Effects of cytokines on nuclear factor-kappa B, cell viability, and synaptic connectivity in a human neuronal cell line.

Maternal infection during pregnancy is associated with increased risk of psychiatric and neurodevelopmental disorders (NDDs). Experimental animal models demonstrate that maternal immune activation (MIA) elevates inflammatory cytokine levels in the maternal and fetal compartments and causes behavioral changes in offspring. Individual cytokines have been shown to modulate neurite outgrowth and synaptic connectivity in cultured rodent neurons, but whether clinically relevant cytokine mixtures similarly modulate neurodevelopment in human neurons is not known. To address this, we quantified apoptosis, neurite outgrowth, and synapse number in the LUHMES human neuronal cell line exposed to varying concentrations of: (1) a mixture of 12 cytokines and chemokines (EMA) elevated in mid-gestational serum samples from mothers of children with autism and intellectual disability; (2) an inflammatory cytokine mixture (ICM) comprised of five cytokines elevated in experimental MIA models; or (3) individual cytokines in ICM. At concentrations that activated nuclear factor-kappa B (NF-κB) in LUHMES cells, EMA and ICM induced caspase-3/7 activity. ICM altered neurite outgrowth, but only at concentrations that also reduced cell viability, whereas ICM reduced synapse number independent of changes in cell viability. Individual cytokines in ICM phenocopied the effects of ICM on NF-κB activation and synaptic connectivity, but did not completely mimic the effects of ICM on apoptosis. These results demonstrate that clinically relevant cytokine mixtures modulate apoptosis and synaptic density in developing human neurons. Given the relevance of these neurodevelopmental processes in NDDs, our findings support the hypothesis that cytokines contribute to the adverse effects of MIA on children.

Linoleic acid-derived metabolites constitute the majority of oxylipins in the rat pup brain and stimulate axonal growth in primary rat cortical neuron-glia co-cultures in a sex-dependent manner.

In adult rats, omega-6 linoleic acid (LA, 18:2n-6) serves as a precursor to oxidized LA metabolites (OXLAMs) known to regulate multiple signaling processes in the brain. However, little is known regarding the levels or role(s) of LA and its metabolites during brain development. To address this gap, fatty acids within various brain lipid pools, and their oxidized metabolites (oxylipins) were quantified in brains from 1-day-old male and female pups using gas chromatography and liquid chromatography coupled to tandem mass spectrometry, respectively. Primary neuron-glia co-cultures derived from postnatal day 0-1 male and female rat neocortex were exposed to vehicle (0.1% ethanol), LA, the OXLAM 13-hydroxyoctadecadienoic acid (13-HODE), or prostaglandin E2 at 10-1000 nM for 48 h to test their effects on neuronal morphology. In both male and female pups, LA accounted for 1-3% of fatty acids detected in brain phospholipids and cholesteryl esters. It was not detected in triacylglycerols, and free fatty acids. Unesterified OXLAMs constituted 47-53% of measured unesterified oxylipins in males and females (vs. ~5-7% reported in adult rat brain). Of these, 13-HODE was the most abundant, accounting for 30-33% of measured OXLAMs. Brain fatty acid and OXLAM concentrations did not differ between sexes. LA and 13-HODE significantly increased axonal outgrowth. Separate analyses of cultures derived from male versus female pups revealed that LA at 1, 50, and 1000 nM, significantly increased axonal outgrowth in female but not male cortical neurons, whereas 13-HODE at 100 nM significantly increased axonal outgrowth in male but not female cortical neurons. prostaglandin E2 did not alter neuronal outgrowth in either sex. This study demonstrates that OXLAMs constitute the majority of unesterified oxylipins in the developing rat brain despite low relative abundance of their LA precursor, and highlights a novel role of LA and 13-HODE in differentially influencing neuronal morphogenesis in the developing male and female brain.

Organophosphorus Pesticides Induce Cytokine Release from Differentiated Human THP1 Cells.

