Restoration of aged hematopoietic cells by their young counterparts through instructive microvesicles release.

This study addresses the potential to reverse age-associated morbidity by establishing methods to restore the aged hematopoietic system. Parabiotic animal models indicated that young secretome could restore aged tissues, leading us to establish a heterochronic transwell system with aged mobilized peripheral blood (MPB), co-cultured with young MPB or umbilical cord blood (UCB) cells. Functional studies and omics approaches indicate that the miRNA cargo of microvesicles (MVs) restores the aged hematopoietic system. The in vitro findings were validated in immune deficient (NSG) mice carrying an aged hematopoietic system, improving aged hallmarks such as increased lymphoid:myeloid ratio, decreased inflammation and cellular senescence. Elevated MYC and E2F pathways, and decreased p53 were key to hematopoietic restoration. These processes require four restorative miRs that target the genes for transcription/differentiation, namely PAX and phosphatase PPMIF. These miRs when introduced in aged cells were sufficient to restore the aged hematopoietic system in NSG mice. The aged MPBs were the drivers of their own restoration, as evidenced by the changes from distinct baseline miR profiles in MPBs and UCB to comparable expressions after exposure to aged MPBs. Restorative natural killer cells eliminated dormant breast cancer cells in vivo, indicating the broad relevance of this cellular paradigm - preventing and reversing age-associated disorders such as clearance of early malignancies and enhanced responses to vaccine and infection.

Mesenchymal Stem Cell-Secreted Extracellular Vesicles Instruct Stepwise Dedifferentiation of Breast Cancer Cells into Dormancy at the Bone Marrow Perivascular Region.

In the bone marrow (BM), breast cancer cells (BCC) can survive in dormancy for decades as cancer stem cells (CSC), resurging as tertiary metastasis. The endosteal region where BCCs exist as CSCs poses a challenge to target them, mostly due to the coexistence of endogenous hematopoietic stem cells. This study addresses the early period of dormancy when BCCs enter BM at the perivascular region to begin the transition into CSCs, which we propose as the final step in dormancy. A two-step process comprises the Wnt-ß-catenin pathway mediating BCC dedifferentiation into CSCs at the BM perivascular niche. At this site, BCCs responded to two types of mesenchymal stem cell (MSC)-released extracellular vesicles (EV) that may include exosomes. Early released EVs began the transition into cycling quiescence, DNA repair, and reorganization into distinct BCC subsets. After contact with breast cancer, the content of EVs changed (primed) to complete dedifferentiation into a more homogeneous population with CSC properties. BCC progenitors (Oct4alo), which are distant from CSCs in a hierarchical stratification, were sensitive to MSC EVs. Despite CSC function, Oct4alo BCCs expressed multipotent pathways similar to CSCs. Oct4alo BCCs dedifferentiated and colocalized with MSCs (murine and human BM) in vivo. Overall, these findings elucidate a mechanism of early dormancy at the BM perivascular region and provide evidence of epigenome reorganization as a potential new therapy for breast cancer. SIGNIFICANCE: These findings describe how the initial process of dormancy and dedifferentiation of breast cancer cells at the bone marrow perivascular niche requires mesenchymal stem cell-derived exosomes, indicating a potential target for therapeutic intervention.

Klebsiella pneumoniae ST307 with blaOXA-181, South Africa, 2014-2016.

Klebsiella pneumoniae sequence type (ST) 307 is an emerging global antimicrobial drug-resistant clone. We used whole-genome sequencing and PCR to characterize K. pneumoniae ST307 with oxacillinase (OXA) 181 carbapenemase across several private hospitals in South Africa during 2014-2016. The South Africa ST307 belonged to a different clade (clade VI) with unique genomic characteristics when compared with global ST307 (clades I-V). Bayesian evolution analysis showed that clade VI emerged around March 2013 in Gauteng Province, South Africa, and then evolved during 2014 into 2 distinct lineages. K. pneumoniae ST307 clade VI with OXA-181 disseminated over a 15-month period within 42 hospitals in 23 cities across 6 northeastern provinces, affecting 350 patients. The rapid expansion of ST307 was most likely due to intrahospital, interhospital, intercity, and interprovince movements of patients. This study highlights the importance of molecular surveillance for tracking emerging antimicrobial clones.

