Recombinant production of Trx-Ib-AMP4 and Trx-E50-52 antimicrobial peptides and antimicrobial synergistic assessment on the treatment of methicillin-resistant Staphylococcus aureus under in vitro and in vivo situations.

PURPOSE: The production of alternative novel antimicrobial agents is considered an efficient way to cope with multidrug resistance among pathogenic bacteria. E50-52 and Ib-AMP4 antimicrobial peptides (AMPs) have illustrated great proven antibacterial effects. The aim of this study was recombinant production of these AMPs and investigation of their synergistic effects on methicillin-resistant Staphylococcus aureus (MRSA). METHOD: At first, the codon optimized sequences of the Ib-AMP4 (UniProt: 024006 (PRO_0000020721), and E50-52 (UniProtKB: P85148) were individually ligated into the pET-32α vector and transformed into E. coli. After the optimization of production and purification steps, the MIC (Minimum inhibitory concentration), time kill and growth kinetic tests of recombinant proteins were determined against MRSA. Finally, the in vivo wound healing efficiency was tested. RESULTS AND CONCLUSION: The recorded MIC of recombinant Trx-Ib-AMP4, Trx-E50-52 against MRSA bacterium were 0.375 and 0.0875 mg/mL respectively. The combination application of the produced AMPs by the checkerboard method confirmed their synergic activity. The results of the time-kill showed sharply decrease of the number of viable cells with over five time reductions in log10 CFU/mL by the combination of Trx-E50-52 and Trx-IbAMP4 at 2 × MIC within 240 min. The growth kinetic results confirmed the combination of Trx-E50-52 and Trx-IbAMP4 had much greater success in the reduction of over 50 % of MRSA suspensions' turbidity within the first hour. Wound healing assay and histological analysis of infected mice treated with Trx-Ib-AMP4 or Trx-E50-52 compared with those treated with a combination of Trx-Ib-AMP4 and Trx-E50-52 showed significant synergic effects.

Anti-inflammatory and antioxidant effect of melatonin on recovery from muscular trauma induced in rats.

In recent decades, the number of people who practice sports has grown exponentially, increasing the number of muscular injuries. Trauma injury occurs when the muscle is exposed to a sudden compression force. Melatonin (MLT) has often been cited in the literature as a potent antioxidant and anti-inflammatory agent. This study was designed to evaluate MLT action on muscle tissue in Wistar rats in an experimental model of muscle trauma. Twenty-eight Wistar rats were used, divided into four groups: CO (Control), CO + MLT (Control + Melatonin), T (Trauma) and T + MLT (Trauma + Melatonin). MLT (20 mg/kg) was administered (ip) daily at dusk until day 7. The trauma occurred on day 1, 2 h before the first MLT application. On day 8, muscle tissue was collected for histological analysis (HE), immunohistochemistry (TNF-α and NFκB), evaluation of oxidative stress through analysis of lipoperoxidation by TBARS and activity of SOD and GPx enzymes, and analysis of nitrites and nitrates. In the evaluation of TBARS and SOD, we observed a significant increase in the T group and a significant decrease in the T + MLT group. In the evaluation of GPx, there was a significant increase in the T group and a significant decrease in the T + MLT group. The histological analysis of muscle tissue revealed structural changes of muscle fibers and inflammatory infiltrate in the T group but a decrease in this damage in the T + MLT group. In the immunohistochemical evaluation, increased expression of TNFα and NFκB proteins in the T group was observed and a significant decrease of this expression in the T + MLT group. MLT was shown to attenuate oxidative damage and to diminish the expression of inflammatory proteins and tissue damage in this experimental model.

Enhanced Cutaneous Wound Healing In Vivo by Standardized Crude Extract of Poincianella pluviosa.

Wound healing is a complex process that involves several biological events, and a delay in this process may cause economic and social problems for the patient. The search continues for new alternative treatments to aid healing, including the use of herbal medicines. Members of the genus Caesalpinia are used in traditional medicine to treat wounds. The related species Poincianella pluviosa (DC.) L.P. Queiroz increases the cell viability of keratinocytes and fibroblasts and stimulates the proliferation of keratinocytes in vitro. The crude extract (CE) from bark of P. pluviosa was evaluated in the wound-healing process in vivo, to validate the traditional use and the in vitro activity. Standardized CE was incorporated into a gel and applied on cutaneous wounds (TCEG) and compared with the formulation without CE (Control) for 4, 7, 10, or 14 days of treatment. The effects of the CE on wound re-epithelialization; cell proliferation; permeation, using photoacoustic spectroscopy (PAS); and proteins, including vascular endothelial growth factor (VEGF), superoxide dismutase 2 (SOD-2) and cyclooxygenase 2 (COX-2) were evaluated. The TCEG stimulated the migration of keratinocytes at day 4 and proliferation on the following days, with a high concentration of cells in metaphase at 7 days. Type I collagen formed more rapidly in the TCEG. PAS showed that the CE had permeated through the skin. TCEG stimulated VEGF at day 4 and SOD-2 and COX-2 at day 7. The results suggest that the CE promoted the regulation of proteins and helped to accelerate the processes involved in healing, promoting early angiogenesis. This led to an increase in the re-epithelialized surface, with significant mitotic activity. Maturation of collagen fibers was also enhanced, which may affect the resistance of the extracellular matrix. PAS indicated a correlation between the rate of diffusion and biological events during the healing process. The CE from P. pluviosa appears promising as an aid in healing.

