Läs källbundna sammanställningar.

Abstract

Nitrile hydratase (NHase, EC 4.2.1.84) is a metalloenzyme which catalyses the conversion of nitriles to amides. The high efficiency and broad substrate range of NHase have led to the successful application of this enzyme as a biocatalyst in the industrial syntheses of acrylamide and nicotinamide and in the bioremediation of nitrile waste. Crystal structures of both cobalt(III)- and iron(III)-dependent NHases reveal an unusual metal binding motif made up from six sequential amino acids and comprising two amide nitrogens from the peptide backbone and three cysteine-derived sulfur ligands, each at a different oxidation state (thiolate, sulfenate and sulfinate). Based on the active site geometry revealed by these crystal structures, we have designed a series of small-molecule ligands which integrate essential features of the NHase metal binding motif into a readily accessible peptide environment. We report the synthesis of ligands based on a pyridine-2,6-dicarboxylic acid scaffold and L-cysteine, L-S-methylcysteine, L-methionine or L-penicillamine. These ligands have been combined with cobalt(III) and iron(III) and tested as catalysts for biomimetic nitrile hydration. The highest levels of activity are observed with the L-penicillamine ligand which, in combination with cobalt(III), converts acetonitrile to acetamide at 1.25 turnovers and benzonitrile to benzamide at 1.20 turnovers.

2.2. Nitrile Hydration Experiments

Ligand 4 returned the best turnover results found with these systems, when combined with Na3[Co(NO2)6] and stirred for 12 h at 25 °C and pH 9 ( shows the reaction outcome, shows a representative chromatogram for this level of turnover). The geminal dimethyl unit adjacent to the thiolate enhances the sub-stoichiometric turnover seen with 1 to a gently catalytic level of 1.25 turnovers. The addition of peroxide diminishes catalytic activity with this ligand (down to 0.7 turnovers), while increasing reaction temperature to 50 °C also lowers activity to 0.7 turnovers.

2.2. Nitrile Hydration Experiments

To investigate the scope of this ligand, a further set of experiments was carried out using a second substrate, the aromatic nitrile benzonitrile 19. Under these conditions the combination of ligand 4 and Na3[Co(NO2)6 also turned over benzonitrile 19 as a substrate, with a similar level of conversion to benzamide 20 (1.2 turnovers). (To aid the miscibility of the benzonitrile in water, a small amount of DMF was added to these reactions: 200 µL in a total reaction volume of 7.2 mL).

Abstract

Neurokinin 1 receptors (NK1R) are overexpressed on several types of important human cancer cells. Substance P (SP) is the most specific endogenous ligand known for NK1Rs. Accordingly,a new SP analogue was synthesized and evaluated for detection of NK1R positive tumors.[6-hydrazinopyridine-3-carboxylic acid (HYNIC)-Tyr8-Met(O)11-SP] was synthesized and radiolabeled with 99mTc using ethylenediamine-N,N'-diacetic acid (EDDA)and Tricine as coligands. Common physicochemical properties of radioconjugate were studied and in-vitro cell line biological tests were accomplished to determine the receptor mediated characteristics. In-vivo biodistribution in normal and tumor bearingnude mice was also assessed. The cold peptide was prepared in high purity (>99%) and radiolabeled with 99mTc at high specific activities (84-112GBq/µmol) with an acceptable labeling yield (>95%). The radioconjugate was stable in-vitro in the presence of human serum and showed 44% protein binding to human serumalbumin. In-vitro cell line studies on U373MG cells showed an acceptable uptake up to 4.91 ± 0.22% with the ratio of 60.21 ± 1.19% for its specific fraction and increasing specific internalization during 4 h. Receptor binding assays on U373MG cells indicated a mean Kd of 2.46 ± 0.43 nM and Bmax of 128925 ± 8145 sites/cell. In-vivo investigations determined the specific tumor uptake in 3.36 percent of injected dose per gram (%ID/g) for U373MG cells and noticeable accumulations of activity in the intestines and lung. Predominant renal excretion pathway was demonstrated.