Keeping it together: absence of genetic variation and DNA incorporation by the predatory bacteria Micavibrio aeruginosavorus and Bdellovibrio bacteriovorus during predation.

The use of predatory bacteria as a potential live therapeutic to control human infection is gaining increased attention. Earlier work with Micavibrio spp. and Bdellovibrio spp. has demonstrated the ability of these predators to control drug-resistant Gram-negative pathogens, Tier-1 select agents and biofilms. Additional studies also confirmed that introducing high doses of the predators into animals does not negatively impact animal well-being and might assist in reducing bacterial burden in vivo. The survival of predators requires extreme proximity to the prey cell, which might bring about horizontal transfer of genetic material, such as genes encoding for pathogenic genetic islands that would indirectly facilitate the spread of genetic material to other organisms. In this study, we examined the genetic makeup of several lab isolates of the predators Bdellovibriobacteriovorus and Micavibrioaeruginosavorus that were cultured repeatedly and stored over a course of 13 years. We also conducted controlled experiments in which the predators were sequentially co-cultured on Klebsiella pneumoniae followed by genetic analysis of the predator. In both cases, we saw little genetic variation and no evidence of horizontally transferred chromosomal DNA from the prey during predator-prey interaction. Culturing the predators repeatedly did not cause any change in predation efficacy.

Inherent X-Linked Genetic Variability and Cellular Mosaicism Unique to Females Contribute to Sex-Related Differences in the Innate Immune Response.

Females have a longer lifespan and better general health than males. Considerable number of studies also demonstrated that, after trauma and sepsis, females present better outcomes as compared to males indicating sex-related differences in the innate immune response. The current notion is that differences in the immuno-modulatory effects of sex hormones are the underlying causative mechanism. However, the field remains controversial and the exclusive role of sex hormones has been challenged. Here, we propose that polymorphic X-linked immune competent genes, which are abundant in the population are important players in sex-based immuno-modulation and play a key role in causing sex-related outcome differences following trauma or sepsis. We describe the differences in X chromosome (ChrX) regulation between males and females and its consequences in the context of common X-linked polymorphisms at the individual as well as population level. We also discuss the potential pathophysiological and immune-modulatory aspects of ChrX cellular mosaicism, which is unique to females and how this may contribute to sex-biased immune-modulation. The potential confounding effects of ChrX skewing of cell progenitors at the bone marrow is also presented together with aspects of acute trauma-induced de novo ChrX skewing at the periphery. In support of the hypothesis, novel observations indicating ChrX skewing in a female trauma cohort as well as case studies depicting the temporal relationship between trauma-induced cellular skewing and the clinical course are also described. Finally, we list and discuss a selected set of polymorphic X-linked genes, which are frequent in the population and have key regulatory or metabolic functions in the innate immune response and, therefore, are primary candidates for mediating sex-biased immune responses. We conclude that sex-related differences in a variety of disease processes including the innate inflammatory response to injury and infection may be related to the abundance of X-linked polymorphic immune-competent genes, differences in ChrX regulation, and inheritance patterns between the sexes and the presence of X-linked cellular mosaicism, which is unique to females.

Trauma-Induced Acute X Chromosome Skewing in White Blood Cells Represents an Immuno-Modulatory Mechanism Unique to Females and a Likely Contributor to Sex-Based Outcome Differences.