The neuroprotective effect of salubrinal in a mouse model of traumatic brain injury.

We have previously reported that mild traumatic brain injury (mTBI) induced cognitive deficits as well as apoptotic changes in the brains of mice. Apoptosis may be caused by severe, prolonged accumulation of misfolded proteins, and protein aggregation in the endoplasmic reticulum (ER stress). In an additional study, we have reported that mTBI activated the pro-apoptotic arm of the integrated stress response (ISR). The main goal of the present study was to test the involvement of the adaptive eIF2α/ATF4 pathway in mTBI-affected brains. Head injury was induced with a noninvasive, closed-head weight drop (30 g) to ICR mice. Salubrinal, the selective phosphatase inhibitor of p-eIF2α, was injected immediately and 24 h after mTBI (1 mg/kg, ip). Y-maze and novel object recognition tests to assess spatial and visual memories, respectively, were conducted either 7 or 30 days post-trauma. Salubrinal administration significantly improved memory deficits following mTBI. Slaubrinal also prevented the elevation of degenerating neurons and the reduction of mature neurons in the cortex (as seen by immunofluorescent staining with Fluoro-Jade-B and NeuN antibodies, 72 h and 1 week post-mTBI, respectively). Western blot analysis revealed that salubrinal prevented the significant reduction in eIF2α and ATF4 phosphorylation in mTBI brains 72 h post-injury. Immunofluorescence staining revealed that although the reduction in p-eIF2α did not reach significance, salubrinal administration elevated it dramatically. Our results show that targeting the translational/adaptive arm of the ISR with salubrinal may serve as a therapeutic strategy for brain damage.

Comfrey root: from tradition to modern clinical trials.

Comfrey (Symphytum officinale L.) has been used over many centuries as a medicinal plant. In particular, the use of the root has a longstanding tradition. Today, several randomised controlled trials have demonstrated the efficacy and safety. Comfrey root extract has been used for the topical treatment of painful muscle and joint complaints. It is clinically proven to relieve pain, inflammation and swelling of muscles and joints in the case of degenerative arthritis, acute myalgia in the back, sprains, contusions and strains after sports injuries and accidents, also in children aged 3 years and older. This paper provides information on clinical trials, non-interventional studies and further literature published on comfrey root till date.

Capsaicin-induced corneal sensory denervation and healing impairment are reversed by NGF treatment.

PURPOSE: We aimed to evaluate the nerve growth factor (NGF) pathway and its influence on corneal healing mechanisms in normal conditions and in an animal model of corneal denervation induced by capsaicin. METHODS: Peripheral sensory damage was induced in rat pups by subcutaneous injection of capsaicin and the effects evaluated by hot-plate test, corneal nerve count, and tear secretion. Corneal damage was induced in capsaicin-treated and -untreated rats by epithelial scraping. Healing rate; NGF pathway (NGF, tyrosine kinase A [TrkA], p75); and the stem cell marker p63 were evaluated by RT-PCR, ELISA, Western blot, and immunohistochemistry. The effects of exogenous NGF administration as eye drop formulation were also tested. RESULTS: Capsaicin treatment induced a significant reduction of peripheral sensitivity, corneal innervation, tear secretion, and corneal healing rate. The ocular effects of capsaicin treatment were associated with an NGF pathway alteration. NGF eye drop treatment aided corneal healing mechanisms through a significant increase in the NGF receptors TrkA and p75, and in the stem cell marker p63. CONCLUSIONS: In this study, we show that an alteration in the NGF pathway is responsible for a delay in corneal healing in an animal model of sensory denervation. Moreover, we show that NGF eye drop administration modulates corneal innervation, epithelial cell healing, and corneal stem cells. These findings may trigger further research on the role of the NGF pathway in limbal stem cell deficiency.

