Intranasally applied human olfactory mucosa neural progenitor cells migrate to damaged brain regions.

Aim: To determine if intranasally administered olfactory mucosa progenitor cells (OMPCs) migrate to damaged areas of brain. Materials & methods: Rowett Nude (RNU) adult rats were injured using the Marmarou model then 2 weeks later received intranasally-delivered human OMPC. After 3 weeks, rats were sacrificed and brain sectioned. The mean distances from the human OMPCs to markers for degenerative neuronal cell bodies (p-c-Jun+), axonal swellings on damaged axons (ß-APP+) and random points in immunostained sections were quantified. One-way ANOVA was used to analyze data. Results: The human OMPCs were seen in specific areas of the brain near degenerating cell bodies and damaged axons. Conclusion: Intranasally delivered human OMPC selectively migrate to brain injury sites suggesting a possible noninvasive stem cell delivery for brain injury.

Phase IIB Randomized Trial on the Use of 4-Aminopyridine in Guillain-Barré Syndrome.

OBJECTIVE: To determine the safety and efficacy of orally delivered 4-aminopyridine (4-AP) in persons with Guillain-Barré Syndrome (GBS) >6 months from initial diagnosis. DESIGN: A randomized, double-blind, placebo-controlled, crossover study. SETTING: Tertiary care clinical outpatient program. PARTICIPANTS: Nineteen participants enrolled (14 male, 5 female; N=19), neurologic impairment secondary to GBS and functional loss on the FIM motor score (stable for ≥12mo) and >3.0 but <5.0 on the American Spinal Injury motor scale. Twelve participants (mean age, 59y; range, 23-77y) completed the study. INTERVENTIONS: A 4-AP dose-escalation study with 8 weeks in each period with a 3-week washout period, followed by 3 months open-label follow-up. MAIN OUTCOME MEASURES: FIM motor score was the primary outcome measure; also evaluated were the American Spinal Injury motor strength score (all limbs), handheld dynamometer, 6-minute walk test, Medical Outcomes Study 12-Item Short Form, Center for Epidemiological Studies Depression scale, Positive and Negative Affect Schedule, pain, GBS disability scale, Jepsen-Taylor Hand Function Test, Minnesota Manual Dexterity Test and Minnesota Rate of Manipulation Test, Get Up and Go Test, McGill Pain Inventory, Craig Handicap Assessment and Reporting Technique, and participant self-evaluation. RESULTS: Seven participants discontinued the study prematurely: 3 because of adverse events, 3 because of travel difficulties or relocation, and 1 because of pretreatment laboratory abnormalities. After removing 3 participants with maximum FIM scores, 4-AP arm trended superior to placebo (P=.065). Patients subjectively could always tell when they were on the active agent usually by tingling sensations or a sense of wellness. No statistically significant differences were found for other outcome measures although there were strong trends. CONCLUSIONS: This study demonstrates the safety of 4-AP in the patient population with GBS as the predominate goal of the study. A trend toward improved function after treatment was noted with most patients electing to stay on the medication after the trial.

Mechanical, topographical and chemical cues combined with physical therapy for peripheral nerve injuries.

Aim: The aim of this paper is to evaluate biomaterial cues combined with physical therapy (PT) on functional recovery in a rat sciatic nerve injury model. Materials & methods: Nerve growth conduits were filled with longitudinally aligned hyaluronic acid fibers and microspheres containing neurotrophic factor (growth factor [GF]). All animals received behavior and functional testing throughout the study, which concluded with measurement of compound muscle action potentials and contractile force of the gastrocnemius muscle. Results & conclusion: Including GF improved recovery of gross motor function and increased sensory pain sensation. During the 4 weeks that animals participated in PT, these groups showed higher static sciatic index scores. Including GF and PT has the potential to improve clinical outcomes following peripheral nerve injury.

The extremely halophilic archaeon Halobacterium salinarum ETD5 from the solar saltern of Sfax (Tunisia) produces multiple halocins.