Sex-related outcome disparities following severe trauma have been demonstrated in human and animal studies; however, sex hormone status could not fully account for the differences. This study tested whether X-linked cellular mosaicism, which is unique to females, could represent a genetically based mechanism contributing to sex-related immuno-modulation following trauma. Serial blood samples collected for routine laboratory tests were analyzed for ChrX inactivation (XCI) ratios in white blood cells. Thirty-nine severely injured (mean ISS 19) female trauma patients on mixed racial and ethnic background were tested for initial (baseline) and trauma-induced changes in XCI ratios and their associations with severity of injury and clinical outcome. At admission, two-thirds of the patients showed XCI-ratio values between one and three, about a third presented skewed XCI ratios (3-7 range) and three patients displayed extremely skewed XCI ratios (8-30 range). Serial blood samples during the clinical course showed additional changes in XCI ratios ranging between 20% and 900% over initial. Increasing XCI ratios during the injury course correlated with the severity of trauma, subsequent need for ventilator support and pneumonia. In contrast, initial XCI ratios did not show correlations with injury severity or clinical complications. Initial XCI ratios showed a positive correlation with age but older patients retained the ability to mount trauma-induced secondary XCI changes. These data show that trauma results in X-linked cell selection in females, which is likely to be driven by polymorphic differences between the parental ChrXs. X-linked white blood cell skewing correlates with injury severity and a complicated postinjury clinical course. Female X-linked cellular mosaicism and its capacity to change dynamically during the injury course compared with the lack of this machinery in males may represent a novel immuno-modulatory mechanism contributing to sex-based outcome differences after injury and infection.

Anti-neurotrophic effects from autoantibodies in adult diabetes having primary open angle glaucoma or dementia.

AIM: To test for anti-endothelial and anti-neurotrophic effects from autoantibodies in subsets of diabetes having open-angle glaucoma, dementia, or control subjects. METHODS: Protein-A eluates from plasma of 20 diabetic subjects having glaucoma or suspects and 34 age-matched controls were tested for effects on neurite outgrowth in rat pheochromocytoma PC12 cells or endothelial cell survival. The mechanism of the diabetic glaucoma autoantibodies' neurite-inhibitory effect was investigated in co-incubations with the selective Rho kinase inhibitor Y27632 or the sulfated proteoglycan synthesis inhibitor sodium chlorate. Stored protein-A eluates from certain diabetic glaucoma or dementia subjects which contained long-lasting, highly stable cell inhibitory substances were characterized using mass spectrometry and amino acid sequencing. RESULTS: Diabetic primary open angle glaucoma (POAG) or suspects (n = 20) or diabetic dementia (n = 3) autoantibodies caused significantly greater mean inhibition of neurite outgrowth in PC12 cells (p < 0.0001) compared to autoantibodies in control diabetic (n = 24) or non-diabetic (n = 10) subjects without glaucoma (p < 0.01). Neurite inhibition by the diabetic glaucoma autoantibodies was completely abolished by 10 µM concentrations of Y27632 (n = 4). It was substantially reduced by 30 mM concentrations of sodium chlorate (n = 4). Peak, long-lasting activity survived storage ×5 years at 0-4°C and was associated with a restricted subtype of Ig kappa light chain. Diabetic glaucoma or dementia autoantibodies (n = 5) caused contraction and process retraction in quiescent cerebral cortical astrocytes effects which were blocked by 5 µM concentrations of Y27632. CONCLUSION: These data suggest that autoantibodies in subsets of adult diabetes having POAG (glaucoma suspects) and/or dementia inhibit neurite outgrowth and promote a reactive astrocyte morphology by a mechanism which may involve activation of the RhoA/p160 ROCK signaling pathway.

Anti-endothelial and anti-neuronal effects from auto-antibodies in subsets of adult diabetes having a cluster of microvascular complications.