Experimental

Labeling of HYNIC-peptide was performed as previously described (-) with 99mTc in the presence of ethylenediamine-N,N'-diacetic acid (EDDA) and tris(hydroxymethyl)methylglycine (Tricine) as coligands using an exchange labeling approach with both present together. In brief, the labeling process was done by adding 10.75 μL of the stock solution (20 μg of peptide), 15 mg of Tricine and 5 mg of EDDA as coligands in 0.5 mL of water. Then, 40 μg SnCl2 (10 μL of freshly prepared 4 mg/mL SnCl2in nitrogen-purged 0.1 M HCl) was added to this solution. Instantly, 30-35 mCi (1110-1295MBq) of freshly eluted sodium pertechnetate (Na99mTcO4) in 0.5 mL normal saline was added to the solution and incubated for 15 min at 95 °C in a shielded container. After cooling down to room temperature, the labeling yield was checked on RP-HPLC.

Methods

Male Wistar rats were divided into five groups (6 rats in each group), namely; control, control+S-methyl cysteine (SMC), high fructose diet (HFD), HFD+SMC and HFD+metformin. The 60% fructose used for 8 weeks and SMC in the dose of 100 mg/kg bw/day/rat was used in the study. The fasting glucose, insulin, insulin resistance, and tumor necrosis factor alpha and erythrocyte enzymatic antioxidants were measured.

Results

Increased levels of plasma glucose, insulin, malondialdehyde, tumor necrosis factor-alpha, and insulin resistance and decreased levels of glutathione, glutathione peroxidase, and catalase were found in rats on a high fructose diet. Oral administration of SMC (100 mg/kg bw/day/rat) for 60 days resulted in significant attenuation of plasma glucose, insulin, tumor necrosis factor–alpha, insulin resistance and improved antioxidant enzyme activities.

Materials and Methods

Group 2: Control-s-methyl-L-cysteine (100 mg/kg bw/day/rat) in aqueous solution orally for 60 days.

Materials and Methods

Group 4: HFD+s-methyl-L-cysteine (100 mg/kg bw/day/rat) in aqueous solution orally for 60 days.

Purpose

Limbic epileptogenesis triggers molecular and cellular events that foster the establishment of aberrant neuronal networks that, in turn, contribute to temporal lobe epilepsy (TLE). Here we have examined hippocampal neuronal network activities in the pilocarpine post-status epilepticus model of limbic epileptogenesis and asked whether or not the docosahexaenoic acid (DHA)-derived lipid mediator, neuroprotectin D1 (NPD1), modulates epileptogenesis.

Methods

Status epilepticus (SE) was induced by intraperitoneal administration of pilocarpine in adult male C57BL/6 mice. To evaluate simultaneous hippocampal neuronal networks, local field potentials were recorded from multi-microelectrode arrays (silicon probe) chronically implanted in the dorsal hippocampus. NPD1 (570 μg/kg) or vehicle was administered intraperitoneally daily for five consecutive days 24 hours after termination of SE. Seizures and epileptiform activity were analyzed in freely-moving control and treated mice during epileptogenesis and epileptic periods. Then hippocampal dendritic spines were evaluated using Golgi-staining.

Results

We found brief spontaneous microepileptiform activity with high amplitudes in the CA1 pyramidal and stratum radiatum in epileptogenesis. These aberrant activities were attenuated following systemic NPD1 administration, with concomitant hippocampal dendritic spine protection. Moreover, NPD1 treatment led to a reduction in spontaneous recurrent seizures.

Introduction

Here we examined the neuroprotective bioactivity of NPD1 on limbic neuronal network activities in the pilocarpine model of TLE [] in freely-moving mice using dorsal hippocampal-implanted multi-microelectrode arrays [, ]. We observed that NPD1 reduces brief spontaneous epileptiform field potentials and dendritic spine loss in the dorsal hippocampus, with resultant limited hippocampal epileptic activity. Understanding the cell-type-specific molecular and cellular events underlying NPD1 bioactivity during limbic epileptogenesis will contribute to development of improved therapies for counteracting neuronal network dysfunctions, thus limiting TLE.

In Vivo NPD1 or Vehicle Treatment

NPD1 and vehicle solutions were prepared according to previous protocols [], and then NPD1 (570 μg/kg) or vehicle was administered i.p. once a day for five consecutive days 24 hours after termination of SE.

