Prokaryotic genomes are substantially diverse, even when from closely related species, with the resulting phenotypic diversity representing a repertoire of adaptations to specific constraints. Within the microbial population, genome content may not be fixed, as changing selective forces favor particular phenotypes; however, organisms well adapted to particular niches may have evolved mechanisms to facilitate such plasticity. The highly diverse Helicobacter pylori is a model for studying genome plasticity in the colonization of individual hosts. For H. pylori, neither point mutation, nor intergenic recombination requiring the presence of multiple colonizing strains, is sufficient to fully explain the observed diversity. Here we demonstrate that H. pylori has extensive, nonrandomly distributed repetitive chromosomal sequences, and that recombination between identical repeats contributes to the variation within individual hosts. That H. pylori is representative of prokaryotes, especially those with smaller (<2 megabases) genomes, that have similarly extensive direct repeats, suggests that recombination between such direct DNA repeats is a widely conserved mechanism to promote genome diversification. P rokaryotes that successfully colonize niches for extended periods of time must be capable of adapting to changing environmental stresses (1). Genomic diversity, caused by spontaneous point mutation, intragenomic rearrangement, and horizontal gene acquisition (2), creates a phenotypically diverse population from which the most-fit variants are selected. Colonization of the primate stomach by Helicobacter pylori is prolonged, usually persisting for decades without host clearance (3). That essentially all H. pylori isolates obtained from unrelated individuals are genetically distinguishable (4), and that diverse subclones have been identified in individual hosts (5-7) suggest that persistence may be facilitated by the organism's ability to adapt to dynamic environments by continuous change (8). HIV and other pathogenic viruses that persistently infect hosts employ similar strategies of ''quasispecies'' development to increase fitness success (9).For H. pylori, neither spontaneous point mutation (10) nor recombination with other bacterial cells (11) is sufficient to explain observed intrahost genetic variation (5, 7). Computational analysis of the fully sequenced H. pylori strain 26695 (12) indicates numerous direct DNA repeats (13, 14); however, these studies do not demonstrate a role for repeats through experimental analyses, provide evidence that these repeat structures are involved in any biologic function, or assess how the nonrandom distribution of repeats may affect the physiology or evolution of the host organism. Because recombination involving direct repeats allows for deletion or duplication of intervening sequences (15), we sought to determine the extent to which H. pylori uses repetitive DNA sequences to generate diversity. Our observations provide evidence that the presence of extensive, nonrandomly distribu...

DNA rearrangement permits bacteria to regulate gene content and expression. In Helicobacter pylori, cagY, which contains an extraordinary number of direct DNA repeats, encodes a surface-exposed subunit of a (type IV) bacterial secretory system. Examining potential DNA rearrangements involving the cagY repeats indicated that recombination events invariably yield in-frame open reading frames, producing alternatively expressed genes. In individual hosts, H. pylori cell populations include strains that produce CagY proteins that differ in size, due to the predicted in-frame deletions or duplications, and elicit minimal or no host antibody recognition. Using repetitive DNA, H. pylori rearrangements in a host-exposed subunit of a conserved bacterial secretion system may permit a novel form of antigenic evasion.

We previously demonstrated that transient stromal cell-derived factor-1 alpha (SDF-1) improved cardiac function when delivered via cell therapy in ischemic cardiomyopathy at a time remote from acute myocardial infarction (MI) rats. We hypothesized that non-viral gene transfer of naked plasmid DNA-expressing hSDF-1 could similarly improve cardiac function. To optimize plasmid delivery, we tested SDF-1 and luciferase plasmids driven by the cytomegalovirus (CMV) promoter with (pCMVe) or without (pCMV) translational enhancers or α myosin heavy chain (pMHC) promoter in a rodent model of heart failure. In vivo expression of pCMVe was 10-fold greater than pCMV and pMHC expression and continued over 30 days. We directly injected rat hearts with SDF-1 plasmid 1 month after MI and assessed heart function. At 4 weeks after plasmid injection, we observed a 35.97 and 32.65% decline in fractional shortening (FS) in control (saline) animals and pMHC-hSDF1 animals, respectively, which was sustained to 8 weeks. In contrast, we observed a significant 24.97% increase in animals injected with the pCMVe-hSDF1 vector. Immunohistochemistry of cardiac tissue revealed a significant increase in vessel density in the hSDF-1-treated animals compared with control animals. Increasing SDF-1 expression promoted angiogenesis and improved cardiac function in rats with ischemic heart failure along with evidence of scar remodeling with a trend toward decreased myocardial fibrosis. These data demonstrate that stand-alone non-viral hSDF-1 gene transfer is a strategy for improving cardiac function in ischemic cardiomyopathy.

