Partial mGlu5 receptor NAM, M-5MPEP, induces rapid and sustained antidepressant-like effects in the BDNF-dependent mechanism and enhances (R)-ketamine action in mice.

BACKGROUND: Partial negative allosteric modulators (NAM) of the metabotropic glutamate 5 (mGlu5) receptor are an excellent alternative to full antagonists and NAMs because they retain therapeutic effects and have a much broader therapeutic window. Here, we investigated whether partial mGlu5 NAM, 2-(2-(3-methoxyphenyl)ethynyl)-5-methylpyridine (M-5MPEP), induced a fast and sustained antidepressant-like effect, characteristic of rapid-acting antidepressant drugs (RAADs) like ketamine, in mice. METHODS: A tail suspension test (TST) was used to investigate acute antidepressant-like effects. Sustained effects were studied 24 h after the four intraperitoneal (ip) administrations using the splash test, designed to measure apathy-like state, the sucrose preference test (SPT), reflecting anhedonia, and the TST. Western blot and ELISA techniques were used to measure brain-derived neurotrophic factor (BDNF) and selected protein levels. METHODS: A tail suspension test (TST) was used to investigate acute antidepressant-like effects. Sustained effects were studied 24 h after the four intraperitoneal (ip) administrations using the splash test, designed to measure apathy-like state, the sucrose preference test (SPT), reflecting anhedonia, and the TST. Western blot and ELISA techniques were used to measure brain-derived neurotrophic factor (BDNF) and selected protein levels. CONCLUSION: Partial mGlu5 receptor NAM, M-5MPEP, induced rapid and sustained antidepressant-like effects in the BDNF-dependent mechanism and enhanced (R)-ketamine action in mice, indicating both substances' convergent mechanisms of action and the possibility of their practical use in treating depression as RAAD.

Impact of endogenous and exogenous nitrogen species on macrophage extracellular trap (MET) formation by bone marrow-derived macrophages.

Macrophage extracellular traps (METs) represent a novel defense mechanism in the antimicrobial arsenal of macrophages. However, mechanisms of MET formation are still poorly understood and this is at least partially due to the lack of reliable and reproducible models. Thus, we aimed at establishing a protocol of MET induction by bone marrow-derived macrophages (BMDMs) obtained from cryopreserved and then thawed bone marrow (BM) mouse cells. We report that BMDMs obtained in this way were morphologically (F4/80+) and functionally (expression of inducible nitric oxide (NO) synthase and NO production) differentiated and responded to various stimuli of bacterial (lipopolysaccharide, LPS), fungal (zymosan) and chemical (PMA) origin. Importantly, BMDMs were successfully casting METs composed of extracellular DNA (extDNA) serving as their backbone to which proteins such as H2A.X histones and matrix metalloproteinase 9 (MMP-9) were attached. In rendered 3D structure of METs, extDNA and protein components were embedded in each other. Since studies had shown the involvement of oxygen species in MET release, we aimed at studying if reactive nitrogen species (RNS) such as NO are also involved in MET formation. By application of NOS inhibitor - L-NAME or nitric oxide donor (SNAP), we studied the involvement of endogenous and exogenous RNS in traps release. We demonstrated that L-NAME halted MET formation upon stimulation with LPS while SNAP alone induced it. The latter phenomenon was further enhanced in the presence of LPS. Taken together, our findings demonstrate that BMDMs obtained from cryopreserved BM cells are capable of forming METs in an RNS-dependent manner.

Scrutinizing Mechanisms of the 'Obesity Paradox in Sepsis': Obesity Is Accompanied by Diminished Formation of Neutrophil Extracellular Traps (NETs) Due to Restricted Neutrophil-Platelet Interactions.

Systemic inflammation is a detrimental condition associated with high mortality. However, obese individuals seem to have higher chances of surviving sepsis. To elucidate what immunological differences exist between obese and lean individuals we studied the course of endotoxemia in mice fed high-fat diet (HFD) and ob/ob animals. Intravital microscopy revealed that neutrophil extracellular trap (NET) formation in liver vasculature is negligible in obese mice in sharp contrast to their lean counterparts (ND). Unlike in lean individuals, neutrophil influx is not driven by leptin or interleukin 33 (IL-33), nor occurs via a chemokine receptor CXCR2. In obese mice less platelets interact with neutrophils forming less aggregates. Platelets transfer from ND to HFD mice partially restores NET formation, and even further so upon P-selectin blockage on them. The study reveals that in obesity the overexaggerated inflammation and NET formation are limited during sepsis due to dysfunctional platelets suggesting their targeting as a therapeutic tool in systemic inflammation.

