Mesenchymal stem cell therapy to promote limb transplant functional recovery.

BACKGROUND: Limb transplantation is a viable option for reconstruction after traumatic limb loss; however, functional recovery can be suboptimal. The aim of this study was to determine whether mesenchymal stem cell (MSC) administration can improve limb transplant functional recovery. METHODS: Orthotopic syngeneic hindlimb transplants were performed in Lewis rats, followed by topical and intravenous injections of syngeneic MSCs (5 × 106 ) or vehicle. Transplanted limb sensory and motor functions were tested by cutaneous pain reaction and walking track analysis, respectively. RESULTS: MSCs expanded ex vivo were CD29+ , CD31- , CD34- , CD44+ , CD45low , CD90+ , MHC Class-I+ , Class-II- , and pluripotent. Greater than 90% of limb transplants survived. At 4 weeks post-transplantation, the mean sensory nerve (tibial, peroneal, or sural) function in MSC (n = 9) and vehicle (n = 9) groups was <0.3 on a scale of Grades 0-3 (0 = No function; 3 = Normal). By 8 weeks, the sensory scores for tibial, peroneal, and sural nerves were 2.2 ± 0.7, 1.2 ± 0.5, and 1.7 ± 0.9 in the vehicle, and 2.6 ± 0.4, 1.0 ± 0.9, and 1.7 ± 0.9 in the MSC group, respectively (n = 9/group). At 4, 8, 16, and 24 weeks, the overall sensory function was higher in MSC group (≥7/group). Sciatic Function Index (SFI), a measure of motor function, could not be calculated because of poor foot prints; therefore, a novel grading system was developed. Bone fusion/vascularization as determined by X-ray films/laser Doppler (≥2 week post-transplantation) were normal (n = 3/group). Gastrocnemius muscle was atrophied (P < 0.05), and flexion contractures were evident by 24 weeks. CONCLUSIONS: Bone marrow-derived MSC therapy appears to improve sensory function recovery in a rat limb transplant model. Published 2016. This article is a U.S. Government work and is in the public domain in the USA Microsurgery 37:222-234, 2017.

Hydrogen sulfide improves resuscitation via non-hibernatory mechanisms in a porcine shock model.

BACKGROUND: Hydrogen sulfide (H2S) has been demonstrated to induce a "suspended animation-like" state in rodent models by reversible inhibition of cellular respiration and marked metabolic suppression and has been proposed as a potential pharmacologic adjunct to resuscitation from shock states. There are few data currently available about the mechanisms and efficacy of H2S in larger animals or humans. We examined H2S as a pharmacologic adjunct to resuscitation in a porcine model of severe traumatic shock. METHODS: Twenty-one adult swine were assigned to three study arms: sham, H2S, and saline vehicle controls (SC). All pigs underwent laparotomy and instrumentation, and the two study arms then underwent a 35% controlled hemorrhage followed by 50 min of truncal ischemia via aortic cross-clamp. H2S (5 mg/kg) or saline was administered immediately before reperfusion, followed by 6 h of resuscitation. Resuscitation requirements, laboratory parameters, end-organ histology, and inflammatory product gene expression (by reverse transcription-polymerase chain reaction) were measured and compared between groups. RESULTS: All animals survived to the 6-h postresuscitation time point. Both treatment arms demonstrated severe shock characterized by fluid and vasopressor requirements, metabolic acidosis, and hypotension compared with sham animals. Animals treated with H2S demonstrated significantly lower resuscitative requirements (total epinephrine 727 versus 3052 µg; P < 0.05), decreased fluid requirements, and lower serum lactate levels (7 versus 10 mmol/L) versus SC. Cardiac output was slightly decreased with H2S treatment but all other hemodynamic and metabolic parameters were equivalent between H2S and C groups. Serum liver and kidney biomarkers were unchanged, but administration of H2S was associated with a significant improvement in histopathologic liver and kidney injury scores compared with SC (both P < 0.05). Both study groups demonstrated significantly increased gene expression of hypoxia-inducible factor 1α and nitric oxide synthase (endogenous nitric oxide synthase, inducible nitric oxide synthase [iNOS]2, iNOS3) relative to sham animals. However, H2S was associated with increased expression of hypoxia-inducible factor 1α and decreased iNOS2 levels compared with SC. CONCLUSIONS: Administration of H2S in a large-animal model of severe traumatic shock resulted in a significant decrease in resuscitative requirements, decreased metabolic acidosis, and less end-organ histologic injury compared with standard resuscitation. H2S did not induce profound metabolic suppression as seen in rodents, and appears to have alternative mechanisms of action in large animals.

Group B streptococcus serotype prevalence in reproductive-age women at a tertiary care military medical center relative to global serotype distribution.