Epidemiologic studies link organophosphorus pesticides (OPs) to increased incidence of asthma. In guinea pigs, OP-induced airway hyperreactivity requires macrophages and TNF-α. Here, we determined whether OPs interact directly with macrophages to alter cytokine expression or release. Human THP1 cells were differentiated into macrophages and then exposed to parathion, chlorpyrifos, or diazinon, or their oxon, phosphate, or phosphorothioate metabolites for 24 hours in the absence or presence of reagents that block cholinergic receptors. TNF-α, IL-1ß, platelet-derived growth factor, and transforming growth factor-ß mRNA and protein were quantified by qPCR and ELISA, respectively. The effects of OPs on NF-κB, acetylcholinesterase, and intracellular calcium were also measured. Parent OPs and their oxon metabolites upregulated cytokine mRNA and stimulated cytokine release. TNF-α release, which was the most robust response, was triggered by parent, but not oxon, compounds. Cytokine expression was also increased by diethyl dithiophosphate but not diethyl thiophosphate or diethyl phosphate metabolites. Parent OPs, but not oxon metabolites, activated NF-κB. Parent and oxon metabolites decreased acetylcholinesterase activity, but comparable acetylcholinesterase inhibition by eserine did not mimic OP effects on cytokines. Consistent with the noncholinergic mechanisms of OP effects on macrophages, pharmacologic antagonism of muscarinic or nicotinic receptors did not prevent OP-induced cytokine expression or release. These data indicate that phosphorothioate OP compounds directly stimulate macrophages to release TNF-α, potentially via activation of NF-κB, and suggest that therapies that target NF-κB may prevent OP-induced airway hyperreactivity.

Mechanisms of organophosphorus pesticide toxicity in the context of airway hyperreactivity and asthma.

Numerous epidemiologic studies have identified an association between occupational exposures to organophosphorus pesticides (OPs) and asthma or asthmatic symptoms in adults. Emerging epidemiologic data suggest that environmentally relevant levels of OPs may also be linked to respiratory dysfunction in the general population and that in utero and/or early life exposures to environmental OPs may increase risk for childhood asthma. In support of a causal link between OPs and asthma, experimental evidence demonstrates that occupationally and environmentally relevant OP exposures induce bronchospasm and airway hyperreactivity in preclinical models. Mechanistic studies have identified blockade of autoinhibitory M2 muscarinic receptors on parasympathetic nerves that innervate airway smooth muscle as one mechanism by which OPs induce airway hyperreactivity, but significant questions remain regarding the mechanism(s) by which OPs cause neuronal M2 receptor dysfunction and, more generally, how OPs cause persistent asthma, especially after developmental exposures. The goals of this review are to 1) summarize current understanding of OPs in asthma; 2) discuss mechanisms of OP neurotoxicity and immunotoxicity that warrant consideration in the context of OP-induced airway hyperreactivity and asthma, specifically, inflammatory responses, oxidative stress, neural plasticity, and neurogenic inflammation; and 3) identify critical data gaps that need to be addressed in order to better protect adults and children against the harmful respiratory effects of low-level OP exposures.

Fc gamma receptors are expressed in the developing rat brain and activate downstream signaling molecules upon cross-linking with immune complex.

BACKGROUND: Exposure of the developing brain to immune mediators, including antibodies, is postulated to increase risk for neurodevelopmental disorders and neurodegenerative disease. It has been suggested that immunoglobulin G-immune complexes (IgG-IC) activate Fc gamma receptors (FcγR) expressed on neurons to modify signaling events in these cells. However, testing this hypothesis is hindered by a paucity of data regarding neuronal FcγR expression and function. METHODS: FcγR transcript expression in the hippocampus, cortex, and cerebellum of neonatal male and female rats was investigated ex vivo and in mixed cultures of primary hippocampal and cortical neurons and astrocytes using quantitative PCR analyses. Expression at the protein level in mixed cultures of primary hippocampal and cortical neurons and astrocytes was determined by immunocytochemistry, western blotting, proteotype analysis, and flow cytometry. The functionality of these receptors was assessed by measuring changes in intracellular calcium levels, Erk phosphorylation, and IgG internalization following stimulation with IgG-immune complexes. RESULTS: FcgrIa, FcgrIIa, FcgrIIb, FcgrIIIa, and Fcgrt transcripts were detectable in the cortex, hippocampus, and cerebellum at postnatal days 1 and 7. These transcripts were also present in primary hippocampal and cortical cell cultures, where their expression was modulated by IFNγ. Expression of FcγRIa, FcγRIIb, and FcγRIIIa, but not FcγRIIa or FcRn proteins, was confirmed in cultured hippocampal and cortical neurons and astrocytes at the single cell level. A subpopulation of these cells co-expressed the activating FcγRIa and the inhibitory FcγRIIb. Functional analyses demonstrated that exposure of hippocampal and cortical cell cultures to IgG-IC increases intracellular calcium and Erk phosphorylation and triggers FcγR-mediated internalization of IgG. CONCLUSIONS: Our data demonstrate that developing neurons and astrocytes in the hippocampus and the cortex express signaling competent FcγR. These findings suggest that IgG antibodies may influence normal neurodevelopment or function via direct interactions with FcγR on non-immune cells in the brain.