On Neutrophil Extracellular Trap (NET) Removal: What We Know Thus Far and Why So Little.

Although neutrophil extracellular traps (NETs) were discovered only 16 years ago, they have already taken us from heaven to hell as we learned that apart from beneficial trapping of pathogens, they cause, or contribute to, numerous disorders. The latter is connected to their persistent presence in the blood or tissue, and we hardly know how they are removed in mild pathophysiological conditions and why their removal is impaired in multiple severe pathological conditions. Herein, we bring together all data available up till now on how NETs are cleared-from engaged cells, their phenotypes, to involved enzymes and molecules. Moreover, we hypothesize on why NET removal is challenged in multiple disorders and propose further directions for studies on NET removal as well as possible therapeutic strategies to have them cleared.

Imaging of Neutrophils and Neutrophil Extracellular Traps (NETs) with Intravital (In Vivo) Microscopy.

As we have learned during recent years, neutrophils are not just simple foot soldiers of the innate immune system with a restricted set of pro-inflammatory functions, and instead, they perform sophisticated functions (some of them only recently discovered) both in innate and adaptive immune responses. Neutrophil behavior and functioning should best be studied in situ, at locations where they are executed in a living organism, especially considering that neutrophils are mobile cells, performing their functions in distal body sites and various organs. For this herein we describe an approach to detect neutrophil presence/behavior in various organs (skin, muscle, liver) of alive mice, that is, intravital imaging/microscopy. We describe all surgeries required prior to imaging and share our methods of detection of neutrophils and neutrophil extracellular traps (NETs).

To NET or not to NET:current opinions and state of the science regarding the formation of neutrophil extracellular traps.

Since the discovery and definition of neutrophil extracellular traps (NETs) 14 years ago, numerous characteristics and physiological functions of NETs have been uncovered. Nowadays, the field continues to expand and novel mechanisms that orchestrate formation of NETs, their previously unknown properties, and novel implications in disease continue to emerge. The abundance of available data has also led to some confusion in the NET research community due to contradictory results and divergent scientific concepts, such as pro- and anti-inflammatory roles in pathologic conditions, demarcation from other forms of cell death, or the origin of the DNA that forms the NET scaffold. Here, we present prevailing concepts and state of the science in NET-related research and elaborate on open questions and areas of dispute.

Differential Time Course of Restoration of Experimentally Depleted Coelomocytes and Fluorophores in the Earthworm Eisenia Andrei.

Stressed earthworms expel coelomic fluid containing several vital cellular and soluble components, thus their post-stress recovery has adaptive value. The present manuscript describes the recovery rates of coelomocytes (amoebocytes and eleocytes) and two fluorophores (riboflavin and 4-methylumbelliferyl ß-D-glucuronide, MUG) after experimental extrusion by electrostimulation. Analyses were conducted at time points (from 0.5 hour to 7 weeks) by a combination of cell counts, spectrofluorimetric measurements of riboflavin and MUG, and fluorescence microscopy. Coelomic fluid retrieved 30 minutes after extrusion contained <10% of the baseline levels of amoebocytes, eleocytes and riboflavin; the depleted levels of these variables were fully restored after 3, 5, and 7 weeks post-extrusion, respectively. Restored eleocytes were richer in riboflavin than the eleocytes of worms electrostimulated at t0. MUG was less severely depleted (to 49% of baseline) than riboflavin, and was restored to the initial level within 1 week post-extrusion. This indicates that MUG, unlike riboflavin, resides mainly within non-coelomocyte cellular location(s); moreover, this fluorophore may be a useful molecular marker for distinguishing even immunologically-compromised E. andrei from closely related composting species.

Exosomes as mediators of intercellular communication: clinical implications.