AIMS: To test autoantibodies from subsets of diabetes with painful neuropathy, maculopathy and nephropathy for effects in neurons. METHODS: Protein-A eluates from plasma of 27 diabetic and 19 age-matched controls were tested for effects on endothelial cell survival, and neurite outgrowth in rat pheochromocytoma PC12 cells. Painful diabetic neuropathy or control autoantibodies were compared for binding to PC12-derived heparan sulfate proteoglycans. The mechanism of the effects from pathologic autoantibodies was investigated by changes in intracellular calcium in endothelial cells, whole cell current in neurons, or using the Rho kinase inhibitor Y27632. RESULTS: Autoantibodies from diabetic patients with maculopathy, nephropathy, and painful neuropathy (n=5) caused significantly greater mean inhibition of neurite outgrowth (p<0.005) than diabetic or control patients with fewer or no complications (n=30). Painful diabetic autoantibodies (3 µg/mL) bound neuronal heparan sulfate proteoglycan (HSPG) more than autoantibodies from diabetic or control subjects without painful neuropathy (p<.0001). Inhibition of PC12 neurite outgrowth by the painful neuropathy antibodies was completely prevented by 1 µM concentrations of Y27632. CONCLUSION: These results suggest anti-endothelial and anti-neuronal effects from auto-antibodies in a subset of diabetic patients with a cluster of microvascular complications.

Creation of a novel peptide with enhanced nuclear localization in prostate and pancreatic cancer cell lines.

BACKGROUND: For improved uptake of oligonucleotide-based therapy, the oligonucleotides often are coupled to peptides that facilitate entry into cells. To this end, novel cell-penetrating peptides (CPPs) were designed for mediating intracellular uptake of oligonucleotide-based therapeutics. The novel peptides were based on taking advantage of the nuclear localization properties of transcription factors in combination with a peptide that would bind putatively to cell surfaces. It was observed that adding a glutamate peptide to the N-terminus of the nuclear localization signal (NLS) of the Oct6 transcription factor resulted in a novel CPP with better uptake and better nuclear colocalization than any other peptide tested. RESULTS: Uptake of the novel peptide Glu-Oct6 by cancer cell lines was rapid (in less than 1 hr, more than 60% of DU-145 cells were positive for FITC), complete (by 4 hr, 99% of cells were positive for FITC), concentration-dependent, temperature-dependent, and inhibited by sodium azide (NaN3). Substitution of Phe, Tyr, or Asn moieties for the glutamate portion of the novel peptide resulted in abrogation of novel CPP uptake; however none of the substituted peptides inhibited uptake of the novel CPP when coincubated with cells. Live-cell imaging and analysis by imaging flow cytometry revealed that the novel CPP accumulated in nuclei. Finally, the novel CPP was coupled to a carboxyfluorescein-labeled synthetic oligonucleotide, to see if the peptide could ferry a therapeutic payload into cells. CONCLUSIONS: These studies document the creation of a novel CPP consisting of a glutamate peptide coupled to the N-terminus of the Oct6 NLS; the novel CPP exhibited nuclear colocalization as well as uptake by prostate and pancreatic cancer cell lines.

Cloning and characterization of the 5' flanking region of the HGFIN gene indicate a cooperative role among p53 and cytokine-mediated transcription factors: relevance to cell cycle regulation.

HGFIN, previously identified as nmb, and its homologs, osteoactivin are single transmembrane protein that is expressed in differentiated immune cells, and are linked to tumor progression. These dichotomous roles suggest that HGFIN could be linked to cell cycle regulation. We hypothesize that HGFIN is linked to different phases of cell cycle regulation via specific transcription factors. This study cloned and analyzed two fragments in the 5' flanking region of HGFIN: HGFIN-RM/2.0: 2.0 kb upstream of Exon 1; HGFIN-RM/1.5: 5' deletion (500 bp) of HGFIN-RM/2.0. Computer analyses indicated that HGFIN has unique upstream sequence with eight potential p53 sites. Electrophoretic mobility shift assay with Cy3-labeled PCR fragments indicated that p53 could interact with fragments encompassing p53 consensus regions. Reporter gene activities with HGFIN-RM/2.0 and HGFIN-RM/1.5 in cells with different p53 levels showed that p53 is relevant to HGFIN activities. Studies with modified T47D in which cytokine production was downregulated, but with p53 level similar to parental line showed synergism between p53 and mediators of cytokine in the regulation of HGFIN. In summary, p53 cooperate with cytokine-mediated transcription factors to regulate the expression of HGFIN.