Mis-trafficking of endosomal urokinase proteins triggers drug-induced glioma nonapoptotic cell death.

5-Benzylglycinyl-amiloride (UCD38B) is the parent molecule of a class of anticancer small molecules that kill proliferative and nonproliferative high-grade glioma cells by programmed necrosis. UCD38B intracellularly triggers endocytosis, causing 40-50% of endosomes containing proteins of the urokinase plasminogen activator system (uPAS) to relocate to perinuclear mitochondrial regions. Endosomal "mis-trafficking" caused by UCD38B in human glioma cells corresponds to mitochondrial depolarization with the release and nuclear translocation of apoptotis-inducing factor (AIF) followed by irreversible caspase-independent cell demise. High-content quantification of immunocytochemical colocalization studies identified that UCD38B treatment increased endocytosis of the urokinase plasminogen activator (uPA), its receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1) into the early and late endosomes by 4- to 5-fold prior to AIF nuclear translocation and subsequent glioma demise. PAI-1 was found to comparably relocate with a subset of early and late endosomes in four different human glioma cell lines after UCD38B treatment, followed by caspase-independent, nonapoptotic cell death. Following UCD38B treatment, the receptor guidance protein LRP-1, which is required for endosomal recycling of the uPA receptor to the plasmalemma, remained abnormally associated with PAI-1 in early and late endosomes. The resultant aberrant endosomal recycling increased the total cellular content of the uPA-PAI-1 protein complex. Reversible inhibition of cellular endocytosis demonstrated that UCD38B bypasses the plasmalemmal uPAS complex and directly acts intracellularly to alter uPAS endocytotic trafficking. UCD38B represents a class of small molecules whose anticancer cytotoxicity is a consequence of causing the mis-trafficking of early and late endosomes containing uPAS cargo and leading to AIF-mediated necrotic cell death.

A novel carboline derivative inhibits nitric oxide formation in macrophages independent of effects on tumor necrosis factor α and interleukin-1ß expression.

Neuropathic pain is a maladaptive immune response to peripheral nerve injury that causes a chronic painful condition refractory to most analgesics. Nitric oxide (NO), which is produced by nitric oxide synthases (NOSs), has been implicated as a key factor in the pathogenesis of neuropathic pain. ß-Carbolines are a large group of natural and synthetic indole alkaloids, some of which block activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), a predominant transcriptional regulator of NOS expression. Here, we characterize the inhibitory effects of a novel 6-chloro-8-(glycinyl)-amino-ß-carboline (8-Gly carb) on NO formation and NF-κB activation in macrophages. 8-Gly carb was significantly more potent than the NOS inhibitor NG-nitro-L-arginine methyl ester in inhibiting constitutive and inducible NO formation in primary rat macrophages. 8-Gly carb interfered with NF-κB-mediated gene expression in differentiated THP1-XBlue cells, a human NF-κB reporter macrophage cell line, but only at concentrations severalfold higher than needed to significantly inhibit NO production. 8-Gly carb also had no effect on tumor necrosis factor α (TNFα)-induced phosphorylation of the p38 mitogen-activated protein kinase in differentiated THP1 cells, and did not inhibit lipopolysaccharide- or TNFα-stimulated expression of TNFα and interleukin-1ß. These data demonstrate that relative to other carbolines and pharmacologic inhibitors of NOS, 8-Gly carb exhibits a unique pharmacological profile by inhibiting constitutive and inducible NO formation independent of NF-κB activation and cytokine expression. Thus, this novel carboline derivative holds promise as a parent compound, leading to therapeutic agents that prevent the development of neuropathic pain mediated by macrophage-derived NO without interfering with cytokine expression required for neural recovery following peripheral nerve injury.