Methods

Eleven pediatric patients with end stage kidney disease underwent bone biopsy, were treated for 8 months with doxercalciferol, and then underwent a second bone biopsy. Bone expression of fibroblast growth factor 23 (FGF23), dentin matrix protein 1 (DMP1), and sclerostin were determined by immunohistochemistry and quantified by Ariol Scanning. Western blot analysis and qRT-PCR was performed on bone abstracts of a subset of study subjects to determine the nature (i.e. size) of FGF23 and DMP1 in bone before and after therapy.

Introduction

FGF23 processing and osteocyte biology appear to change with a progressive decline in kidney function. Although circulating FGF23 undergoes cleavage in patients with normal kidney function and in those with mild CKD,[] the majority of circulating FGF23 in dialysis patients is in its full-length form [] and changes in circulating mineral ion and hormone concentrations may play a significant role in osteocytic protein expression as CKD advances. Current data suggest that increasing PTH levels suppress osteocytic sclerostin expression;[] however, bone sclerostin is increased in CKD.[] Circulating phosphorus and PTH both increase FGF23 concentrations[, ] and therapies used to treat renal osteodystrophy, in addition to controlling secondary hyperparathyroidism and bone turnover, likely also alter osteocytic protein expression, potentially via changes in mineral metabolism. Indeed, vitamin D sterol therapy suppresses PTH while increasing circulating FGF23 levels dramatically in patients with end-stage kidney disease;[] however, the effect of this form of therapy on skeletal expression of FGF23, sclerostin, and DMP1 in the context of advanced kidney disease and secondary hyperparathyroidism remains unknown. Thus, in order to assess the interplay between these three skeletal proteins and parameters of mineral metabolism during active vitamin D sterol therapy in advanced CKD, the current study evaluated iliac crest bone tissue in pediatric dialysis patients before and after 8 months of therapy with 1α-(OH)vitamin D2 (doxercalciferol).

Study Subjects

Eleven pediatric patients with end-stage kidney disease (ESKD) treated with peritoneal dialysis who had bone biopsies before and 8 months after the initiation of doxercalciferol therapy were candidates for the study. Active vitamin D sterol therapy was withheld during the 4 weeks preceding the initial bone biopsy [] and high turnover renal osteodystrophy, as defined by an increase in bone formation rate and/or the presence of marrow fibrosis on the initial biopsy, was a pre-requisite for study participation. Exclusion criteria included: previous history of poor medication compliance; parathyroidectomy within the past 12 months; concurrent treatment immunosuppressive agents or growth hormone; or bone pathology not related to secondary hyperparathyroidism.

Patient Demographic Data, Biochemical Values, and Bone Histomorphometry at Baseline and After 8 Months of Therapy

Eleven pediatric dialysis patients (7 male and 4 female/ 9 Hispanic, 1 white, and 1 black) met entry criteria, agreed to participate, and were included in the study. The average age was 15.8 + 0.8 years and BMI was 21.7 + 2.2 kg/m2. Subjects had been on dialysis for an average of 1.1 + 0.4 years. All of the study participants had some residual urine output. During the course of therapy, subjects received an average of 19.3 + 3.8 mcg of doxercalciferol per week. Calcitriol levels increased by 10.9 + 5.0 pg/ml and doxercalciferol doses correlated with the change in circulating calcitriol concentration (r = 0.55, p = 0.08). Baseline and final biochemical parameters are shown in . At baseline, serum calcium levels were 8.6 + 0.2 mg/dl and serum phosphorus concentrations were 6.3 + 0.4 mg/dl; neither parameter changed with doxercalciferol therapy. Plasma PTH, alkaline phosphatase, and FGF23 levels were elevated at baseline. Overall, 6 patients achieved PTH levels within the target range while values remained elevated in the remainder due to the development of hypercalcemia and hyperphosphatemia. While PTH and alkaline phosphatase values did not change with therapy, FGF23 concentrations increased and final values were 1696 (565, 3973) RU/ml (p<0.05 from baseline).

DT3 protected mice from lethal doses of gamma radiation

DT3 (75 mg/kg) or vehicle was administered to CD2F1 mice 24 h before total-body irradiation (TBI) at various radiation doses as indicated in “Materials and Methods,” and 30-day survival data were used to evaluate the DT3 dose reduction factor (DRF, N = 20 mice/group) [,]. The probit lines in are drawn with percent mortality data. The LD50/30 radiation dose was 8.94 Gy for the vehicle-treated mice and 11.44 Gy for DT3-treated animals (p<0.01). The probit line for DT3-treated mice was significantly (p<0.01) shifted to the right compared with vehicle, yielding a DRF (95% confidence limits) of 1.28 (1.20–1.36). These data confirm that DT3 administration significantly protects mice from lethal doses of gamma-radiation [].