A functional interaction between RHA and Ubc9, an E2-like enzyme specific for Sumo-1.

RNA helicase A (RHA) is a member of the DEAH helicase family of proteins. Recent studies imply the role of RHA in the regulation of the topology of chromatin DNA, which could influence diverse nuclear processes such as transcription activity of the chromatin DNA and chromosome condensation. We previously reported that Ubc9, an E2-like enzyme specific for small ubiquitin-like modifier 1 (Sumo-1), is required for the interaction between RHA and topoisomerase IIalpha. Here, we describe that Ubc9 is a novel factor that functionally interacts with RHA and activates the transcription activity of RHA, measured in the CREB-mediated pathway. We demonstrate that the N-terminal domain of RHA, encompassing amino acid residues 1-137, is sufficient for its interaction with Ubc9. Our data also show that interaction with Ubc9 leads to the Sumo-1 conjugation of RHA both in vitro and in vivo. However, the catalytic activity of Ubc9 seems to be dispensable for the transcription activation activity of RHA. Our observation suggests multiple roles for Ubc9 in the regulation of the RHA function.

Functional analysis of the subunits of the chromatin assembly factor RSF.

The human ISWI-containing factor RSF (for remodeling and spacing factor) is composed of two subunits: the ATPase hSNF2H and p325 (Rsf-1), a protein encoded by a novel human gene. We previously showed that RSF mediates nucleosome deposition and generates regularly spaced nucleosome arrays. Here we report the characterization of the largest subunit of RSF, Rsf-1. We found that Rsf-1 is a highly acidic protein containing a plant homology domain. The present study includes the cloning of Rsf-1, the preparation of recombinant RSF, and the dissection of the role of each subunit in the chromatin assembly reaction. The sequence of the gene for Rsf-1 includes a recently characterized cDNA, HBXAP; postulated to be involved in the transcriptional regulation of the hepatitis B virus. HBXAP actually contains a 252-amino-acid truncation of the amino terminus of Rsf-1. Finally, comparison of HBXAP and Rsf-1 properties shows that they are functionally different.

Signaling of monocytic differentiation by a non-hypercalcemic analog of vitamin D3, 1,25(OH)2-5,6 trans-16-ene-vitamin D3, involves nuclear vitamin D receptor (nVDR) and non-nVDR-mediated pathways.

Exposure of leukemia cells to the physiologically active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25D3) normalizes their phenotype to cells that resemble mature monocytes. One of the earliest detectable events in this process is an upregulation of the nuclear receptor for 1,25D3, the vitamin D receptor (nVDR). In contrast, the novel analog of 1,25D3, 1,25-dihydroxy-5,6 trans-16-ene-vitamin D3 (5,6-16D3), which has recently been reported to have low calcium-mobilizing activity in vivo, rapidly induced the expression of CD14, CD11b, and monocyte-specific esterase (MSE), classical markers of the mature monocyte, but upregulated nVDR expression less than 1,25D3. This upregulation was shown to be the result of altered degradation of the nVDR protein, while the levels of nVDR mRNA were constant. Knock-out of nVDR transcriptional activity by a decoy VDRE double-stranded deoxyoligonucleotide, markedly abrogated 1,25D3-induced differentiation, but incompletely inhibited 5,6-16D3-induced differentiation. These findings suggest that the unique ability of 5,6-16D3 to induce cell differentiation but not systemic hypercalcemia, may be due to the activation of pathways which initiate differentiation independently of nVDR.