The extremely halophilic archaeon Halobacterium salinarum strain ETD5 was previously isolated from the solar saltern of Sfax (Tunisia) and shown to encode and express halocin S8. The Hbt. salinarum ETD5 culture supernatant was shown here to exhibit high antimicrobial activity against several halophilic archaea and bacteria of different genera, showing a cross-domain inhibition. The antimicrobial activity was destroyed by proteases, thus pointing to halocins. A bioguided purification procedure was applied using two chromatography steps and antimicrobial assays directed against Halorubrum chaoviator ETR14. In-gel screening assay showed the presence of two antimicrobial bands of approximately 8 and 14 kDa, for which characterization was investigated by N-terminal sequencing and mass spectrometry. The full-length form of halocin S8 that contains 81 amino acids and differs from the 36 amino acid short-length halocin S8 previously described from an uncharacterized haloarchaeon S8a, was identified in the 8 kDa halocin band. A novel halocin that we termed halocin S14 was found in the 14 kDa band. It exhibits amino acid sequence identities with the N-terminally truncated region of the archaeal Mn-superoxide dismutase. These results show that Hbt. salinarum ETD5 produces multiple halocins, a feature that had not been described until now in the domain Archaea.

Haloarcula sebkhae sp. nov., an extremely halophilic archaeon from Algerian hypersaline environment.

A halophilic organism, SWO25T, was isolated from water sampled in Algeria at the salt lake (sebkha) of Ouargla. The novel strain stained Gram-negative, and cells were pleomorphic with a red pigmentation. Strain SWO25T grew optimally at 35-45 °C, at pH 6.0-8.0 and 0.05-0.25 M MgCl2 concentrations. Cells were extremely halophilic, with optimal growth at 4.3-5.1 M NaCl. The predominant membrane polar lipids were C20C20 glycerol diether derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate, phosphatidylglycerol sulfate, triglycosyl diether and diglycosyl diether. The major respiratory menaquinone component was MK-8. Cells were highly tolerant to the presence of decane and isooctane in the growth medium. Chemotaxonomic properties supported the assignment of strain SWO25T to the genus Haloarcula. The DNA G+C content was 61.1mol%. DNA-DNA hybridization and phylogenetic analyses of the 16S rRNA and rpoB' genes showed that strain SWO25T is distinct from known Haloarcula species. Based on phenotypic, chemotaxonomic, genotypic and phylogenetic data, we describe a novel species of the genus Haloarcula, for which the name Haloarculasebkhae sp. nov. is proposed. The type strain is SWO25T (=CIP 110583T=JCM 19018T).

Halocin C8: an antimicrobial peptide distributed among four halophilic archaeal genera: Natrinema, Haloterrigena, Haloferax, and Halobacterium.

Halophilic archaea thrive in hypersaline ecosystems and produce antimicrobial peptides (AMPs) named halocins. AMPs are essential effectors of microbial interactions in natural ecosystems. Halocin C8 is a 7.4 kDa peptide produced by Natrinema sp. AS7092. Surrounded by genes involved in regulation and transport, the halC8 gene encodes a precursor, processed into the mature halocin and an immunity protein, protecting the producing strain against its halocin. This feature constitutes a unique property of halocin C8, as known AMPs and their immunity proteins are generally encoded on distinct ORFs in an operon. By complementary in silico and PCR-based approaches, the presence of halC8 in halophilic archaea collected from various parts of the world was evidenced. The full-length halC8 gene is restricted and consistently found in the genomes of strains belonging to the phylogenetically related genera Natrinema and Haloterrigena, along with transport and regulation genes. Functional expression of halC8 was demonstrated by RT-PCR and antimicrobial assays. Active halocin C8 was shown to contain five disulphide bridges, presumably conferring a compact structure resistant to harsh environmental conditions. In other archaeal genera, Haloferax and Halobacterium, genes encoding halocin C8 with diverging immunity protein moiety were evidenced. A phylogenetic analysis of halocin C8 sequences was conducted.

