Immunotherapeutic treatment of inflammation in mice exposed to methamphetamine.

Introduction: Currently, there are no FDA-approved medications to treat methamphetamine addiction, including the inflammatory, neurotoxic, and adverse neuropsychiatric effects. We have shown that partial (p)MHC class II constructs (i.e., Recombinant T-cell receptor Ligand - RTL1000), comprised of the extracellular α1 and ß1 domains of MHC class II molecules linked covalently to myelin oligodendrocyte glycoprotein (MOG)-35-55 peptide, can address the neuroimmune effects of methamphetamine addiction through its ability to bind to and down-regulate CD74 expression, block macrophage migration inhibitory factor (MIF) signaling, and reduce levels of pro-inflammatory chemokine ligand 2 (CCL2). The present study evaluated the effects of our third-generation pMHC II construct, DRmQ, on cognitive function and concentration of inflammatory cytokines in the frontal cortex, a region critical for cognitive functions such as memory, impulse control, and problem solving. Methods: Female and male C57BL/6J mice were exposed to methamphetamine (or saline) via subcutaneous (s.c.) injections administered four times per day every other day for 14 days. Following methamphetamine exposure, mice received immunotherapy (DRmQ or ibudilast) or vehicle s.c. injections daily for five days. Cognitive function was assessed using the novel object recognition test (NORT). To evaluate the effects of immunotherapy on inflammation in the frontal cortex, multiplex immunoassays were conducted. ANOVA was used to compare exploration times on the NORT and immune factor concentrations. Results: Post hoc analysis revealed increased novel object exploration time in MA-DRmQ treated mice, as compared to MA-VEH treated mice (non-significant trend). One-way ANOVA detected a significant difference across the groups in the concentration of macrophage inflammatory protein-2 (MIP-2) (p = 0.03). Post hoc tests indicated that mice treated with methamphetamine and DRmQ or ibudilast had significantly lower levels of MIP-2 in frontal cortex, as compared to mice treated with methamphetamine and vehicle (p > 0.05). Discussion: By specifically targeting CD74, our DRQ constructs can block the signaling of MIF, inhibiting the downstream signaling and pro-inflammatory effects that contribute to and perpetuate methamphetamine addiction.

Circulating fibrillin fragment concentrations in patients with and without aortic pathology.

Objective: Fragments of fibrillin-1 and fibrillin-2 will be detectable in the plasma of patients with aortic dissections and aneurysms. We sought to determine whether the plasma fibrillin fragment levels (PFFLs) differ between patients with thoracic aortic pathology and those presenting with nonaortic chest pain. Methods: PFFLs were measured in patients with thoracic aortic aneurysm (n = 27) or dissection (n = 28). For comparison, patients without aortic pathology who had presented to the emergency department with acute chest pain (n = 281) were categorized into three groups according to the cause of the chest pain: ischemic cardiac chest pain; nonischemic cardiac chest pain; and noncardiac chest pain. The PFFLs were measured using a sandwich enzyme-linked immunosorbent assay. Results: Fibrillin-1 fragments were detectable in all patients and were lowest in the ischemic cardiac chest pain group. Age, sex, and the presence of hypertension were associated with differences in fibrillin-1 fragment levels. Fibrillin-2 fragments were detected more often in the thoracic aneurysm and dissection groups than in the emergency department chest pain group (P < .0001). Patients with aortic dissection demonstrated a trend toward increased detectability (P = .051) and concentrations (P = .06) of fibrillin-2 fragments compared with patients with aortic aneurysms. Analysis of specific antibody pairs identified fibrillin-1 B15-HRP26 and fibrillin-2 B205-HRP143 as the most informative in distinguishing between the emergency department and aortic pathology groups. Conclusions: Patients with thoracic aortic dissections demonstrated elevated plasma fibrillin-2 fragment levels (B205-HRP143) compared with patients presenting with ischemic or nonischemic cardiac chest pain and increased fibrillin-1 levels (B15-HRP26) compared with patients with ischemic cardiac chest pain. Investigation of fibrillin-1 and fibrillin-2 fragment generation might lead to diagnostic, therapeutic, and prognostic advances for patients with thoracic aortic dissection.

Novel MPDZ/MUPP1 transgenic and knockdown models confirm Mpdz's role in ethanol withdrawal and support its role in voluntary ethanol consumption.

Association studies implicate multiple PDZ domain protein (MPDZ/MUPP1) sequence and/or expression in risk for alcoholism in humans and ethanol withdrawal (EW) in mice, but confirmation has been hindered by the dearth of targeted genetic models. We report the creation of transgenic (MPDZ-TG) and knockout heterozygote (Mpdz(+/-) ) mice, with increased (2.9-fold) and decreased (53%) target expression, respectively. Both models differ in EW compared with wild-type littermates (P ≤ 0.03), providing compelling evidence for an inverse relationship between Mpdz expression and EW severity. Additionally, ethanol consumption is reduced up to 18% (P = 0.006) in Mpdz(+/-) , providing the first evidence implicating Mpdz in ethanol self-administration.