BACKGROUND: Group B Streptococcus (GBS) serotype (Ia, Ib, II-IX) correlates with pathogen virulence and clinical prognosis. Epidemiological studies of seroprevalence are an important metric for determining the proportion of serotypes in a given population. The purpose of this study was to evaluate the prevalence of individual GBS serotypes at Madigan Healthcare System (Madigan), the largest military tertiary healthcare facility in the Pacific Northwestern United States, and to compare seroprevalences with international locations. METHODS: To determine serotype distribution at Madigan, we obtained GBS isolates from standard-of-care anogenital swabs from 207 women of indeterminate gravidity between ages 18-40 during a five month interval. Serotype was determined using a recently described molecular method of polymerase chain reaction by capsular polysaccharide synthesis (cps) genes associated with pathogen virulence. RESULTS: Serotypes Ia, III, and V were the most prevalent (28%, 27%, and 17%, respectively). A systematic review of global GBS seroprevalence, meta-analysis, and statistical comparison revealed strikingly similar serodistibution at Madigan relative to civilian-sector populations in Canada and the United States. Serotype Ia was the only serotype consistently higher in North American populations relative to other geographic regions (p < 0.005). The number of non-typeable isolates was significantly lower in the study (p < 0.005). CONCLUSION: This study establishes PCR-based serotyping as a viable strategy for GBS epidemiological surveillance. Our results suggest that GBS seroprevalence remains stable in North America over the past two decades.

Correction: The orphan nuclear receptor Nr4a1 mediates perinatal neuroinflammation in a murine model of preterm labor.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The orphan nuclear receptor Nr4a1 mediates perinatal neuroinflammation in a murine model of preterm labor.

Prematurity is associated with perinatal neuroinflammation and injury. Screening for genetic modulators in an LPS murine model of preterm birth revealed the upregulation of Nr4a1, an orphan nuclear transcription factor that is normally absent or limited in embryonic brains. Concurrently, Nr4a1 was downregulated with magnesium sulfate (MgSO4) and betamethasone (BMTZ) treatments administered to LPS exposed dams. To understand the role of Nr4a1 in perinatal brain injury, we compared the preterm neuroinflammatory response in Nr4a1 knockout (KO) versus wild type (wt) mice. Key inflammatory factors Il1b, Il6 and Tnf, and Iba1+ microglia were significantly lower in Nr4a1 KO versus wt brains exposed to LPS in utero. Treatment with MgSO4/BMTZ mitigated the neuroinflammatory process in wt but not Nr4a1 KO brains. These results correspond with a reduction in cerebral hemorrhage in wt but not mutant embryos from dams given MgSO4/BMTZ. Further analysis with Nr4a1-GFP-Cre × tdTomato loxP reporter mice revealed that the upregulation of Nr4a1 with perinatal neuroinflammation occurs in the cerebral vasculature. Altogether, this study implicates Nr4a1 in the developing vasculature as a potent mediator of neuroinflammatory brain injury that occurs with preterm birth. It is also possible that MgSO4/BMTZ mitigates this process by direct or indirect inhibition of Nr4a1.

Characterization of the 16S-23S internal transcribed spacer among 34 higher plants: suitability for interspecific plastid transformation.

Biomanufacturing by chloroplast transgene expression has the potential to produce significant amounts of biopharmaceuticals, endow plants with novel commercial or humanitarian capabilities, enhance phytoremediation methods and harden plants against adverse environments. Plastid bioengineering exploits the phenomenon of homologous recombination to specifically integrate heterologous sequences into the plastid genome. Previous research suggests the plastid genome 16S-23S internal transcribed spacer provides an advantageous integration site for transgene expression. To characterize the suitability of the 16S-23S region for interspecific recombination, we developed primers against conserved plastid sequences and amplified approximately 2.6 kb from 25 plant species. We analyzed the amplicons with nine species from Genbank for homeology, phylogenetic relationships, potential to form chimeric rDNA elements disruptive to translational/replication systems, and the potential number of recombination events for various minimal essential processing segments (MEPS) lengths. Multiple sequence alignment of the 34 species revealed considerable conservation, with identities exceeding 95% among the angiosperms. Substitutions were statistically clustered, generally in noncoding sites, although proposed functional elements such as the OriA region and 3' terminus of the 16S rRNA exhibited unexpected variation. The nonrandom distribution of substitutions undermines the established, statistical method of estimating the number of recombination initiation sites. This finding is further substantiated by comparing statistical estimates of the number of MEPS sites to a direct count at three different MEPS lengths. We frame this in silico analysis in terms of the potential of the 16S-23S region as a target for interspecific transformation, and describe a 'primer-to-plastid' system to rapidly generate species-specific flanking regions for transformation vectors.