Rat primary cortical cell tri-culture to study effects of amyloid-beta on microglia function.

Introduction: The etiology and progression of sporadic Alzheimer's Disease (AD) have been studied for decades. One proposed mechanism is that amyloid-beta (Aß) proteins induce neuroinflammation, synapse loss, and neuronal cell death. Microglia play an especially important role in Aß clearance, and alterations in microglial function due to aging or disease may result in Aß accumulation and deleterious effects on neuronal function. However, studying these complex factors in vivo , where numerous confounding processes exist, is challenging, and until recently, in vitro models have not allowed sustained culture of microglia, astrocytes and neurons in the same culture. Here, we employ a tri-culture model of rat primary neurons, astrocytes, and microglia and compare it to co-culture (neurons and astrocytes) and mono-culture enriched for microglia to study microglial function (i.e., motility and Aß clearance) and proteomic response to exogenous Aß. Methods: We established cortical co-culture (neurons and astrocytes), tri-culture (neurons, astrocytes, and microglia), and mono-culture (microglia) from perinatal rat pups. On days in vitro (DIV) 7 - 14, the cultures were exposed to fluorescently-labeled Aß (FITC-Aß) particles for varying durations. Images were analyzed to determine the number of FITC-Aß particles after specific lengths of exposure. A group of cells were stained for ßIII-tubulin, GFAP, and Iba1 for morphological analysis via quantitative fluorescence microscopy. Cytokine profiles from conditioned media were obtained. Live-cell imaging with images acquired every 5 minutes for 4 hours was employed to extract microglia motility parameters (e.g., Euclidean distance, migration speed, directionality ratio). Results and discussion: FITC-Aß particles were more effectively cleared in the tri-culture compared to the co-culture. This was attributed to microglia engulfing FITC-Aß particles, as confirmed via epifluorescence and confocal microscopy. Adding FITC-Aß significantly increased the size of microglia, but had no significant effect on neuronal surface coverage or astrocyte size. Analysis of the cytokine profile upon FITC-Aß addition revealed a significant increase in proinflammatory cytokines (TNF-α, IL-1α, IL-1ß, IL-6) in tri-culture, but not co-culture. In addition, Aß addition altered microglia motility marked by swarming-like motion with decreased Euclidean distance yet unaltered speed. These results highlight the importance of cell-cell communication in microglia function (e.g., motility and Aß clearance) and the utility of the tri-culture model to further investigate microglia dysfunction in AD.

Developmental exposure to the Fox River PCB mixture modulates behavior in juvenile mice.

Developmental exposures to PCBs are implicated in the etiology of neurodevelopmental disorders (NDDs). This observation is concerning given the continued presence of PCBs in the human environment and the increasing incidence of NDDs. Previous studies reported that developmental exposure to legacy commercial PCB mixtures (Aroclors) or single PCB congeners found in Aroclors caused NDD-relevant behavioral phenotypes in animal models. However, the PCB congener profile in contemporary human samples is dissimilar to that of the legacy Aroclors, raising the question of whether human-relevant PCB mixtures similarly interfere with normal brain development. To address this question, we assessed the developmental neurotoxicity of the Fox River Mixture (FRM), which was designed to mimic the congener profile identified in fish from the PCB-contaminated Fox River that constitute a primary protein source in the diet of surrounding communities. Adult female C57BL/6 J mouse dams (8-10 weeks old) were exposed to vehicle (peanut oil) or FRM at 0.1, 1.0, or 6.0 mg/kg/d in their diet throughout gestation and lactation, and neurodevelopmental outcomes were assessed in their pups. Ultrasonic vocalizations (USVs) and measures of general development were quantified at postnatal day (P) 7, while performance in the spontaneous alternation task and the 3-chambered social approach/social novelty task was assessed on P35. Triiodothyronine (T3) and thyroxine (T4) were quantified in serum collected from the dams when pups were weaned and from pups on P28 and P35. Developmental exposure to FRM did not alter pup weight or body temperature on P7, but USVs were significantly decreased in litters exposed to FRM at 0.1 or 6.0 mg/kg/d in the maternal diet. FRM also impaired male and female pups' performance in the social novelty task. Compared to sex-matched vehicles, significantly decreased social novelty was observed in male and female pups in the 0.1 and 6.0 mg/kg/d dose groups. FRM did not alter performance in the spontaneous alternation or social approach tasks. FRM increased serum T3 levels but decreased serum T4 levels in P28 male pups in the 1.0 and 6.0 mg/kg/d dose groups. In P35 female pups and dams, serum T3 levels decreased in the 6.0 mg/kg/d dose group while T4 levels were not altered. Collectively, these findings suggest that FRM interferes with the development of social communication and social novelty, but not memory, supporting the hypothesis that contemporary PCB exposures pose a risk to the developing brain. FRM had sex, age, and dose-dependent effects on serum thyroid hormone levels that overlapped but did not perfectly align with the FRM effects on behavioral outcomes. These observations suggest that changes in thyroid hormone levels are not likely the major factor underlying the behavioral deficits observed in FRM-exposed animals.