A cannabinoid type 2 receptor agonist attenuates blood-brain barrier damage and neurodegeneration in a murine model of traumatic brain injury.

After traumatic brain injury (TBI), inflammation participates in both the secondary injury cascades and the repair of the CNS, both of which are influenced by the endocannabinoid system. This study determined the effects of repeated treatment with a cannabinoid type 2 receptor (CB(2) R) agonist on blood-brain barrier integrity, neuronal degeneration, and behavioral outcome in mice with TBI. We also looked for the presence of a prolonged treatment effect on the macrophage/microglial response to injury. C57BL/6 mice underwent controlled cortical impact (CCI) and received repeated treatments with a CB(2) R agonist, 0-1966, or vehicle. After euthanasia at 6 hr or 1, 2, 3, or 7 days postinjury, brains were removed for histochemical analysis. Blood-brain barrier permeability changes were evaluated by using sodium fluorescein (NaF). Perilesional degenerating neurons, injury volumes, and macrophage/microglia cells were quantified by stereological methods. Rota-rod and open-field testing were performed to evaluate motor function and natural exploratory behavior in mice. 0-1966 Treatment resulted in a significant reduction in NaF uptake and number of degenerating neurons compared with the vehicle-treated group. 0-1966-Treated mice demonstrated improvement on rota-rod and open-field testing compared with vehicle-treated mice. These changes in CCI mice treated with 0-1966 were associated with a prolonged reduction in macrophage/microglia cell counts. In conclusion, repeated treatments with a CB(2) R agonist, 0-1966, result in attenuated blood-brain barrier disruption and neuronal degeneration. In addition, repeated treatment with 0-1966 shows prolonged treatment effects on behavior and the macrophage/microglia cell response over several days.

Post-surgical scalp wounds with exposed bone treated with a plant-derived wound therapeutic.

OBJECTIVE: To evaluate the efficacy of a plant-derived wound dressing, a mixture of hypericum oil (Hypericum perforatum) and neem oil (Azadirachta indica), in scalp wounds with exposed bone. METHOD: A retrospective review was conducted of all patients presenting with scalp wounds with exposed bone following the excision of skin tumours and treated with a plant-derived wound dressings (1 Primary Wound Dressing; Phytoceuticals AG), from January to July 2011. Time to healing, wound size, area of exposed bone, ease of handling, pain and complications were evaluated. RESULTS: Nine consecutive patients were analysed retrospectively. The patients' mean age was 81.2 ± 8.5 years (63-90 years), with a mean wound size of 13.2 ± 6.8cm(2) (0.4-22.6cm(2)) and 6.8 ± 6.5cm(2) (0.3-20.7cm(2)) of exposed bone. The time to complete healing by secondary intention was 4-20 weeks. A rapid induction of granulation tissue was observed, which covered the entire exposed bone surface in six out of nine cases (67%) after 4 weeks, and showed a reduction in the mean area of exposed bone of 95%. Dressing change was easy and without pain and there were no complications. CONCLUSION: This retrospective, non-controlled analysis suggests that ONE is a very simple to use, safe and potentially effective therapy for the treatment of scalp wounds with exposed bone. DECLARATION OF INTEREST: There were no external sources of funding for this study. The authors have no conflict of interest to declare.

Escin/diethylammonium salicylate/heparin combination gels for the topical treatment of acute impact injuries: a randomised, double blind, placebo controlled, multicentre study.

OBJECTIVES: To investigate the clinical efficacy and safety of escin-containing gels in the topical treatment of blunt impact injuries. METHODS: Competitors in soccer, handball, or karate competitions were enrolled within two hours of sustaining a strain, sprain, or contusion and treated three times with the trial gel within a period of eight hours. Patients were randomised to three parallel groups consisting of two active treatment gels, containing escin (1% or 2%), 5% diethylammonium salicylate, and 5000 IU heparin, or placebo gel. Tenderness produced by pressure was measured at 0 (baseline), 1, 2, 3, 4, 6, and 24 hours after enrollment (within two hours of the injury). Tenderness was defined as the amount of pressure (measured by a calibrated caliper at the centre of the injury) that first produced a pain reaction as reported by the patient. RESULTS: A total of 158 patients were enrolled; 156 were evaluated in the intention to treat analysis. The primary efficacy variable was the area under the curve for tenderness over a six hour period. The gel preparations containing 1% and 2% escin were significantly more effective (a priori ordered hypotheses testing controlling the multiple alpha = 5% significance level) than placebo (p(1) = 0.0001 and p(2) = 0.0002 respectively). The treatment effects were 5.7 kp h/cm(2) (95% confidence interval (CI) 2.9 to 8.5) and 5.9 kilopond (kp) h/cm(2) (95% CI 2.9 to 8.8) between 1% escin and placebo and between 2% escin and placebo respectively. These results were supported by secondary efficacy variables. The time to reach the baseline contralateral tenderness value (resolution of pain) at the injured site was shorter in the treatment groups than in the placebo group (p<0.0001). Both active gel preparations produced more rapid pain relief than the placebo gel. No relevant differences were detected between the two active gels. The safety and tolerability of the escin-containing gels were excellent. CONCLUSIONS: Escin/diethylammonium salicylate/heparin combination gel preparations are effective and safe for the treatment of blunt impact injuries.