Baclofen Solution for Low-Volume Therapeutic Delivery.

OBJECTIVES: Baclofen is a zwitterion molecule where increased ions in the excipient increase the solubility. We developed baclofen in a stable solution similar to cerebrospinal fluid (CSF) without bicarbonate and proteins to improve the solubility of the baclofen and to reduce the potential toxicity to the central nervous system (CNS) and subarachnoid space. The objective is to develop a solution of baclofen wherein baclofen is solubilized in a multivalent physiological ion solution such as artificial cerebrospinal fluid (aCSF) at a concentration from 2 mg/cc to 10 mg/cc. METHODS: First, to determine the solubility of Baclofen in aCSF, solubility was determined at six different pH levels at 37°C, by the addition of aCSF to a known amount of Baclofen. The final concentrations were confirmed by high performance liquid chromatography (HPLC) analysis. Second, the stability of Baclofen at 4 mg/cc investigated in a test manufacturing batch utilizing standard methods of production of 1500 20 cc vials inverted for 18 months at 25°C at 60% humidity. The stability and purity of the baclofen was verified at 18 months by HPLC analysis. RESULTS: Baclofen was initially soluble between pH of 6-8 above 7 mg/cc but fell back to 6.3-5.8 mg/cc level with time. Baclofen produced in vials with inversion were noted to be stable at 4 mg/cc at 18 months with less than 2% breakdown of the baclofen in solution. CONCLUSION: Baclofen is much more soluble in artificial CSF than normal saline. The artificial CSF may also be less toxic to the subarachnoid space than saline.

Antagonistic interactions and production of halocin antimicrobial peptides among extremely halophilic prokaryotes isolated from the solar saltern of Sfax, Tunisia.

Thirty-five extremely halophilic microbial strains isolated from crystallizer (TS18) and non-crystallizer (M1) ponds in the Sfax solar saltern in Tunisia were examined for their ability to exert antimicrobial activity. Antagonistic assays resulted in the selection of eleven strains that displayed such antimicrobial activity and they were further characterized. Three cases of cross-domain inhibition (archaea/bacteria or bacteria/archaea) were observed. Four archaeal strains exerted antimicrobial activity against several other strains. Three strains, for which several lines of evidence suggested the antimicrobial activity was, at least in part, due to peptide/protein agents (Halobacterium salinarum ETD5, Hbt. salinarum ETD8, and Haloterrigena thermotolerans SS1R12), were studied further. Optimal culture conditions for growth and antimicrobial production were determined. Using DNA amplification with specific primers, sequencing and RT-PCR analysis, Hbt. salinarum ETD5 and Hbt. salinarum ETD8 were shown to encode and express halocin S8, a hydrophobic antimicrobial peptide targeting halophilic archaea. Although the gene encoding halocin H4 was amplified from the genome of Htg. thermotolerans SS1R12, no transcript could be detected and the antimicrobial activity was most likely due to multiple antimicrobial compounds. This is also the first report that points to four different strains isolated from different geographical locations with the capacity to produce identical halocin S8 proteins.

A hemocyanin-derived antimicrobial peptide from the penaeid shrimp adopts an alpha-helical structure that specifically permeabilizes fungal membranes.