Gamma secretase activity modulates BMP-7-induced dendritic growth in primary rat sympathetic neurons.

Autonomic dysfunction has been observed in Alzheimer's disease (AD); however, the effects of genes involved in AD on the peripheral nervous system are not well understood. Previous studies have shown that presenilin-1 (PSEN1), the catalytic subunit of the gamma secretase (γ-secretase) complex, mutations in which are associated with familial AD function, regulates dendritic growth in hippocampal neurons. In this study, we examined whether the γ-secretase pathway also influences dendritic growth in primary sympathetic neurons. Using immunoblotting and immunocytochemistry, molecules of the γ-secretase complex, PSEN1, PSEN2, PEN2, nicastrin and APH1a, were detected in sympathetic neurons dissociated from embryonic (E20/21) rat sympathetic ganglia. Addition of bone morphogenetic protein-7 (BMP-7), which induces dendrites in these neurons, did not alter expression or localization of γ-secretase complex proteins. BMP-7-induced dendritic growth was inhibited by siRNA knockdown of PSEN1 and by three γ-secretase inhibitors, γ-secretase inhibitor IX (DAPT), LY-411575 and BMS-299897. These effects were specific to dendrites and concentration-dependent and did not alter early downstream pathways of BMP signaling. In summary, our results indicate that γ-secretase activity enhances BMP-7 induced dendritic growth in sympathetic neurons. These findings provide insight into the normal cellular role of the γ-secretase complex in sympathetic neurons.

Oxidized linoleic acid metabolites regulate neuronal morphogenesis in vitro.

Linoleic acid (LA, 18:2n-6) is an essential nutrient for optimal infant growth and brain development. The effects of LA in the brain are thought to be mediated by oxygenated metabolites of LA known as oxidized LA metabolites (OXLAMs), but evidence is lacking to directly support this hypothesis. This study investigated whether OXLAMs modulate key neurodevelopmental processes including axon outgrowth, dendritic arborization, cell viability and synaptic connectivity. Primary cortical neuron-glia co-cultures from postnatal day 0-1 male and female rats were exposed for 48h to the following OXLAMs: 1) 13-hydroxyoctadecadienoic acid (13-HODE); 2) 9-hydroxyoctadecadienoic acid (9-HODE); 3) 9,10-dihydroxyoctadecenoic acid (9,10-DiHOME); 4) 12(13)-epoxyoctadecenoic acid (12(13)-EpOME); 5) 9,10,13-trihydroxyoctadecenoic acid (9,10,13-TriHOME); 6) 9-oxo-octadecadienoic acid (9-OxoODE); and 7) 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME). Axonal outgrowth, evaluated by Tau-1 immunostaining, was increased by 9-HODE, but decreased by 12,13-DiHOME in male but not female neurons. Dendrite arborization, evaluated by MAP2B-eGFP expression, was affected by 9-HODE, 9-OxoODE, and 12(13)-EpOME in male neurons and, by 12(13)-EpOME in female neurons. Neither cell viability nor synaptic connectivity were significantly altered by OXLAMs. Overall, this study shows select OXLAMs modulate neuron morphology in a sex-dependent manner, with male neurons being more susceptible.

