The spleen as a possible source of serine protease inhibitors and migrating monocytes required for liver regeneration after 70% resection in mice.

Introduction: The role of the immune system in liver repair is fundamentally complex and most likely involves the spleen. The close connection between the two organs via the portal vein enables delivery of splenic cytokines and living cells to the liver. This study evaluates expression of inflammation-related genes and assesses the dynamics of monocyte-macrophage and lymphocyte populations of the spleen during the recovery from 70% hepatectomy in mice. Methods: The study used the established mouse model of 70% liver volume resection. The animals were sacrificed 24 h, 72 h or 7 days post-intervention and splenic tissues were collected for analysis: Clariom™ S transcriptomic assay, immunohistochemistry for proliferation marker Ki-67 and macrophage markers, and flow cytometry for lymphocyte and macrophage markers. Results: The loss and regeneration of 70% liver volume affected the cytological architecture and gene expression profiles of the spleen. The tests revealed significant reduction in cell counts for Ki-67+ cells and CD115+ macrophages on day 1, Ly6C + cells on days 1, 3 and 7, and CD3+CD8+ cytotoxic lymphocytes on day 7. The transcriptomic analysis revealed significant activation of protease inhibitor genes Serpina3n, Stfa2 and Stfa2l1 and decreased expression of cell cycle regulatory genes on day 1, mirrored by inverse dynamics observed on day 7. Discussion and conclusion: Splenic homeostasis is significantly affected by massive loss in liver volume. High levels of protease inhibitors indicated by increased expression of corresponding genes on day 1 may play an anti-inflammatory role upon reaching the regenerating liver via the portal vein. Leukocyte populations of the spleen react by a slow-down in proliferation. A transient decrease in the local CD115+ and Ly6C+ cell counts may indicate migration of splenic monocytes-macrophages to the liver.

Spleen regeneration after subcutaneous heterotopic autotransplantation in a mouse model.

BACKGROUND: Splenectomy may lead to severe postoperative complications, including sepsis and cancers. A possible solution to this problem is heterotopic autotransplantation of the spleen. Splenic autografts rapidly restore the regular splenic microanatomy in model animals. However, the functional competence of such regenerated autografts in terms of lympho- and hematopoietic capacity remains uncertain. Therefore, this study aimed to monitor the dynamics of B and T lymphocyte populations, the monocyte-macrophage system, and megakaryocytopoiesis in murine splenic autografts. METHODS: The model of subcutaneous splenic engraftment was implemented in C57Bl male mice. Cell sources of functional recovery were studied using heterotopic transplantations from B10-GFP donors to C57Bl recipients. The cellular composition dynamics were studied by immunohistochemistry and flow cytometry. Expression of regulatory genes at mRNA and protein levels was assessed by real-time PCR and Western blot, respectively. RESULTS: Characteristic splenic architecture is restored within 30 days post-transplantation, consistent with other studies. The monocyte-macrophage system, megakaryocytes, and B lymphocytes show the highest rates, whereas the functional recovery of T cells takes longer. Cross-strain splenic engraftments using B10-GFP donors indicate the recipient-derived cell sources of the recovery. Transplantations of scaffolds populated with splenic stromal cells or without them afforded no restoration of the characteristic splenic architecture. CONCLUSIONS: Allogeneic subcutaneous transplantation of splenic fragments in a mouse model leads to their structural recovery within 30 days, with full reconstitution of the monocyte-macrophage, megakaryocyte and B lymphocyte populations. The circulating hematopoietic cells provide the likely source for the cell composition recovery.

Regulation of Intersubunit Interactions in Homotetramer of Glyceraldehyde-3-Phosphate Dehydrogenases upon Its Immobilization in Protein-Kappa-Carrageenan Gels.

Polysaccharides, being biocompatible and biodegradable polymers, are highly attractive as materials for protein delivery systems. However, protein-polysaccharide interactions may lead to protein structural transformation. In the current study, we analyze the structural adjustment of a homotetrameric protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), upon its interactions with both flexible coil chain and the rigid helix of κ-carrageenan. FTIR spectroscopy was used to probe the secondary structures of both protein and polysaccharide. Electrostatically driven protein-polysaccharide interactions in dilute solutions resulted in an insoluble complex formation with a constant κ-carrageenan/GAPDH ratio of 0.2, which amounts to 75 disaccharide units per mole of protein tetramer. Upon interactions with both coiled and helical polysaccharides, a weakening of the intersubunit interactions was revealed and attributed to a partial GAPDH tetramer dissociation. In turn, protein distorted the helical conformation of κ-carrageenan when co-gelled. Molecular modeling showed the energy favorable interactions between κ-carrageenan and GAPDH at different levels of oligomerization. κ-Carrageenan binds in the region of the NAD-binding groove and the S-loop in OR contact, which may stabilize the OP dimers. The obtained results highlight the mutual conformational adjustment of oligomeric GAPDH and κ-carrageenan upon interaction and the stabilization of GAPDH's dissociated forms upon immobilization in polysaccharide gels.