Methods

This was a double-blind randomized cross over study conducted in a clinical research unit in United Kingdom. Forty-eight individual who has self reported sensitivity to aspartame were compared to 48 age and gender matched aspartame non-sensitive individuals. They were given aspartame (100mg)-containing or control snack bars randomly at least 7 days apart. The main outcome measures were acute effects of aspartame measured using repeated ratings of 14 symptoms, biochemistry and metabonomics.

Results

Aspartame sensitive and non-sensitive participants differed psychologically at baseline in handling feelings and perceived stress. Sensitive participants had higher triglycerides (2.05 ± 1.44 vs. 1.26 ± 0.84mmol/L; p value 0.008) and lower HDL-C (1.16 ± 0.34 vs. 1.35 ± 0.54 mmol/L; p value 0.04), reflected in 1H NMR serum analysis that showed differences in the baseline lipid content between the two groups. Urine metabonomic studies showed no significant differences. None of the rated symptoms differed between aspartame and control bars, or between sensitive and control participants. However, aspartame sensitive participants rated more symptoms particularly in the first test session, whether this was placebo or control. Aspartame and control bars affected GLP-1, GIP, tyrosine and phenylalanine levels equally in both aspartame sensitive and non-sensitive subjects.

Study Design

Aspartame (100mg, equivalent to that consumed in a can of diet soda and less than the Acceptable Daily Intake maximum (ADI) recommended by FDA of 40mg/kg body weight/day) was incorporated into cold pressed cereal bars (Campden BRI, United Kingdom). In a prior taste trial, 40 healthy controls could not distinguish between aspartame or control bars. Randomisation was performed by Campden BRI. The study was unblinded only after all of the analyses had been undertaken.

Study Procedures

Following an 8 hour overnight fast participants were given the aspartame or control bar, and crossed over at least 1 week apart. Bars were consumed within 5 minutes. Standard questionnaires were used to assess psychological condition at baseline, and 14 symptoms were rated repeatedly over 4 hours after consuming bars (see section “”). Blood samples were taken at baseline and at 4 hours: urine specimens were provided at baseline, at 4, 12 and 24 hours.

Abstract

Ergothioneine is a histidine thio-derivative isolated in 1909. In ergothioneine biosynthesis, the combination of a mononuclear non-heme iron enzyme catalyzed oxidative C-S bond formation reaction and a PLP-mediated C-S lyase (EgtE) reaction results in a net sulfur transfer from cysteine to histidine side-chain. This demonstrates a new sulfur transfer strategy in the biosynthesis of sulfur-containing natural products. Due to difficulties associated with the overexpression of Mycobacterium smegmatis EgtE protein, the proposed EgtE functionality remained to be verified biochemically. In this study, we have successfully overexpressed and purified M. smegmatis EgtE enzyme and evaluated its activities under different in vitro conditions: C-S lyase reaction using either thioether or sulfoxide as a substrate in the presence or absence of reductants. Results from our biochemical characterizations support the assignment of sulfoxide 4 as the native EgtE substrate and the involvement of a sulfenic acid intermediate in the ergothioneine C-S lyase reaction.

Results

We sub-cloned M. smegmatis EgtE gene into the pASK-IBA3+ vector and the protein was overexpressed in the E. coli BL21(DE3) strain. The expressed EgtE was then purified using Strep-Tactin resin to near homogeneity (higher than 95% based on the analysis of the SDS-PAGE, ). Furthermore, as suggested by EgtE sequence analysis, its UV-visible spectra are consistent with the presence of a PLP cofactor (). The pH-dependence of the EgtE internal aldimine electronic absorption was also studied. In PLP-containing proteins, the PLP-Lysine conjugate can exist in a few different forms: enolimine (9), ketoenamine (10) or deprotonated ketoenamine (11),,,,,,,. For the isolated EgtE protein at pH 7.5, it has an absorption feature near 420 nm (), which is consistent with the presence of ketoenamine (10) tautomer of the protonated Schiff base. As the pH changes from 6.0 to 10.0, the UV-vis spectra () do not show distinct changes in the overall absorption features and the maximal absorption wavelengths. Thus, for EgtE, the internal aldimine is present mainly in the form of 10. The amount of PLP per EgtE monomer was also determined after it was released by 0.2 M NaOH treatment. The isolated EgtE has 0.8 mole PLP per mole of EgtE monomer.