Endogenous adenosine and secondary injury after chest trauma.

BACKGROUND: No previous studies have examined actions of adenosine or related compounds after blunt chest trauma, but we have shown that the prototype adenosine-regulating agent, acadesine (aminoimidazole carboxamide ribonucleotide [AICAR]), has multiple favorable anti-inflammatory actions after other forms of trauma, ischemia, hemorrhage, and sepsis; and that a progressive inflammatory response in the contralateral (uninjured) lung after unilateral blunt chest trauma is caused (in part) by activation and sequestration of circulating leukocytes (white blood cells [WBCs]). Thus, we hypothesized that AICAR would ameliorate WBC-dependent, secondary pathophysiologic changes after blunt chest trauma. METHODS: Mongrel pigs (28+/-1 kg, n = 21) were anesthetized, mechanically ventilated, and injured on the right chest (pulmonary contusion) with a captive bolt gun. Either AICAR (1 mg/kg + 0.2 mg/kg/min) or its saline vehicle were administered for a 12-hour period, beginning 15 minutes before injury. RESULTS: Injury caused a three- to fourfold increase in bronchoalveolar lavage (BAL) WBC counts, 10- to 20-fold increases in BAL protein, and 200% increases in lung edema as measured by wet-dry ratio (all p < 0.05), in both the injured (right) and the noninjured (left) lungs. With AICAR versus saline, BAL WBC counts, lung myeloperoxidase levels, and systemic hemodynamics were similar. However, the increases in BAL protein were attenuated by 30% to 50% (p < 0.14, NS) and edema was reduced (p < 0.05) in both lungs. Furthermore, oxygenation, hypercapnia, acidosis (all p < 0.05), and survival were improved (9 of 10 vs. 4 of 11, p < 0.04). CONCLUSION: Pretreatment with AICAR before experimental pulmonary contusion ameliorates the trauma-induced destruction of the alveolar capillary membrane, and attenuates the delayed secondary injury in the contralateral uninjured lung, by a mechanism that may be independent of leukocytes. Endogenous adenosine could have a role in the pathophysiologic response after blunt chest injury, with potential sites of action including the endothelium and alveolar macrophage. Adenosine-regulating agents may have therapeutic potential after blunt chest injury, but further studies are needed in clinically relevant models, with administration begun at the time of resuscitation.

[Bromelain in blunt injuries of the locomotor system. A study of observed applications in general practice]. / Bromelain bei stumpfen Verletzungen des Bewegungsapparats. Eine Anwendungsbeobachtungsstudie aus der Praxis.

METHOD: In an open case observation study involving patients with blunt injuries to the musculoskeletal system, the efficacy and tolerability of high-dose Bromelain POS, a plant-derived enzyme preparation, were investigated. The investigating physician was an orthopedic surgeon who, in addition to the usual therapeutic measures, treated 59 of his patients with the bromelaine preparation. The duration of the application was determined by the nature and severity of the lesion, and varied between one and three weeks. The test criteria were swelling, pain at rest and during movement, and tenderness. These parameters were evaluated on the day of the injury and on five subsequent dates. RESULTS: Treatment with bromelaine resulted in a clear reduction in all four parameters tested. Both swelling and the symptoms of pain had improved appreciably at all evaluation time points as compared with baseline. The tolerability of the preparation was very good, and patient compliance was correspondingly high.

The effect of long-term high-dose naloxone infusion in experimental blunt spinal cord injury.

The effect of long-term continuous subcutaneous infusion of naloxone on blunt spinal cord injury in the rat was assessed using four tests of neurological function, seven histological categories, and two electrophysiological measures. All four neurological function tests showed a trend toward improvement in naloxone-treated animals: the degree of improvement was statistically significant in two of the four categories. A significant reduction in myelin sheath edema was found in the naloxone-treated animals. Although there was a decrease in corticomotor-evoked potentials complexity following injury, there was no significant difference in naloxone-treated animals. Somatosensory-evoked potentials were significantly increased in amplitude and latency in naloxone-treated animals. This increase was most apparent at 60 min: no difference was found by 3 weeks postinjury. These results confirm earlier reports that naloxone can ameliorate the functional neurological deficits of spinal cord injury. Naloxone also produces alterations in the somatosensory-evoked responses in the early phase of treatment and significantly reduces myelin sheath edema.