Transient Stimulation with Psychoplastogens Is Sufficient to Initiate Neuronal Growth.

Cortical neuron atrophy is a hallmark of depression and includes neurite retraction, dendritic spine loss, and decreased synaptic density. Psychoplastogens, small molecules capable of rapidly promoting cortical neuron growth, have been hypothesized to produce long-lasting positive effects on behavior by rectifying these deleterious structural and functional changes. Here we demonstrate that ketamine and LSD, psychoplastogens from two structurally distinct chemical classes, promote sustained growth of cortical neurons after only short periods of stimulation. Furthermore, we show that psychoplastogen-induced cortical neuron growth can be divided into two distinct epochs: an initial stimulation phase requiring TrkB activation and a growth period involving sustained mTOR and AMPA receptor activation. Our results provide important temporal details concerning the molecular mechanisms by which next-generation antidepressants produce persistent changes in cortical neuron structure, and they suggest that rapidly excreted psychoplastogens might still be effective neurotherapeutics with unique advantages over compounds like ketamine and LSD.

Glioma-associated microglia/macrophages augment tumorigenicity in canine astrocytoma, a naturally occurring model of human glioma.

BACKGROUND: Glioma-associated microglia/macrophages (GAMs) markedly influence glioma progression. Under the influence of transforming growth factor beta (TGFB), GAMs are polarized toward a tumor-supportive phenotype. However, neither therapeutic targeting of GAM recruitment nor TGFB signaling demonstrated efficacy in glioma patients despite efficacy in preclinical models, underscoring the need for a comprehensive understanding of the TGFB/GAM axis. Spontaneously occurring canine gliomas share many features with human glioma and provide a complementary translational animal model for further study. Given the importance of GAM and TGFB in human glioma, the aims of this study were to further define the GAM-associated molecular profile and the relevance of TGFB signaling in canine glioma that may serve as the basis for future translational studies. METHODS: GAM morphometry, levels of GAM-associated molecules, and the canonical TGFB signaling axis were compared in archived samples of canine astrocytomas versus normal canine brain. Furthermore, the effect of TGFB on the malignant phenotype of canine astrocytoma cells was evaluated. RESULTS: GAMs diffusely infiltrated canine astrocytomas. GAM density was increased in high-grade tumors that correlated with a pro-tumorigenic molecular signature and upregulation of the canonical TGFB signaling axis. Moreover, TGFB1 enhanced the migration of canine astrocytoma cells in vitro. CONCLUSIONS: Canine astrocytomas share a similar GAM-associated immune landscape with human adult glioma. Our data also support a contributing role for TGFB1 signaling in the malignant phenotype of canine astrocytoma. These data further support naturally occurring canine glioma as a valid model for the investigation of GAM-associated therapeutic strategies for human malignant glioma.

Mast cell repopulation of the peritoneal cavity: contribution of mast cell progenitors versus bone marrow derived committed mast cell precursors.

BACKGROUND: Mast cells have recently gained new importance as immunoregulatory cells that are involved in numerous pathological processes. One result of these processes is an increase in mast cell numbers at peripheral sites. This study was undertaken to determine the mast cell response in the peritoneal cavity and bone marrow during repopulation of the peritoneal cavity in rats. RESULTS: Two mast cell specific antibodies, mAb AA4 and mAb BGD6, were used to distinguish the committed mast cell precursor from more mature mast cells. The peritoneal cavity was depleted of mast cells using distilled water. Twelve hours after distilled water injection, very immature mast cells could be isolated from the blood and by 48 hours were present in the peritoneal cavity. At this same time the percentage of mast cells in mitosis increased fourfold. Mast cell depletion of the peritoneal cavity also reduced the total number of mast cells in the bone marrow, but increased the number of mast cell committed precursors. CONCLUSIONS: In response to mast cell depletion of the peritoneal cavity, a mast cell progenitor is released into the circulation and participates in repopulation of the peritoneal cavity, while the committed mast cell precursor is retained in the bone marrow.

Identification of Psychoplastogenic N,N-Dimethylaminoisotryptamine (isoDMT) Analogues through Structure-Activity Relationship Studies.