Mutations of Francisella novicida that alter the mechanism of its phagocytosis by murine macrophages.

Infection with the bacterial pathogen Francisella tularensis tularensis (F. tularensis) causes tularemia, a serious and debilitating disease. Francisella tularensis novicida strain U112 (abbreviated F. novicida), which is closely related to F. tularensis, is pathogenic for mice but not for man, making it an ideal model system for tularemia. Intracellular pathogens like Francisella inhibit the innate immune response, thereby avoiding immune recognition and death of the infected cell. Because activation of inflammatory pathways may lead to cell death, we reasoned that we could identify bacterial genes involved in inhibiting inflammation by isolating mutants that killed infected cells faster than the wild-type parent. We screened a comprehensive transposon library of F. novicida for mutant strains that increased the rate of cell death following infection in J774 macrophage-like cells, as compared to wild-type F. novicida. Mutations in 28 genes were identified as being hypercytotoxic to both J774 and primary macrophages of which 12 were less virulent in a mouse infection model. Surprisingly, we found that F. novicida with mutations in four genes (lpcC, manB, manC and kdtA) were taken up by and killed macrophages at a much higher rate than the parent strain, even upon treatment with cytochalasin D (cytD), a classic inhibitor of macrophage phagocytosis. At least 10-fold more mutant bacteria were internalized by macrophages as compared to the parent strain if the bacteria were first fixed with formaldehyde, suggesting a surface structure is required for the high phagocytosis rate. However, bacteria were required to be viable for macrophage toxicity. The four mutant strains do not make a complete LPS but instead have an exposed lipid A. Interestingly, other mutations that result in an exposed LPS core were not taken up at increased frequency nor did they kill host cells more than the parent. These results suggest an alternative, more efficient macrophage uptake mechanism for Francisella that requires exposure of a specific bacterial surface structure(s) but results in increased cell death following internalization of live bacteria.

5-HT2C and GABAB receptors influence handling-induced convulsion severity in chromosome 4 congenic and DBA/2J background strain mice.

Progress towards elucidating the underlying genetic variation for susceptibility to complex central nervous system (CNS) hyperexcitability states has just begun. Genetic mapping analyses suggest that a gene(s) on mid-chromosome 4 has pleiotropic effects on multiple CNS hyperexcitability states in mice, including alcohol and barbiturate withdrawal and convulsions elicited by chemical and audiogenic stimuli. We recently identified Mpdz within this chromosomal region as a gene that influences alcohol and barbiturate withdrawal convulsions. Mpdz encodes the multi-PDZ domain protein (MPDZ). Currently, there is limited information available about the mechanism by which MPDZ influences drug withdrawal and/or other CNS hyperexcitability states, but may involve its interaction with 5-HT2C and/or GABAB receptors. One of the most useful tools we have developed thus far is a congenic strain that possesses a segment of chromosome 4 from the C57BL/6J (donor) mouse strain superimposed on a genetic background that is >99% from the DBA/2J strain. The introduced segment spans the Mpdz gene. Here, we demonstrate that handling-induced convulsions are less severe in congenic vs. background strain mice in response to either a 5-HT2C receptor antagonist (SB242084) or a GABAB receptor agonist (baclofen), but not a GABAA receptor channel blocker (pentylenetetrazol). These data suggest that allelic variation in Mpdz, or a linked gene, influences SB242084- and baclofen-enhanced convulsions. Our results are consistent with the hypothesis that Mpdz's effects on CNS hyperexcitability, including alcohol and barbiturate withdrawal, involve MPDZ interaction with 5-HT2C and/or GABAB receptors. However, additional genes reside within the congenic interval and may also influence CNS hyperexcitability.

The syntaxin binding protein 1 gene (Stxbp1) is a candidate for an ethanol preference drinking locus on mouse chromosome 2.

BACKGROUND: We previously mapped a quantitative trait locus (QTL) for ethanol preference drinking to mouse chromosome 2 (mapped with high confidence, LOD = 15.5, p = 3 x 10(-16)). The specific gene(s) in the QTL interval responsible for phenotypic variation in ethanol preference drinking has not been identified. METHODS: In the current study, we investigated the association of the syntaxin binding protein 1 gene (Stxbp1) with ethanol preference drinking and other ethanol traits using a panel of B6 x D2 (BXD) recombinant inbred (RI) strains derived from the C57BL/6J (B6) and DBA/2J (D2) inbred mouse strains. Confirmation analyses for ethanol consumption and withdrawal were performed using a large B6D2 F2 cross, short-term selected lines derived from the B6 and D2 progenitor strains, and standard inbred strains. RESULTS: BXD RI strain analysis detected provisional associations between Stxbp1 molecular variants and ethanol consumption, as well as severity of acute ethanol withdrawal, ethanol-conditioned taste aversion, and ethanol-induced hypothermia. Confirmation analyses using three independent genetic models supported the involvement of Stxbp1 in ethanol preference drinking but not in ethanol withdrawal. CONCLUSIONS: Stxbp1 encodes a Sec1/Munc18-type protein essential for vesicular neurotransmitter release. The present study provides supporting evidence for the involvement of Stxbp1 in ethanol preference drinking.