BackgroundStromal cell-derived factor-1 (SDF-1) promotes tissue repair through mechanisms of cell survival, endogenous stem cell recruitment, and vasculogenesis. Stromal Cell-Derived Factor-1 Plasmid Treatment for Patients with Heart Failure (STOP-HF) is a Phase II, double-blind, randomized, placebo-controlled trial to evaluate safety and efficacy of a single treatment of plasmid stromal cell-derived factor-1 (pSDF-1) delivered via endomyocardial injection to patients with ischaemic heart failure (IHF).MethodsNinety-three subjects with IHF on stable guideline-based medical therapy and left ventricular ejection fraction (LVEF) ≤40%, completed Minnesota Living with Heart Failure Questionnaire (MLWHFQ) and 6-min walk distance (6 MWD), were randomized 1 : 1 : 1 to receive a single treatment of either a 15 or 30 mg dose of pSDF-1 or placebo via endomyocardial injections. Safety and efficacy parameters were assessed at 4 and 12 months after injection. Left ventricular functional and structural measures were assessed by contrast echocardiography and quantified by a blinded independent core laboratory. Stromal Cell-Derived Factor-1 Plasmid Treatment for Patients with Heart Failure was powered based on change in 6 MWD and MLWHFQ at 4 months.ResultsSubject profiles at baseline were (mean ± SD): age 65 ± 9 years, LVEF 28 ± 7%, left ventricular end-systolic volume (LVESV) 167 ± 66 mL, N-terminal pro brain natriuretic peptide (BNP) (NTproBNP) 1120 ± 1084 pg/mL, MLWHFQ 50 ± 20 points, and 6 MWD 289 ± 99 m. Patients were 11 ± 9 years post most recent myocardial infarction. Study injections were delivered without serious adverse events in all subjects. Sixty-two patients received drug with no unanticipated serious product-related adverse events. The primary endpoint was a composite of change in 6 MWD and MLWHFQ from baseline to 4 months follow-up. The primary endpoint was not met (P = 0.89). For the patients treated with pSDF-1, there was a trend toward an improvement in LVEF at 12 months (placebo vs. 15 mg vs. 30 mg ΔLVEF: −2 vs. −0.5 vs. 1.5%, P = 0.20). A pre-specified analysis of the effects of pSDF-1 based on tertiles of LVEF at entry revealed improvements in EF and LVESV from lowest-to-highest LVEF. Patients in the first tertile of EF (<26%) that received 30 mg of pSDF-1 demonstrated a 7% increase in EF compared with a 4% decrease in placebo (ΔLVEF = 11%, P = 0.01) at 12 months. There was also a trend towards improvement in LVESV, with treated patients demonstrating an 18.5 mL decrease compared with a 15 mL increase for placebo at 12 months (ΔLVESV = 33.5 mL, P = 0.12). The change in end-diastolic and end-systolic volume equated to a 14 mL increase in stroke volume in the patients treated with 30 mg of pSDF-1 compared with a decrease of −11 mL in the placebo group (ΔSV = 25 mL, P = 0.09). In addition, the 30 mg-treated cohort exhibited a trend towards improvement in NTproBNP compared with placebo at 12 months (−784 pg/mL, P = 0.23).ConclusionsThe blinded placebo-controlled STOP-HF trial demonstrated the safety of a single ...