Ketamine, N,N-dimethyltryptamine (DMT), and other psychoplastogens possess enormous potential as neurotherapeutics due to their ability to potently promote neuronal growth. Here, we report the first-ever structure-activity relationship study with the explicit goal of identifying novel psychoplastogens. We have discovered several key features of the psychoplastogenic pharmacophore and used this information to develop N,N-dimethylaminoisotryptamine (isoDMT) psychoplastogens that are easier to synthesize, have improved physicochemical properties, and possess reduced hallucinogenic potential as compared to their DMT counterparts.

The low affinity IgG receptor Fc gamma RIIB contributes to the binding of the mast cell specific antibody, mAb BGD6.

The mast cell specific monoclonal antibody, mAb BGD6, is a mast cell lineage marker [Jamur, M.C., Grodzki, A.C., Berenstein, E.H., Hamawy, M.M., Siraganian, R.P., Oliver, C., 2005. Identification and characterization of undifferentiated mast cells in mouse bone marrow. Blood 105, 4282-4289]. In rat basophilic leukemia (RBL-2H3) cells, mAb BGD6 precipitates cell-surface proteins of approximately 110 and 40-60 kDa. An expression cloning strategy was used to identify proteins that interact with mAb BGD6. A RBL-2H3 cDNA library in plasmids was transfected into PEAK cells, which do not bind mAb BGD6, and positive cells were selected with mAb BGD6. The plasmids recovered from the positive cells were amplified; retransfected into PEAK cells and after several screening cycles a positive clone was identified. This clone showed almost complete identity to Fc gamma RIIB (CD32), the low affinity IgG receptor. However, in contrast to the sequence in GenBank, this clone had an insert of 141 bp which codes for a longer isoform of this molecule with an extra 47 aa in its cytoplasmic domain. In RBL-2H3 cells both isoforms were expressed, with higher expression of the shorter form. The mechanism of binding of mAB BGD6 on both RBL-2H3 and CD32 transfected PEAK cells was then examined. Intact mAb BGD6 bound to both RBL-2H3 and CD32 expressing PEAK cells, but F(ab')(2) fragments bound only to RBL-2H3 cells demonstrating that mAb BGD6 binds to Fc gamma RIIB only through its Fc portion. On RBL-2H3 cells, the Fab of an anti-CD32 mAb partially inhibited the binding of intact mAb BGD6. The binding pattern of mAb BGD6 inhibited with anti-CD32 resembled that of the F(ab')(2) fragment of the antibody suggesting that the Fc portion of mAb BGD6 contributes to its binding on cells that have Fc gamma RIIB. These results are consistent with a model where mAb BGD6 binds through its Fab portion to a approximately 110 kDa protein and the Fc tail interacts with Fc gamma RIIB (CD32).

The Organophosphorus Pesticide Chlorpyrifos Induces Sex-Specific Airway Hyperreactivity in Adult Rats.

Occupational and environmental exposures to organophosphorus pesticides (OPs) are associated with increased incidence of asthma and other pulmonary diseases. Although the canonical mechanism of OP neurotoxicity is inhibition of acetylcholinesterase (AChE), it was previously reported that the OP chlorpyrifos (CPF) causes airway hyperreactivity (AHR) in guinea pigs at levels that do not inhibit lung or brain AChE. The guinea pig is considered to have inherently hyperresponsive airways, thus, cross-species validation is needed to confirm relevance to humans. Additionally, sex differences in asthma incidence have been demonstrated in the human population, but whether OP-induced AHR is sex-dependent has not been systematically studied in a preclinical model. In this study, 8-week old male and female Sprague Dawley rats were administered CPF at doses causing comparable AChE inhibition in whole lung homogenate (30 mg/kg in males, 7 mg/kg in females, sc) prior to assessing pulmonary mechanics in response to electrical stimulation of the vagus nerves at 24 h, 48 h, 72 h, 7 d or 14 d post-exposure in males, and 24 h or 7 d post-exposure in females. CPF significantly potentiated vagally induced airway resistance and tissue elastance at 7 d post-exposure in males, and at 24 h and 7 d post-exposure in females. These effects occurred independent of significant AChE inhibition in cerebellum, blood, trachealis, or isolated airway, suggesting that AChE independent OP-induced airway hyperreactivity is a cross-species phenomenon. These findings have significant implications for assessing the risk posed by CPF, and potentially other OPs, to human health and safety.