Abstract

All-trans-Retinal (ATR) is a photosensitizer, serving as the chromophore for depolarizing and hyperpolarizing light-sensitive ion channels and pumps (opsins), recently employed as fast optical actuators. In mammalian optogenetic applications (in brain and heart), endogenous ATR availability is not considered a limiting factor, yet it is unclear how ATR modulation may affect the response to optical stimulation. We hypothesized that exogenous ATR may improve light responsiveness of cardiac cells modified by Channelrhodopsin2 (ChR2), hence lowering the optical pacing energy. In virally-transduced (Ad-ChR2(H134R)-eYFP) light-sensitive cardiac syncytium in vitro, ATR supplements ≤2 μM improved cardiomyocyte viability and augmented ChR2 membrane expression several-fold, while >4 μM was toxic. Employing integrated optical actuation (470 nm) and optical mapping, we found that 1–2 μM ATR dramatically reduced optical pacing energy (over 30 times) to several μW/mm2, lowest values reported to date, but also caused action potential prolongation, minor changes in calcium transients and no change in conduction. Theoretical analysis helped explain ATR-caused reduction of optical excitation threshold in cardiomyocytes. We conclude that cardiomyocytes operate at non-saturating retinal levels, and carefully-dosed exogenous ATR can enhance the performance of ChR2 in cardiac cells and yield energy benefits over orders of magnitude for optogenetic stimulation.

Cardiomyocyte viability upon ChR2 expression and ATR treatment

Initial exploration of dosing with exogenous ATR revealed a narrow window of viable concentrations (). ATR supplementation greater than 4 μM was toxic to the cardiomyocytes, regardless of whether they expressed ChR2 or not, and caused complete cell death above 8 μM. However, ATR supplements less than 4 μM appeared to paradoxically improve cell viability even though control samples were quite healthy (): CM with 1 and 2 μM ATR supplements had only 0.1 × and 0.15 × the dead cell amount of CM control (virtually eliminating dead cells, i.e. <2% cell death), p ≪ 0.001 for both, and ChR2-CM with 1 and 2 μM ATR supplements had only 0.38 × and 0.45 × the dead cell amount of CM control, p < 0.01 for both. Intermediate ATR concentration of 4 μM increased cell death compared to cells without ATR supplementation, significant in both CM (1.8 × increase) and ChR-CM cells (4.4 × increase), p < 0.01 for both. Furthermore, it is worth noting that when ATR was present, the ChR2-CM samples had consistently lower viability than the CM samples at the same ATR, even though this was not the case for non-supplemented samples.

ChR2 expression with ATR supplementation

Since ChR2 stabilization/degradation had been reported to be dependent on ATR availability, we examined ChR2 expression efficiency (by eYFP reporter) in the viable range of ATR supplements, i.e. 0–4 μM. Employing previously reported infection methods without the addition of exogenous ATR, stable and robust transgene expression (>98%) was achieved within 48 hours, (, middle row, first image). We confirmed that in confocal image acquisition of fluorescence (eYFP), greater ATR concentrations did not result in an increase in autofluorescence (, top row). ATR supplementation in ChR2-CM samples led to significant enhancement of ChR2 expression, indicated by the saturation of eYFP fluorescence at the higher doses, despite our attempt to select a gain that maximizes the dynamic range and accommodates all ATR dosing groups (, middle row). Higher resolution images and lowered gain to optimize imaging of the ChR2-CM samples with 4 μM are also shown ( bottom row). Image analysis of eYFP-positive pixels (normalized per cell) showed 3.8 ×, 5 × and 5.9 × increase in ChR2 expression by the addition of 1, 2, and 4 μM ATR compared to ChR2-CM without retinal, p ≪ 0.001 in all cases (). The eYFP fluorescence indicates the total number of expressed channels, including non-functional channelopsins without ATR, therefore the increases shown in represent an upper bound for functional ChR2 channels. We assume that the data indicate a proportionally increasing number of stabilized functional ChR2 channels (fraction of the total reported channels), though increase in actual ChR2 current may be more modest.