Using the cre-lox recombination system to assess functional impairment caused by amino acid substitutions in yeast proteins.

A method was developed to assess the functional significance of a sequence motif in yeast Upf3p, a protein required for nonsense-mediated mRNA decay (NMD). The motif lies at the edge of the Upf3p-Upf2p interaction domain, but at the same time resembles the canonical leucine-rich nuclear export sequence (NES) found in proteins that bind Crm1p exportin. To test the function of the putative NES, site-directed mutations that cause substitutions of conserved NES-A residues were first selected to identify hypermorphic alleles. Next, a portable Crm1p-binding NES from HIV-1 Rev protein that functions in yeast was fused en masse to the C-terminus of variant Upf3 proteins using loxP sites recognized by bacterial cre-recombinase. Finally, variant Upf3-Rev proteins that were functional in NMD were selected and examined for the types of amino acid substitutions present in NES-A. The mutational analysis revealed that amino acid substitutions in the Upf3 NES impair both nuclear export and the Upf2p-Upf3p interaction, both of which are required for Upf3p to function in NMD. The method described in this report could be modified for the genetic analysis of a variety of portable protein domains.

Mpdz is a quantitative trait gene for drug withdrawal seizures.

Physiological dependence and associated withdrawal episodes can constitute a powerful motivational force that perpetuates drug use and abuse. Using robust behavioral models of drug physiological dependence in mice, positional cloning, and sequence and expression analyses, we identified an addiction-relevant quantitative trait gene, Mpdz. Our findings provide a framework to define the protein interactions and neural circuit by which this gene's product (multiple PDZ domain protein) affects drug dependence, withdrawal and relapse.

Nuclear import of Upf3p is mediated by importin-alpha/-beta and export to the cytoplasm is required for a functional nonsense-mediated mRNA decay pathway in yeast.

Upf3p, which is required for nonsense-mediated mRNA decay (NMD) in yeast, is primarily cytoplasmic but accumulates inside the nucleus when UPF3 is overexpressed or when upf3 mutations prevent nuclear export. Upf3p physically interacts with Srp1p (importin-alpha). Upf3p fails to be imported into the nucleus in a temperature-sensitive srp1-31 strain, indicating that nuclear import is mediated by the importin-alpha/beta heterodimer. Nuclear export of Upf3p is mediated by a leucine-rich nuclear export sequence (NES-A), but export is not dependent on the Crm1p exportin. Mutations identified in NES-A prevent nuclear export and confer an Nmd(-) phenotype. The addition of a functional NES element to an export-defective upf(-) allele restores export and partially restores an Nmd(+) phenotype. Our findings support a model in which the movement of Upf3p between the nucleus and the cytoplasm is required for a fully functional NMD pathway. We also found that overexpression of Upf2p suppresses the Nmd(-) phenotype in mutant strains carrying nes-A alleles but has no effect on the localization of Upf3p. To explain these results, we suggest that the mutations in NES-A that impair nuclear export cause additional defects in the function of Upf3p that are not rectified by restoration of export alone.

Congenic mapping of alcohol and pentobarbital withdrawal liability loci to a <1 centimorgan interval of murine chromosome 4: identification of Mpdz as a candidate gene.

Risk for onset of alcoholism is related to genetic differences in acute alcohol withdrawal liability. We previously mapped a locus responsible for 26% of the genetic variance in acute alcohol withdrawal convulsion liability to a >35 centimorgan (cM) interval of murine chromosome 4. Here, we narrow the position of this locus to a <1 cM interval (approximately 1.8 megabase, containing 15 genes and/or predicted genes) using a combination of novel, interval-specific congenic strains and recombinant progeny testing. We report the development of a small-donor-segment congenic strain, which confirms capture of a gene affecting alcohol withdrawal within the <1 cM interval. We also confirm a pentobarbital withdrawal locus within this interval, suggesting that the same gene may influence predisposition to physiological dependence on alcohol and a barbiturate. This congenic strain will be invaluable for determining whether this interval also harbors a gene(s) underlying other quantitative trait loci mapped to chromosome 4, including loci affecting voluntary alcohol consumption, alcohol-induced ataxia, physical dependence after chronic alcohol exposure, and seizure response to pentylenetetrazol or an audiogenic stimulus. To date, Mpdz, which encodes the multiple PSD95/DLG/ZO-1 (PDZ) domain protein (MPDZ), is the only gene within the interval shown to have allelic variants that differ in coding sequence and/or expression. Sequence analysis of 15 standard inbred mouse strains identifies six Mpdz haplotypes that predict three MPDZ protein variants. These analyses, and evidence using interval-specific congenic lines, show that alcohol withdrawal severity is genetically correlated with MPDZ status, indicating that MPDZ variants may influence alcohol withdrawal liability.