BACKGROUND: Hemocyanins are respiratory proteins with multiple functions. In diverse crustaceans hemocyanins can release histidine-rich antimicrobial peptides in response to microbial challenge. In penaeid shrimp, strictly antifungal peptides are released from the C-terminus of hemocyanins. METHODS: The three-dimensional structure of the antifungal peptide PvHCt from Litopenaeus vannamei was determined by NMR. Its mechanism of action against the shrimp pathogen Fusarium oxysporum was investigated using immunochemistry, fluorescence and transmission electron microscopy. RESULTS: PvHCt folded into an amphipathic α-helix in membrane-mimicking media and displayed a random conformation in aqueous environment. In contact with F. oxysporum, PvHCt bound massively to the surface of fungal hyphae without being imported into the cytoplasm. At minimal inhibitory concentrations, PvHCt made the fungal membrane permeable to SYTOX-green and fluorescent dextran beads of 4 kDa. Higher size beads could not enter the cytoplasm. Therefore, PvHCt likely creates local damages to the fungal membrane. While the fungal cell wall appeared preserved, gradual degeneration of the cytoplasm most often resulting in cell lysis was observed in fungal spores and hyphae. In the remaining fungal cells, PvHCt induced a protective response by the formation of daughter hyphae. CONCLUSION: The massive accumulation of PvHCt at the surface of fungal hyphae and subsequent insertion into the plasma membrane disrupt its integrity as a permeability barrier, leading to disruption of internal homeostasis and fungal death. GENERAL SIGNIFICANCE: The histidine-rich antimicrobial peptide PvHCt derived from shrimp hemocyanin is a strictly antifungal peptide, which adopts an amphipathic α-helical structure, and selectively binds to and permeabilizes fungal cells.

Antimicrobial peptides and proteins in the face of extremes: Lessons from archaeocins.

Archaeocins are ribosomally-synthesized antimicrobial peptides or proteins produced by archaea. Halocins and sulfolobicins are produced by archaea belonging to the order Halobacteriales (Euryarchaeota) and Sulfolobales (Crenarchaeota), respectively. These weapons contribute helping the producer to prosper in spite of the microbial warfare. Given the fact that many archaea thrive in various extreme environments, archaeocins are challenged with inhospitable and destructive environmental conditions. Their structural features and mechanisms of action, which could be original, mostly remain to be deciphered. This review summarizes the present knowledge on halocins and sulfolobicins, the two classes of archaeocins that have been evidenced until now, and brings light on aspects of emerging research such as their ecological role or potential applications. Other antimicrobial compounds produced by archaea are also considered.

Characterization of leucocin B-KM432Bz from Leuconostoc pseudomesenteroides isolated from boza, and comparison of its efficiency to pediocin PA-1.

A bacteriocin-producing bacterium was isolated from boza and identified as Leuconostoc pseudomesenteroides KM432Bz. The antimicrobial peptide was purified and shown to be identical to other class IIa bacteriocins: leucocin A from Leuconostoc gelidum UAL-187 and Leuconostoc pseudomesenteroides QU15 and leucocin B from Leuconostoc carnosum Ta11a. The bacteriocin was named leucocin B-KM432Bz. Leucocin B-KM432Bz gene cluster encodes the bacteriocin precursor (lcnB), the immunity protein (lcnI) and the dedicated export machinery (lcnD and lcnE). A gene of unknown and non-essential function (lcnC), which is interrupted by an insertion sequence of the IS30 family, is localized between lcnB and lcnD. The activity of leucocin B-KM432Bz requires subunit C of the EII(t) Man mannose permease, which is the receptor for entry into target cells. The determination of the minimum inhibitory concentrations revealed the lowest values for leucocin B-KM432Bz over Listeria strains, with 4 to 32 fold better efficiency than pediocin PA-1.

Isolation and characterization of environmental bacteria capable of extracellular biosorption of mercury.

Accumulation of toxic metals in the environment represents a public health and wildlife concern. Bacteria resistant to toxic metals constitute an attractive biomass for the development of systems to decontaminate soils, sediments, or waters. In particular, biosorption of metals within the bacterial cell wall or secreted extracellular polymeric substances (EPS) is an emerging process for the bioremediation of contaminated water. Here the isolation of bacteria from soil, effluents, and river sediments contaminated with toxic metals permitted the selection of seven bacterial isolates tolerant to mercury and associated with a mucoid phenotype indicative of the production of EPS. Inductively coupled plasma-optical emission spectroscopy and transmission electron microscopy in conjunction with X-ray energy dispersive spectrometry revealed that bacteria incubated in the presence of HgCl2 sequestered mercury extracellularly as spherical or amorphous deposits. Killed bacterial biomass incubated in the presence of HgCl2 also generated spherical extracellular mercury deposits, with a sequestration capacity (40 to 120 mg mercury per g [dry weight] of biomass) superior to that of live bacteria (1 to 2 mg mercury per g [dry weight] of biomass). The seven strains were shown to produce EPS, which were characterized by Fourier transform-infrared (FT-IR) spectroscopy and chemical analysis of neutral-carbohydrate, uronic acid, and protein contents. The results highlight the high potential of Hg-tolerant bacteria for applications in the bioremediation of mercury through biosorption onto the biomass surface or secreted EPS.