Abstract

All-trans-Retinal (ATR) is a photosensitizer, serving as the chromophore for depolarizing and hyperpolarizing light-sensitive ion channels and pumps (opsins), recently employed as fast optical actuators. In mammalian optogenetic applications (in brain and heart), endogenous ATR availability is not considered a limiting factor, yet it is unclear how ATR modulation may affect the response to optical stimulation. We hypothesized that exogenous ATR may improve light responsiveness of cardiac cells modified by Channelrhodopsin2 (ChR2), hence lowering the optical pacing energy. In virally-transduced (Ad-ChR2(H134R)-eYFP) light-sensitive cardiac syncytium in vitro, ATR supplements ≤2 μM improved cardiomyocyte viability and augmented ChR2 membrane expression several-fold, while >4 μM was toxic. Employing integrated optical actuation (470 nm) and optical mapping, we found that 1–2 μM ATR dramatically reduced optical pacing energy (over 30 times) to several μW/mm2, lowest values reported to date, but also caused action potential prolongation, minor changes in calcium transients and no change in conduction. Theoretical analysis helped explain ATR-caused reduction of optical excitation threshold in cardiomyocytes. We conclude that cardiomyocytes operate at non-saturating retinal levels, and carefully-dosed exogenous ATR can enhance the performance of ChR2 in cardiac cells and yield energy benefits over orders of magnitude for optogenetic stimulation.

Functional electrophysiology effects of ChR2 expression and ATR supplementation in cardiomyocytes

We further examined the effects of ATR supplementation on CM electrophysiology by quantifying action potentials, calcium transients, and conduction properties. We compared optically-measured (via Di-4-ANBDQBS) action potential durations (APDs) of ChR2-CM to CM with the same treatment, . At zero ATR, CM and ChR2-CM showed overlapping AP and similar APD80 of 260 ms and 270 ms, respectively; reassuring of the benign nature of the opsin expression and optogenetic activation. Addition of 1 μM ATR significantly prolonged the APD80 in ChR2-CMs by 44% (p < 0.05) compared to ChR2-CM without ATR (). Similarly, at zero ATR, no significant differences were seen in calcium transient morphology between the CM and ChR2-CM groups across the tested conditions (); CTD80 of CM and ChR2-CM were 552 ms and 530 ms, respectively (). Supplementation with ATR of 1, 2, and 4 μM in control CM led to: 13.8% (p < 0.05), 13.3% (p < 0.05), and 4% (n.s.) prolongation of CTD80, respectively. In ChR2-CM samples, exogenous ATR supplementation also led to small but significant prolongation of CTD80 by approximately 10.9%, 11.6%, and non-significant 10% for the same tested concentrations (). Examination of propagation and activation maps for the different ChR2-CM groups () showed that ATR supplementation at 1 and 2 μM yielded smooth and fast propagation with similar CV to ChR2-CM control: 19.1 cm/s, 17.0 cm/s, and 17.0 cm/s for 0, 1, and 2 μM, respectively, while 4 μM resulted in small conduction disturbances (likely due to increased number of dead myocytes) and a drop in CV to 14.7 cm/s (). In control CM, ATR had no effect on conduction velocity.

Functional electrophysiology effects of ChR2 expression and ATR supplementation in cardiomyocytes

Overall, low concentrations of ATR (up to 2 μM) helped improve survival of cardiomyocytes in standard culturing conditions, did not alter conduction and resulted in significant APD prolongation but only slight prolongation of intracellular calcium transients in the ChR2-expressing cardiomyocytes.

Abstract

Resolvins of the D-series are specialized pro-resolving lipid mediators that regulate cellular response by orchestrating resolution networks involved in host responses to injury and infection. Here, endogenous resolvin D4 was identified in human tissues and found to persist late into the resolution phase of acute murine Staphylococcus aureus infections. Completion of the first total synthesis of resolvin D4 established the absolute stereochemical configuration of RvD4 confirmed by matching with endogenous RvD4 from resolving exudates in dorsal pouch S. aureus infections. In vivo, RvD4 (ng/mouse) reduced neutrophilic infiltration (~40%) and enhanced uptake of apoptotic PMN (51%) by human dermal fibroblasts at concentrations as low as 0.1 nM. These results establish the complete stereochemistry of RvD4 as 4S,5R,17S-trihydroxydocosa-6E,8E,10Z,13Z,15E,19Z-hexaenoic acid and its novel pro-resolving actions in S. aureus infections as well as its potent ability to stimulate clearance of apoptotic cells by skin fibroblasts.