Dimeric Lectin Chimeras as Novel Candidates for Gb3-Mediated Transcytotic Drug Delivery through Cellular Barriers.

Receptor-mediated transcytosis is an elegant and promising strategy for drug delivery across biological barriers. Here, we describe a novel ligand-receptor pair based on a dimeric, engineered derivative of the Pseudomonas aeruginosa lectin LecA, here termed Di-LecA, and the host cell glycosphingolipid Gb3. We characterized the trafficking kinetics and transcytosis efficiencies in polarized Gb3-positive and -negative MDCK cells using mainly immunofluorescence in combination with confocal microscopy. To evaluate the delivery capacity of dimeric LecA chimeras, EGFP was chosen as a fluorescent model protein representing macromolecules, such as antibody fragments, and fused to either the N- or C-terminus of monomeric LecA using recombinant DNA technology. Both LecA/EGFP fusion proteins crossed cellular monolayers in vitro. Of note, the conjugate with EGFP at the N-terminus of LecA (EGFP-LecA) showed a higher release rate than the conjugate with EGFP at the C-terminus (LecA-EGFP). Based on molecular dynamics simulations and cross-linking studies of giant unilamellar vesicles, we speculate that EGFP-LecA tends to be a dimer while LecA-EGFP forms a tetramer. Overall, we confidently propose the dimeric LecA chimeras as transcytotic drug delivery tools through Gb3-positive cellular barriers for future in vivo tests.

Spleen regeneration after subcutaneous heterotopic autotransplantation in a mouse model

BACKGROUND: Splenectomy may lead to severe postoperative complications, including sepsis and cancers. A possible solution to this problem is heterotopic autotransplantation of the spleen. Splenic autografts rapidly restore the regular splenic microanatomy in model animals. However, the functional competence of such regenerated autografts in terms of lympho- and hematopoietic capacity remains uncertain. Therefore, this study aimed to monitor the dynamics of B and T lymphocyte populations, the monocyte-macrophage system, and megakaryocytopoiesis in murine splenic autografts. METHODS: The model of subcutaneous splenic engraftment was implemented in C57Bl male mice. Cell sources of functional recovery were studied using heterotopic transplantations from B10-GFP donors to C57Bl recipients. The cellular composition dynamics were studied by immunohistochemistry and flow cytometry. Expression of regulatory genes at mRNA and protein levels was assessed by real-time PCR and Western blot, respectively. RESULTS: Characteristic splenic architecture is restored within 30 days post-transplantation, consistent with other studies. The monocyte-macrophage system, megakaryocytes, and B lymphocytes show the highest rates, whereas the functional recovery of T cells takes longer. Cross-strain splenic engraftments using B10-GFP donors indicate the recipient-derived cell sources of the recovery. Transplantations of scaffolds populated with splenic stromal cells or without them afforded no restoration of the characteristic splenic architecture. CONCLUSIONS: Allogeneic subcutaneous transplantation of splenic fragments in a mouse model leads to their structural recovery within 30 days, with full reconstitution of the monocyte-macrophage, megakaryocyte and B lymphocyte populations. The circulating hematopoietic cells provide the likely source for the cell composition recovery.

Identification of a New Antimicrobial, Desertomycin H, Utilizing a Modified Crowded Plate Technique.

The antibiotic-resistant bacteria-associated infections are a major global healthcare threat. New classes of antimicrobial compounds are urgently needed as the frequency of infections caused by multidrug-resistant microbes continues to rise. Recent metagenomic data have demonstrated that there is still biosynthetic potential encoded in but transcriptionally silent in cultivatable bacterial genomes. However, the culture conditions required to identify and express silent biosynthetic gene clusters that yield natural products with antimicrobial activity are largely unknown. Here, we describe a new antibiotic discovery scheme, dubbed the modified crowded plate technique (mCPT), that utilizes complex microbial interactions to elicit antimicrobial production from otherwise silent biosynthetic gene clusters. Using the mCPT as part of the antibiotic crowdsourcing educational program Tiny EarthTM, we isolated over 1400 antibiotic-producing microbes, including 62 showing activity against multidrug-resistant pathogens. The natural product extracts generated from six microbial isolates showed potent activity against vancomycin-intermediate resistant Staphylococcus aureus. We utilized a targeted approach that coupled mass spectrometry data with bioactivity, yielding a new macrolactone class of metabolite, desertomycin H. In this study, we successfully demonstrate a concept that significantly increased our ability to quickly and efficiently identify microbes capable of the silent antibiotic production.

pH-Sensitive fluorophores from locked GFP chromophores by a non-alternant analogue of the photochemical meta effect.