Targeted ablation of mesenteric projecting sympathetic neurons reduces the hemodynamic response to pain in conscious, spinal cord-transected rats.

Individuals with spinal cord injuries above thoracic level 6 (T(6)) experience episodic bouts of life-threatening hypertension as part of a condition termed autonomic dysreflexia. The paroxysmal hypertension can be caused by a painful stimulus below the level of the injury. Targeted ablation of mesenteric projecting sympathetic neurons may reduce the severity of autonomic dysreflexia by reducing sympathetic activity. Therefore, cholera toxin B subunit (CTB) conjugated to saporin (SAP; a ribosomal inactivating protein that binds to and inactivates ribosomes) was injected into the celiac ganglion to test the hypothesis that targeted ablation of mesenteric projecting sympathetic neurons reduces the pressor response to pain in conscious, spinal cord-transected rats. Nine Sprague-Dawley male rats underwent a spinal cord transection between thoracic vertebrae 4 and 5. Following recovery (5 wk), all rats were instrumented with a radio telemetry device for recording arterial pressure and bilateral catheters in the gluteus maximus muscles for the infusion of hypertonic saline (hNa(+)Cl(-)). Subsequently, the hemodynamic responses to intramuscular injection of hNa(+)Cl(-) (100 microl and 250 microl, in random order) were determined. Following the experiments in the no celiac ganglia injected condition (NGI), rats received injections of CTB-SAP (n = 5) or CTB (n = 3) into the celiac ganglia. CTB-SAP rats, compared with NGI and CTB rats, had reduced pressor responses to hNa(+)Cl(-). Furthermore, the number of stained neurons in the celiac ganglia and spinal cord (segments T(6)-T(12)), was reduced in CTB-SAP rats. Thus, CTB-SAP retrogradely transported from the celiac ganglia is effective at ablating mesenteric projecting sympathetic neurons and reducing the pressor response to pain in spinal cord-transected rats.

Olfactory mucosal autografts and rehabilitation for chronic traumatic spinal cord injury.

BACKGROUND/OBJECTIVE: Basic science advances in spinal cord injury (SCI) are leading to novel clinical approaches. The authors report a prospective, uncontrolled pilot study of the safety and outcomes of implanting olfactory mucosal autografts (OMA) in 20 patients with chronic, sensorimotor complete or motor complete SCI. METHODS: Seven paraplegic and 13 tetraplegic subjects (17 men and 3 women; 19-37 years old) who sustained a traumatic SCI 18 to 189 months previously (mean = 49 months) were enrolled. Preoperative rehabilitation that emphasized lower extremity stepping using either overground walking training or a robotic weight-supported treadmill training was provided for 25 to 39 hours per week for a median of 4 months at 3 sites. No change in ASIA Impairment Scale (AIS) motor scores for the lower extremities or AIS grades of completeness was found. OMAs were transplanted into 1.3- to 4-cm lesions at C4-T12 neurological levels after partial scar removal. Therapy was continued postoperatively. Preoperative and postoperative assessments included AIS scores and classification, electromyography (EMG) of attempted voluntary contractions, somatosensory evoked potentials (SSEP), urodynamic studies with sphincter EMG, spinal cord magnetic resonance imaging (MRI), and otolaryngology and psychology evaluations. The Functional Independence Measure (FIM) and Walking Index for Spinal Cord Injury (WISCI) were obtained in 13 patients. RESULTS: All patients survived and recovered olfaction. One patient was rehospitalized for aseptic meningitis. Minor adverse events occurred in 4 others. The mean duration of follow-up was 27.7 months (range = 12-45 months). By MRI, the lesion site was filled in all patients with no neoplastic overgrowth or syringomyelia. AIS grades improved in 11 of 20 patients, 6 (A --> C), 3 (B --> C), and 2 (A --> B), and declined in 1 (B --> A). Improvements included new voluntary EMG responses (15 patients) and SSEPs (4 patients). Scores improved in the FIM and WISCI (13/13 tested), and urodynamic responses improved in 5 patients. CONCLUSION: OMA is feasible, relatively safe, and possibly beneficial in people with chronic SCI when combined with postoperative rehabilitation. Future controlled trials may need to include a lengthy and intensive rehabilitation arm as a control.