Stem cell therapy for the prevention and treatment of cardiac dysfunction holds significant promise for patients with ischemic heart disease. Excitingly early clinical studies have demonstrated safety and some clinical feasibility, while at the same time studies in the laboratory have investigated mechanisms of action and strategies to optimize the effects of regenerative cardiac therapies. One of the key pathways that has been demonstrated critical in stem cell-based cardiac repair is (stromal cell-derived factor-1) SDF-1:CXCR4. SDF-1:CXCR4 has been shown to affect stem cell homing, cardiac myocyte survival and ventricular remodeling in animal studies of acute myocardial infarction and chronic heart failure. Recently released clinical data suggest that SDF-1 alone is sufficient to induce cardiac repair. Most importantly, studies like those on the SDF-1:CXCR4 axis have suggested mechanisms critical for cardiac regenerative therapies that if clinical investigators continue to ignore will result in poorly designed studies that will continue to yield negative results. Gene Therapy (2012) 19, 583-587; doi:10.1038/gt.2012.32Keywords: acute myocordial infarction; regenerative medicine; stem cells; chemokines INTRODUCTIONOver a decade ago we hypothesized that stem cell-based repair of ischemic tissue is a natural response to tissue injury but that it is clinically inefficient because of the short-lived nature of the molecular signals regulating the process, not a lack of stem cells. At that time it was of great interest to us that stem cells in the blood stream homed to the myocardium in animal models of acute myocardial infarction (AMI), but not in models of chronic ischemic cardiomyopathy. These observations led us to investigate potential regulators of stem cell homing. We eventually identified stromal cell-derived factor-1 (SDF-1, aka CXCL12) as the key regulator of stem cell migration to sites of tissue injury. 1 More recently studies have extended the relevance of the SDF-1:CXCR4 axis by demonstrating its critical importance in cardiogenic specification during development. 2 As that initial observation we have demonstrated that transient engineered-cell-based 3 or plasmid-based 4 overexpression of SDF-1 in ischemic cardiomyopathy improved cardiac function. Furthermore, we have demonstrated that delivery of mesenchymal stem cells engineered to overexpress SDF-1 at the time of AMI leads to improvement in cardiac function. 5 Research by our laboratory and others have demonstrated that mechanism of action of SDF-1 overexpression in AMI and chronic heart failure (CHF) are clearly multifactorial including both systemic and direct trophic effects. [4][5][6] The initiation of endogenous stem cell-based repair appears blunted because of the natural short-term expression of SDF-1 at the time of AMI. 1 Furthermore, there appears to be commonalities associated with the mechanisms of action of SDF-1 in AMI and CHF as well as some distinct differences. We were the first to show that SDF-1 leads to the recruitment of cardiac st...

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Proper wound diagnosis and management is an increasingly important clinical challenge and is a large and growing unmet need. Pressure ulcers, hard-to-heal wounds, and problematic surgical incisions are emerging at increasing frequencies. At present, the wound-healing industry is experiencing a paradigm shift towards innovative treatments that exploit nanotechnology, biomaterials, and biologics. Our study utilized an alginate hydrogel patch to deliver stromal cell-derived factor-1 (SDF-1), a naturally occurring chemokine that is rapidly overexpressed in response to tissue injury, to assess the potential effects SDF-1 therapy on wound closure rates and scar formation. Alginate patches were loaded with either purified recombinant human SDF-1 protein or plasmid expressing SDF-1 and the kinetics of SDF-1 release were measured both in vitro and in vivo in mice. Our studies demonstrate that although SDF-1 plasmid-and protein-loaded patches were able to release therapeutic product over hours to days, SDF-1 protein was released faster (in vivo K d 0.55 days) than SDF-1 plasmid (in vivo K d 3.67 days). We hypothesized that chronic SDF-1 delivery would be more effective in accelerating the rate of dermal wound closure in Yorkshire pigs with acute surgical wounds, a model that closely mimics human wound healing. Wounds treated with SDF-1 protein (n = 10) and plasmid (n = 6) loaded patches healed faster than sham (n = 4) or control (n = 4). At day 9, SDF-1-treated wounds significantly accelerated wound closure (55.0 ± 14.3% healed) compared to nontreated controls (8.2 ± 6.0%, p < 0.05). Furthermore, 38% of SDF-1-treated wounds were fully healed at day 9 (vs. none in controls) with very little evidence of scarring. These data suggest that patch-mediated SDF-1 delivery may ultimately provide a novel therapy for accelerating healing and reducing scarring in clinical wounds.

To dissect genetically the regulation of NorA, a multidrug transporter of Staphylococcus aureus, we analyzed the differential expression of the norA promoter using a transcriptional fusion with a ␤-lactamase reporter gene. Expression studies with an arlS mutant revealed that the norA promoter is ArlS dependent. The arlR-arlS locus was shown to code for a two-component regulatory system. The protein ArlR has strong similarity to response regulators, and ArlS has strong similarity to protein histidine kinases. We have also analyzed the 350-bp region upstream of the Shine-Dalgarno sequence of norA by gel mobility shift experiments. It was shown that only the 115-bp region upstream of the promoter was necessary for multiple binding of an 18-kDa protein.From transcriptional fusions, we have localized four different putative boxes of 6 bp, which appear to play a role in the binding of the 18-kDa protein and in the up-regulation of norA expression in the presence of the arlS mutation. Furthermore, the gel mobility shift of the 18-kDa protein was modified in the presence of the arlS mutation, and the arlS mutation altered the growth-phase regulation of NorA. These results indicate that expression of norA is modified by a two-component regulatory system.