We report the synthesis and characterization of a pH-sensitive fluorescence switch based on a conformationally-locked green fluorescent protein (GFP) chromophore. The chromophore differs from difluoroboryl-locked parent by the addition of a titratable alcohol group on the imidazolinone ring. The chromophore is fluorescent at pH ≤ 5, but becomes non-fluorescent at higher pH, where the substituent is ionized. We use a quantum chemical model to show that the mechanism of the fluorescence turn-off is electronically analogous to photochemical meta effects in aryl-containing systems.

Prostaglandins and prostaglandin receptor antagonism in migraine.

Human models of headache may contribute to understanding of prostaglandins' role in migraine pathogenesis. The current thesis investigated the migraine triggering effect of prostaglandin E2 (PGE2) in migraine patients without aura, the efficacy of a novel EP4 receptor antagonist, BGC20-1531, in prevention of PGE2-induced headache and the ability of prostaglandin F2α (PGF2α) to trigger headache without any vasodilatation in healthy volunteers. All studies were designed as double-blind, placebo-controlled, cross-over experiments, where PGE2/PGF2α or saline were infused over 20-25 min. In the study with EP4 receptor antagonist healthy volunteers were pre-treated with two different doses of BGC20-1531 or placebo followed by PGE2 infusion over 25 min. The headache data were collected during the whole study day, whereas the possible vascular changes were measured during the in-hospital phase of 1.5 h. The infusion of PGE2 caused the immediate migraine-like attacks and vasodilatation of the middle cerebral artery in migraine patients without aura. The highly specific and potent EP4 receptor antagonist, BGC20-1531, was not able to attenuate PGE2-induced headache and vasodilatation of both intra- and extra-cerebral arteries. The intravenous infusion of PGF2α did not induce headache or statistically significant vasoconstriction of cerebral arteries in healthy volunteers. Novel data on PGE2-provoked immediate migraine-like attacks suggest that PGE2 may be one of the important final products in the pathogenesis of migraine. The lack of efficacy of EP4 receptor antagonist suggests that a single receptor blockade is not sufficient to block PGE2 responses, hence EP2 receptor should be investigated as a potential drug target for the treatment of migraine. The absence of headache during the PGF2α infusion demonstrates that vasodilating properties are necessary for the induction of headache and migraine.

Prostaglandins in migraine: update.

PURPOSE OF REVIEW: This review presents recent findings on the role of prostaglandins in migraine pathophysiology. RECENT FINDINGS: Experimental studies have shown that prostaglandins are distributed in the trigeminal-vascular system and its receptors are localized in the trigeminal ganglion and the trigeminal nucleus caudalis. Prostaglandins were found in smooth muscles of cranial arteries, and functional studies in vivo showed that prostaglandins induced dilatation of cranial vessels. Human studies showed that intravenous infusion of vasodilating prostaglandins such as prostaglandin E2 (PGE2), prostaglandin I2 (PGI2) and prostaglandin D2 (PGD2) induced headache and dilatation of intra-cranial and extra-cranial arteries in healthy volunteers. In contrast, infusion of non-dilating prostaglandin F2α (PGF2α) caused no headache or any vascular responses in cranial arteries. PGE2 and PGI2 triggered migraine-like attacks in migraine patients without aura, accompanied by dilatation of the intra-cerebral and extra-cerebral arteries. A novel EP4 receptor antagonist could not prevent PGE2-induced headache in healthy volunteers. SUMMARY: Recent in-vitro/in-vivo data demonstrated presence and action of prostaglandins within the trigeminal pain pathways. Migraine induction after intravenous administration of PGE2 and PGI2 suggests a specific blockade of their receptors, EP and IP respectively, as a new potential drug target for the acute treatment of migraine.

Prostaglandin E(2) induces immediate migraine-like attack in migraine patients without aura.

