induced by vaccination was identified in all seven patients. No patients developed adverse autoimmune events or other side effects. Compared to matched controls, progression-free survival was 2.9 times longer in vaccinated patients (median 694 vs. 236 days, p = 0.0018, log-rank test).

Conclusion

These findings suggest that vaccination against glioblastoma stem cells is safe, well-tolerated, and may prolong progression-free survival.     

Homocysteine Levels in Patients with Schizophrenia on Clozapine Monotherapy

We tested the hypothesis that homocysteine levels are higher in blood of schizophrenic subjects on clozapine monotherapy than in healthy controls and they correlate with anthropometric measurements, laboratory tests and results of bioimpedance analysis of body composition. Data for 24 subjects with schizophrenia treated with clozapine and 24 age- and sex-matched healthy volunteers was analyzed. Regarding the whole group, homocysteine levels were significantly higher in men (17.0 ± 3.4 vs. 12.1 ± 4.0 μmol/L, p = 0.009). Homocysteine levels correlated with waist circumference (R = 0.58, p = 0.003), waist-to-hip ratio (R = 0.57, p = 0.003), basal metabolic rate (R = 0.48, p = 0.01), lean body mass [kg] (R = 0.53, p = 0.008), body water [L] (R = 0.53, p = 0.008) and triglycerides (R = 0.57, p = 0.003). There were no significant differences of homocysteine levels for impaired fasting glucose, abdominal obesity, obesity/overweight, and dyslipidemia. Homocysteine levels did not correlate with age, treatment duration, clozapine dose, weight, body mass index, abdominal circumference, blood pressure, total body fat, cholesterol, high density lipoproteins, low density lipoproteins, uric acid, calcium, glucose, insulin, homoeostasis model assessment of insulin resistance 1, and homoeostasis model assessment of insulin resistance 2. We did not find significant differences in blood homocysteine levels between subjects with schizophrenia and controls. Association with waist circumference may support homocysteine role as an important cardiovascular risk factor. Association with lean weight may explain why men have higher levels of homocysteine than women.     

Salt Tolerance in Astragalus cicer Microsymbionts: The Role of Glycine Betaine in Osmoprotection

In this study, we have investigated intrinsic salt tolerance of Astragalus cicer microsymbionts (USDA3350, ACMP18) and the role of exogenous glycine betaine in osmoprotection in these bacteria. Salt stress was imposed by NaCl concentrations ranging from 0.5 to 2 %. A. cicer mesorhizobia were capable of tolerating up to 2 % sodium chloride with a population count that was inversely proportional to the salt content. When the extracellular concentration of NaCl was raised to 2 %, the generation time of the UDSA3350 strain in the mid-exponential phase of growth was 3.9-times greater than that in the no-salt control medium, whereas the ACMP18 strain survived under the same conditions but did not multiply. Application of 1 mM glycine betaine into the salt-stressed rhizobium cultures increased the number of culturable bacteria, pointing out that this molecule was involved in restoration of osmotic balance. The decline in A. cicer symbiont viability in the medium with sodium chloride and the osmoprotective role of glycine betaine for these bacteria was confirmed in the experiment using the live/dead Bac Light Bacterial Vibility Kit. Data presented in this study showed the presence of proU-like genes in the genomes of A. cicer rhizobia with high sequence similarity to the genes of the ProU-like system in Sinorhizobium meliloti and the proU operon of Escherichia coli.     

Genetic Diversity of Bile Salt Hydrolases Among Human Intestinal Bifidobacteria

Attachment of Brachyspira hyodysenteriae to intestinal epithelial cell lines and its possible mediation by outer membrane proteins (OMPs) of the spirochete were examined. Different B. hyodysenteriae serotypes were shown to adhere to rat and swine intestinal epithelial cells (IEC-18 and IPEC-J2) in vitro but not to the human rectal tumor cell line (HRT-18). Adherence of strain B204 to IPEC-J2 cells was reduced by rOMP-specific antisera in amounts of 29 % (anti-rBhlp29.7), 59 % (anti-rBhlp16), 70 % (anti-rBhmp39h), and 74 % (anti-rBhmp39h), respectively. By use of pooled antisera against Bhlp16 and Bhmp39f inhibition rates of the other serotypes ranged from 53 to 91 %. In a western blot assay OMPs of all serotypes but one were detected by the respective rOMP antisera. Altogether the results indicated that OMPs of B. hyodysenteriae displayed a serotype overlapping antigenicity and mediated adherence of the spirochetes to animal cell cultures.