Oxygen tension modulates differentiation and primary macrophage functions in the human monocytic THP-1 cell line.

The human THP-1 cell line is widely used as an in vitro model system for studying macrophage differentiation and function. Conventional culture conditions for these cells consist of ambient oxygen pressure (∼20% v/v) and medium supplemented with the thiol 2-mercaptoethanol (2-ME) and serum. In consideration of the redox activities of O2 and 2-ME, and the extensive experimental evidence supporting a role for reactive oxygen species (ROS) in the differentiation and function of macrophages, we addressed the question of whether culturing THP-1 cells under a more physiologically relevant oxygen tension (5% O2) in the absence of 2-ME and serum would alter THP-1 cell physiology. Comparisons of cultures maintained in 18% O2versus 5% O2 indicated that reducing oxygen tension had no effect on the proliferation of undifferentiated THP-1 cells. However, decreasing the oxygen tension to 5% O2 significantly increased the rate of phorbol ester-induced differentiation of THP-1 cells into macrophage-like cells as well as the metabolic activity of both undifferentiated and PMA-differentiated THP-1 cells. Removal of both 2-ME and serum from the medium decreased the proliferation of undifferentiated THP-1 cells but increased metabolic activity and the rate of differentiation under either oxygen tension. In differentiated THP-1 cells, lowering the oxygen tension to 5% O2 decreased phagocytic activity, the constitutive release of ß-hexosaminidase and LPS-induced NF-κB activation but enhanced LPS-stimulated release of cytokines. Collectively, these data demonstrate that oxygen tension influences THP-1 cell differentiation and primary macrophage functions, and suggest that culturing these cells under tightly regulated oxygen tension in the absence of exogenous reducing agent and serum is likely to provide a physiologically relevant baseline from which to study the role of the local redox environment in regulating THP-1 cell physiology.

IFNγ Increases M2 Muscarinic Receptor Expression in Cultured Sympathetic Neurons.

M2 muscarinic receptors are expressed on both parasympathetic and sympathetic nerve endings where they function as autoinhibitory receptors to limit release of acetylcholine and norepinephrine, respectively. M2 muscarinic receptor expression on parasympathetic nerves is decreased by viral infection and by gamma-interferon (IFNγ) and increased by dexamethasone; and these effects are of clinical relevance in the etiology and treatment of asthma. Whether IFNγ and dexamethasone similarly modulate M2 receptor expression on sympathetic nerves is not known. To address this question, we examined the effects of IFNγ and dexamethasone on M2 receptor expression at the mRNA and protein level in primary cultures of sympathetic neurons dissociated from the rat superior cervical ganglia (SCG). Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that neither IFNγ nor dexamethasone altered M2 receptor transcript levels. However, western blot analyses demonstrated that IFNγ, but not dexamethasone, increases M2 receptor protein expression in sympathetic neurons. Increased expression did not significantly alter subcellular localization of M2 receptors in sympathetic neurons as determined using immunocytochemistry. These findings indicate that M2 receptors are differentially regulated in different types of autonomic neurons, and they suggest a novel mechanism by which IFNγ may contribute to airway hyperreactivity in viral-induced asthma.

Introduction to the purification of antibodies.

Antibodies are a powerful and essential tool in scientific laboratories being used in an array of applications such as immuno-histochemistry, immunobloting, immunoprecipitation and enzyme-linked immunosorbent assays (ELISA). The different sources for antibodies include polyclonal antisera from immunized animals and monoclonal antibodies from cells in culture or from ascites in animals. Both polyclonal and monoclonal antibodies have their advantages, and or disadvantages, but in general the production of monoclonal antibodies is more time consuming and requires tissue culture facilities and skills. The use of either monoclonal or polyclonal antibodies in some of the applications may require that the antibody is in a purified form. They can be purified by a variety of methods described in the next few chapters. The availability of commercially available kits primarily designed for the purification of IgG and IgM classes of antibodies derived from all common animal species should also be mentioned.