Benefits of early anti-inflammatory medication following acute ankle injury.

A total of 122 patients presenting with acute ankle injuries within 6 h of injury were entered into a double-blind study. Treatment consisted of a standardized regimen of physiotherapy and elastic support for all patients, who were then randomized into two groups. One group received immediate ibuprofen (2400 mg/day) while the other group received placebo medication for 48 h, followed by ibuprofen (2400 mg/day) from the 3rd day onwards, i.e. delayed antiinflammatory treatment. Assessments were made by means of a daily diary and also by clinical and radiological examination. The immediate treatment group demonstrated more rapid recovery by day 7 in terms of regression of swelling (P less than 0.01) and clinician's impression of severity (P less than 0.05). This group also tended to consider their ankle more able to bear weight at this stage (P = 0.05). In comparison with an earlier study, in which the only active treatment was an elastic support, a greater percentage of patients recovered earlier in the present study. The incidence of side-effects was low. Immediate high-dose ibuprofen is therefore recommended as treatment for moderate to severe acute ankle injuries.

[Immunomodulating action of myelopeptides in severe closed experimental craniocerebral injury in rats]. / Immunomoduliruiushchee deistvie mielopeptidov pri tiazheloi zakrytoi éksperimental'noi cherepno-mozgovoi travme u krys.

An immunocorrective effect of myelopeptides (MP) isolated from pig bone marrow cell culture supernatant in the early posttraumatic period in rats with severe cranial injury has been assessed. MP administration prevented cellular devastation of thymus and bone marrow, as well as spleen hyperplasia. The most marked MP effect was observed within the first 24 h after its administration. MP affected the functional and migration properties of both the entire population of lymphoid cells and individual subpopulations. MP had a pronounced protective effect against Staph. aureus infection during cranial trauma. MP completely prevented the death of animals and reduced more than 3-fold Staph. aureus persistence in the organism of animals. Anti-stress and protective effects of MP open vast prospects for their therapeutic and preventive application in the clinical practice.

The effect of topical cyclosporin A on the rabbit cornea. A clinical and electron microscopic study.

To investigate the effect of topical cyclosporin A (CSA) on the rabbit cornea, both eyes of 80 animals were treated with CSA 2% eye drops over a 3-week period under various conditions. CSA was dissolved in castor oil or ethanol 13.8 vol. %. Slit-lamp, scanning and transmission electron microscopic examinations were performed. CSA 2% in castor oil given 5 times daily showed no damage to either the corneal epithelium or endothelium. In contrast the solutions containing ethanol revealed considerable epithelial cell damage.

[The effect of proteolytic enzymes (traumanase) on posttraumatic edema]. / Die Wirkung proteolytischer Enzyme (traumanase) auf das posttraumatische Odem.

The edema producing property of a proteolytic enzyme (bromelain), which was parenterally or intraduodenally applied, was investigated in a traumatically induced hindleg edema in rats. Under standardized conditions the hindlegs were squeezed by a wringer and swelling was volumetrically measured. Whereas after enteral application of bromelain a significant reduction of the edema could be observed, the parenteral application only resulted in a minimal therapeutic effect. Although enterally applied enzymes are thought to be degraded in the gut, the better results were obtained after enteral administration of bromelain. This supports the observation that also enzymes can be absorbed by the gut without loosing their biological properties.

[Effect of various kanamycin dosage forms in experimental nonpenetrating infected injuries to the cornea]. / Vliianie razlichnykh lekarstvennykh form kanamitsina pri nepronikaiushchikh infitsirovannykh povrezhdeniiakh rogovitsy v éksperimente.

Animals with experimental non-penetrating local wound of the cornea of the type of erosion infected with highly virulent strains of Staph. aureus were treated at various periods, i.e. in 15 minutes, 2, 6 and 9 hours with kanamycin solution (10000 gamma/ml) and kanamycin imbibized films. The therapeutic effect of the kanamycin solution on the non-penetrating infected wound of the cornea was higher when the treatment was started at earlier periods after infection with Staph. aureus. At later periods when the inflammatory process was highly developed the effect of the kanamycin solution on the wound was lower. As for the use of the kanamycin films the regularity was the reverse: they had a higher therapeutic effect in pronounced inflammation and highly developed infection of the eye.