Isolation and characterization of two members of the siderophore-microcin family, microcins M and H47.

In this paper we provide the first biochemical evidence of the existence of a family of structure-related antimicrobial peptides, the siderophore-microcins, in the Enterobacteriaceae family. We isolated and characterized two novel siderophore-microcins, MccM and MccH47, previously characterized through genetic studies. MccM and MccH47 were expressed from several Escherichia coli strains containing the microcin gene clusters. The spectra of their bactericidal activities were found to be restricted to some species of the Enterobacteriaceae. MccM and MccH47 were unable to inhibit the growth of strains carrying mutations in the fepA, cir, and fiu genes, which showed the requirement of the iron-catecholate receptors for their recognition. The MccM and MccH47 peptide moieties contain 77 and 60 residues, respectively, and are derived from the microcin precursors McmA and MchB, respectively. In addition, both peptides carried a C-terminal posttranslational modification containing a salmochelin-like siderophore moiety also found in MccE492 (X. Thomas et al., J. Biol. Chem., 279:28233-28242, 2004). Interestingly, when MccM was isolated from E. coli Nissle 1917, which lacks the two genes necessary for modification biosynthesis, it was devoid of posttranslational modification. Those two genes could be complemented by their homologues from the MccH47 gene cluster, thereby showing their functional interchangeability between at least two members of the siderophore-microcin family. Finally, from the sequence analysis of the MccE492 gene cluster, we hypothesized the existence of an additional member of the siderophore-microcin family. Therefore, we propose that the siderophore-microcin family contains five representatives.

Targeted ablation of cardiac sympathetic neurons reduces resting, reflex and exercise-induced sympathetic activation in conscious rats.

Cholera toxin B subunit conjugated to saporin (SAP, a ribosomal inactivating protein that binds to and inactivates ribosomes) was injected in both stellate ganglia to evaluate the physiological response to targeted ablation of cardiac sympathetic neurons. Resting cardiac sympathetic activity (cardiac sympathetic tonus), exercise-induced sympathetic activity (heart rate responses to graded exercise), and reflex sympathetic activity (heart rate responses to graded doses of sodium nitroprusside, SNP) were determined in 18 adult conscious Sprague-Dawley male rats. Rats were randomly divided into the following three groups (n = 6/group): 1) control (no injection), 2) bilateral stellate ganglia injection of unconjugated cholera toxin B (CTB), and 3) bilateral stellate ganglia injection of cholera toxin B conjugated to SAP (CTB-SAP). CTB-SAP rats, compared with control and CTB rats, had reduced cardiac sympathetic tonus and reduced heart rate responses to graded exercise and graded doses of SNP. Furthermore, the number of stained neurons in the stellate ganglia and spinal cord (segments T(1)-T(4)) was reduced in CTB-SAP rats. Thus CTB-SAP retrogradely transported from the stellate ganglia is effective at ablating cardiac sympathetic neurons and reducing resting, exercise, and reflex sympathetic activity. Additional studies are required to further characterize the physiological responses to this procedure as well as determine if this new approach is safe and efficacious for the treatment of conditions associated with excess sympathetic activity (e.g., autonomic dysreflexia, hypertension, heart failure, and ventricular arrhythmias).