BACKGROUND: Prostaglandin E(2) (PGE(2)) has been suggested to play an important role in the pathogenesis of migraine. In the present experiment we investigated if an intravenous infusion of PGE(2) would induce migraine-like attacks in patients with migraine. METHODS: Twelve patients with migraine without aura were randomly allocated to receive 0.4 µg/kg/min PGE(2) (Prostin(®)E2, dinoprostone) or placebo over 25 minutes in a two-way, crossover study. Headache intensity was recorded on a verbal rating scale, middle cerebral artery blood flow velocity (V(MCA)) was measured by transcranial Doppler (TCD) and diameter of the superficial temporal artery (STA) was obtained by c-series scan (Dermascan C). RESULTS: In total, nine migraine patients (75%) experienced migraine-like attacks after PGE(2) compared to none after placebo (p = 0.004). Seven out of 9 (58%) patients reported the migraine-like attacks during the immediate phase (0-90 min) (p = 0.016). Only two patients experienced the delayed migraine-like attacks several hours after the PGE(2) infusion stop (p = 0.500). The V(MCA) decreased during the PGE(2) infusion (p = 0.005) but there was no significant dilatation of the STA (p = 0.850). CONCLUSION: The migraine-like attacks during, and immediately after, the PGE(2) infusion contrast with those found in previous provocation studies, in which the other pharmacological compounds triggered the delayed migraine-like attacks several hours after the infusion. We suggest that PGE(2) may be one of the important final products involved in the generation of migraine attacks.

Pro-inflammatory and vasoconstricting prostanoid PGF2α causes no headache in man.

BACKGROUND: During two decades of migraine provocation studies with naturally occurring signalling molecules, vasodilators such as prostaglandin E(2), prostaglandin I(2) (prostacyclin) and prostaglandin D(2) were shown to be able to induce headache in man. To elucidate the role of inflammation and vasodilatation in the generation of headache, we investigated whether the pro-inflammatory and vasoconstricting prostanoid prostaglandin F(2α) (PGF(2α)) would cause headache in a human model of headache. METHODS: Twelve healthy volunteers were randomly allocated to receive 3.5 µg/kg/min PGF(2α) or placebo over 20 min in a two-way crossover study. We recorded headache intensity on a verbal rating scale, middle cerebral artery blood flow velocity (V(MCA)) and the diameters of the superficial temporal artery (STA) and radial artery (RA). RESULTS: We found no difference in the area under the curve (AUC) for immediate headache (0-90 min) between PGF(2α) and placebo (p = 0.144). The McNemar's test showed no difference in the incidence of immediate and delayed headache between verum and placebo (p = 0.500 and p = 1.000, respectively). There was no difference in V(MCA) (p = 0.776) and in the diameter of the STA (p = 0.460) or RA (p = 0.780) between PGF(2α) and placebo. CONCLUSION: The present study shows that PGF(2α), unlike vasodilating prostaglandins, does not provoke headache. We suggest that the vasodilating abilities of prostaglandins are important for the induction of experimental headache in healthy volunteers.

The pharmacological effect of BGC20-1531, a novel prostanoid EP4 receptor antagonist, in the prostaglandin E2 human model of headache.

Using a human Prostaglandin E(2) (PGE(2)) model of headache, we examined whether a novel potent and selective EP(4) receptor antagonist, BGC20-1531, may prevent headache and dilatation of the middle cerebral (MCA) and superficial temporal artery (STA). In a three-way cross-over trial, eight healthy volunteers were randomly allocated to receive 200 and 400 mg BGC20-1531 and placebo, followed by a 25-min infusion of PGE(2). We recorded headache intensity on a verbal rating scale, MCA blood flow velocity and STA diameter. There was no difference in headache response or prevention of the dilation of the MCA or the STA (P > 0.05) with either dose of BGC20-1531 relative to placebo, although putative therapeutic exposures were not reached in all volunteers. In conclusion, these data suggest that the other EP receptors may be involved in PGE(2) induced headache and dilatation in normal subjects.

Metabolism of melatonin by cytochrome P450s in rat liver mitochondria and microsomes.

In the present study we provide direct evidence for the involvement of rat microsomal cytochrome P450s in melatonin O-demethylation and hydroxylation at two different positions: 2 and 6, as well as generation of N(1)-acetyl-N(2)-formyl-5-methoxy-kynuramine (AFMK) and two unknown products. Moreover, we found that mitochondrial cytochrome P450s also converts melatonin into AFMK, N-acetylserotonin, 2-hydroxymelatonin, 6-hydroxymelatonin and the same two unknown products. Eadie-Hofstee plots for 6-hydroxylation and O-demethylation reactions were curvilinear for all tested fractions, suggestive of involvement of at least two components, one with a high affinity and low capacity, and another with a low affinity and high capacity. Mitochondrial cytochrome P450s exhibited higher affinity (suggesting lower K(m) value) and higher V(max) for melatonin 6-hydroxylation and O-demethylation for both high-affinity and low-affinity components as compared with microsomal enzymes. The intrinsic clearance for melatonin hydroxylation by high- and low-affinity components displayed the highest values in all tested fractions, indicating that both mitochondrial and microsomal cytochrome P450s metabolize melatonin principally by 6-hydroxylation, with O-demethylation representing a minor metabolic pathway.