RvD4 stops neutrophil infiltration in sterile peritonitis and S. aureus skin infection

With the biological activity of RvD4 confirmed, we next assessed its in vivo actions in S. aureus-mediated infection probing its action as well as temporal formation. To determine its role in bacterial killing and host protection during S. aureus infections, RvD4 was administered at 200 ng/mouse concomitantly with a self-limited bacterial inoculation (105 c.f.u) into the dorsal pouch of male FVB mice. Resolution indices were then measured. RvD4 potently reduced neutrophilic infiltration by ~40% at Tmax (12 h) and shortened the resolution interval in mice treated with RvD4 from 6 to 3 hr (). Analysis by histochemical staining of dorsal pouch lining from mice administered vehicle or RvD4 gave lower PMN levels in the cavity in mice treated with RvD4 (). Treatment with RvD4 also significantly lowered bacterial counts when compared to vehicle control (). In addition, RvD4 enhanced the phagocytosis of S. aureus by isolated mouse macrophages by ~40% at 10nM (data not shown). RvD4 also regulated inflammation-initiating eicosanoids in the inflammatory exudates including LTB4 and PGD2 by 68 and 48% respectively, demonstrating potent anti-inflammatory actions in vivo ().

RvD4 exerts pro-resolving actions with human neutrophils and macrophages

To further elucidate its host-protective actions, we sought to establish the potent anti-inflammatory and pro-resolving actions of RvD4 with isolated human leukocytes. RvD4 (0.01 nM to 10 nM) promoted bacterial phagocytosis by human macrophages (12–18%) to a similar extent as the pro-resolving mediator RvD3 (). RvD4 also promoted the phagocytosis of opsonized zymosan A at concentrations as low as 0.1–10 nM, significantly enhancing macrophage uptake by 40–60% above vehicle controls ().

RvD4 exerts pro-resolving actions with human neutrophils and macrophages

The clearance of apoptotic PMN during inflammation is a cellular hallmark in resolution. To determine RvD4’s role in the clearance of apoptotic neutrophils we assessed whether RvD4 promoted macrophage efferocytosis. RvD4 statistically significantly increased macrophage efferocytosis of apoptotic neutrophils by 40–50% at concentrations as low as 0.1–10 nM (). These results suggest that RvD4 enhances human macrophage functions in clearing cellular debris and microbial particles. Together these results provide evidence that RvD4 is pro-resolving with human phagocytes.

RvD4 exerts pro-resolving actions with human fibroblasts: Comparison with RvD1 and MaR1

Given that RvD4 displayed potent pro-resolving actions in skin infection, we investigated whether it also acts on human dermal fibroblasts. RvD4 potently promoted the clearance of apoptotic PMN by human fibroblasts () by ~59% at 1 nM. Of the SPM tested, RvD4 and RvD1 at 1 nM gave the highest increase in apoptotic cell uptake (~52 and 57%). These findings demonstrate new action of SPM such as RvD1 and MaR1 (7R,14S-dihydroxydocosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid) in resolution and that RvD4 displays potent actions with human dermal fibroblasts ().

Discussion

In the present report we establish the complete stereochemical assignment of RvD4 as 4S,5R,17S-trihydroxydocosa-6E,8E,10Z,13Z,15E,19Z-hexaenoic acid by matching endogenous material isolated from purulent inflammatory exudates with synthetic compound () made by total organic synthesis (). RvD4 was temporally regulated during S. aureus infection as well as remained at bioactive levels (~ pM) in the late stages of resolution (). Administration of RvD4 in vivo counter regulated inflammatory initiating eicosanoids in S. aureus infection and shortened resolution indices during infection (), concepts on which the edifice of resolution is based. Administration of RvD4 in both sterile inflammation and microbial infection gave a reduction in neutrophil recruitment in vivo ( and ). RvD4 also enhanced macrophage responses with human macrophages and enhanced fibroblast responses with human fibroblasts in efferocytosis of apoptotic PMN and sterile zymosan (). Furthermore RvD4 was found to be present in a number of tissues across species suggesting its utility through evolutionary conservation ().