Cells of multicellular organisms exchange informative signals by diverse mechanisms. Recent findings uncovered the special role of extracellular vesicles, especially exosomes, in intercellular communication. Exosomes, present in all tested human bodily fluids, carry various functional compounds including proteins, lipids, and diverse RNA molecules. The composition of exosome cargo in vivo is likely formed by a regulated selection of specific components and can express the current status of the exosome-secreting cell. Therefore, particular emphasis is now placed on the extremely high potential of exosomes as essentially noninvasive prognostic and diagnostic biomarkers, but also as therapeutic nanocarriers, especially after the discovery that their cargo as well as cell-targeting specificity could be shaped in vitro. In addition, targeting the exosomes mediating pathological intercellular communication may also express high therapeutic potential. Hence, numerous studies are conducted to explore the profile and function of exosomes and their cargo in health and disease and to shape their properties to facilitate their clinical application. The present review summarizes the current knowledge on the role of exosomes in different physiological and pathological mechanisms of intercellular communication with a particular focus on the use of exosomes in the diagnosis and treatment of various inflammatory, cardiovascular, metabolic, and neurodegenerative disorders as well as malignant neoplasms.

Effects of anesthetic compounds on responses of earthworms to electrostimulation.

Earthworms play an important role in biomedical research, and some surgical procedures require anesthesia. Anesthetic treatments used so far usually induce convulsive body movements connected with extrusion of coelomocyte-containing coelomic fluid that may affect experimental results. Extensive movements connected with the expulsion of coelomic fluid are exploited by immunologists as a method of harvesting immunocompetent coelomocytes from worms subjected to mild electrostimulation (4.5V). The aim of the investigations was to find anesthetic drugs without unintentional coelomocyte depletion. Experiments were performed on adult specimens of Dendrobaena veneta, the coelomocytes of which consist of amoebocytes and riboflavin-storing eleocytes. Earthworm mobility was filmed and extrusion of coelomocytes was quantified by detection of eleocyte-derived riboflavin in immersion fluid. Treatments included earthworms (1) immersed either in physiological saline (controls) or in a solution of one of the tested anesthetic drugs; (2) electrostimulated immediately after anesthesia, and (3) electrostimulated a second time after a 1-hour recovery period. The well-established fish and amphibian anesthetic agent MS-222 induced coelomocyte expulsion. In contrast, solutions of the mammalian local anesthetic drug, prilocaine hydrochloride (0.25-0.5%, 5-10 min) caused temporal earthworm immobilization followed by recovery, thus showing utility as an efficient earthworm anesthetic.

Coelomocyte-derived fluorescence and DNA markers of composting earthworm species.

Supravital species identification of morphologically similar syntopic earthworms inhabiting dung and compost heaps or those from commercial cultures is difficult. The aim of the studies was to find out non-invasive species-specific markers for proper segregation of earthworm species from a dense mixed colony of waste decomposers. Worms were segregated according to external characteristics into Eisenia andrei, Eisenia fetida, and Dendrobaena veneta, and left for reproduction and analysis of non-invasively retrieved coelomocyte-containing coelomic fluid and/or species-specific partial sequences of cytochrome c oxidase subunit I (COI) gene in DNA extracted from amputated tail tips of adults and their offspring. Flow cytometric analysis of coelomocyte samples revealed that amount of nuclear DNA increases in order D. veneta ≪ E. andrei < E. fetida, and intensity of eleocyte-derived fluorescence is lower in D. veneta than in Eisenia spp. Spectrofluorimetry of coelomocyte lysates revealed that the amount of eleocyte-stored riboflavin is significantly lower in coelomocyte lysates from D. veneta than from Eisenia spp., and the emission peak of X-fluorophore is much more distinct in D. veneta than in Eisenia spp. Coelomic fluid of E. andrei exhibits a very distinct spectra of MUG fluorophore which are absent in D. veneta and in the majority of E. fetida, while some E. fetida possess MUG-like fluorophore. Sequences of the COI gene in the DNA of the worms from the mixed colony and their offspring confirmed species identity. In conclusion, species-specific coelomocyte-derived markers may be a useful complement to morphological and DNA-based taxonomy during studies on syntopic earthworms.