Manganese-enhanced magnetic resonance imaging reveals increased DOI-induced brain activity in a mouse model of schizophrenia.

Maternal infection during pregnancy increases the risk for schizophrenia in offspring. In rodent models, maternal immune activation (MIA) yields offspring with schizophrenia-like behaviors. None of these behaviors are, however, specific to schizophrenia. The presence of hallucinations is a key diagnostic symptom of schizophrenia. In mice, this symptom can be defined as brain activation in the absence of external stimuli, which can be mimicked by administration of hallucinogens. We find that, compared with controls, adult MIA offspring display an increased stereotypical behavioral response to the hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI), an agonist for serotonin receptor 2A (5-HT2AR). This may be explained by increased levels of 5-HT2AR and downstream signaling molecules in unstimulated MIA prefrontal cortex (PFC). Using manganese-enhanced magnetic resonance imaging to identify neuronal activation elicited by DOI administration, we find that, compared with controls, MIA offspring exhibit a greater manganese (Mn(2+)) accumulation in several brain areas, including the PFC, thalamus, and striatum. The parafascicular thalamic nucleus, which plays the role in the pathogenesis of hallucinations, is activated by DOI in MIA offspring only. Additionally, compared with controls, MIA offspring demonstrate higher DOI-induced expression of early growth response protein 1, cyclooxygenase-2, and brain-derived neurotrophic factor in the PFC. Chronic treatment with the 5-HT2AR antagonist ketanserin reduces DOI-induced head twitching in MIA offspring. Thus, the MIA mouse model can be successfully used to investigate activity induced by DOI in awake, behaving mice. Moreover, manganese-enhanced magnetic resonance imaging is a useful, noninvasive method for accurately measuring this type of activity.

XTX301, a Tumor-Activated Interleukin-12 Has the Potential to Widen the Therapeutic Index of IL12 Treatment for Solid Tumors as Evidenced by Preclinical Studies.

IL12 is a proinflammatory cytokine, that has shown promising antitumor activity in humans by promoting the recruitment and activation of immune cells in tumors. However, the systemic administration of IL12 has been accompanied by considerable toxicity, prompting interest in researching alternatives to drive preferential IL12 bioactivity in the tumor. Here, we have generated XTX301, a tumor-activated IL12 linked to the human Fc protein via a protease cleavable linker that is pharmacologically inactivated by an IL12 receptor subunit beta 2 masking domain. In vitro characterization demonstrates multiple matrix metalloproteases, as well as human primary tumors cultured as cell suspensions, can effectively activate XTX301. Intravenous administration of a mouse surrogate mXTX301 demonstrated significant tumor growth inhibition (TGI) in inflamed and non-inflamed mouse models without causing systemic toxicities. The superiority of mXTX301 in mediating TGI compared with non-activatable control molecules and the greater percentage of active mXTX301 in tumors versus other organs further confirms activation by the tumor microenvironment-associated proteases in vivo. Pharmacodynamic characterization shows tumor selective increases in inflammation and upregulation of immune-related genes involved in IFNγ cell signaling, antigen processing, presentation, and adaptive immune response. XTX301 was tolerated following four repeat doses up to 2.0 mg/kg in a nonhuman primate study; XTX301 exposures were substantially higher than those at the minimally efficacious dose in mice. Thus, XTX301 has the potential to achieve potent antitumor activity while widening the therapeutic index of IL12 treatment and is currently being evaluated in a phase I clinical trial.

Maternal immune activation yields offspring displaying mouse versions of the three core symptoms of autism.

The core symptoms of autism are deficits in social interaction and language, and the presence of repetitive/stereotyped behaviors. We demonstrate that behaviors related to these symptoms are present in a mouse model of an environmental risk factor for autism, maternal infection. We stimulate the maternal immune system by injecting the viral mimic poly(I:C) during pregnancy, and analyze the social and communicative behaviors of the offspring. In one test, young pups respond to a brief separation from the mother with ultrasonic vocalizations (USVs). We find that, compared to pups born to saline-injected mothers, pups born to maternal immune activation (MIA) mothers produce a lower rate of USVs in the isolation test starting at day 8. The quality of the vocalizations is also different; analysis of sound spectrograms of 10 day-old pups shows that male pups from MIA mothers emit significantly fewer harmonic and more complex and short syllables. These communication differences are also apparent in adult offspring. Compared to controls, adult MIA males emit significantly fewer USVs in response to social encounters with females or males, and display reduced scent marking in response to female urine. Regarding a second autism symptom, MIA males display decreased sociability. In a third test of characteristic autism behaviors, MIA offspring exhibit increased repetitive/stereotyped behavior in both marble burying and self-grooming tests. In sum, these results indicate that MIA yields male offspring with deficient social and communicative behavior, as well as high levels of repetitive behaviors, all of which are hallmarks of autism.

An experimental model of anti-PD-1 resistance exhibits activation of TGFß and Notch pathways and is sensitive to local mRNA immunotherapy.

Immune checkpoint blockade elicits durable anti-cancer responses in the clinic, however a large proportion of patients do not benefit from treatment. Several mechanisms of innate and acquired resistance to checkpoint blockade have been defined and include mutations of MHC I and IFNγ signaling pathways. However, such mutations occur in a low frequency of patients and additional mechanisms have yet to be elucidated. In an effort to better understand acquired resistance to checkpoint blockade, we generated a mouse tumor model exhibiting in vivo resistance to anti-PD-1 antibody treatment. MC38 tumors acquired resistance to PD-1 blockade following serial in vivo passaging. Lack of sensitivity to PD-1 blockade was not attributed to dysregulation of PD-L1 or ß2M expression, as both were expressed at similar levels in parental and resistant cells. Similarly, IFNγ signaling and antigen processing and presentation pathways were functional in both parental and resistant cell lines. Unbiased gene expression analysis was used to further characterize potential resistance mechanisms. RNA-sequencing revealed substantial differences in global gene expression, with tumors resistant to anti-PD-1 displaying a marked reduction in expression of immune-related genes relative to parental MC38 tumors. Indeed, resistant tumors exhibited reduced immune infiltration across multiple cell types, including T and NK cells. Pathway analysis revealed activation of TGFß and Notch signaling in anti-PD-1 resistant tumors, and activation of these pathways was associated with poorer survival in human cancer patients. While pharmacological inhibition of TGFß and Notch in combination with PD-1 blockade decelerated tumor growth, a local mRNA-based immunotherapy potently induced regression of resistant tumors, resulting in complete tumor remission, and resensitized tumors to treatment with anti-PD-1. Overall, this study describes a novel anti-PD-1 resistant mouse tumor model and underscores the role of two well-defined signaling pathways in response to immune checkpoint blockade. Furthermore, our data highlights the potential of intratumoral mRNA therapy in overcoming acquired resistance to PD-1 blockade.

Synthetic peptide SLTCLVKGFY competes with beta-endorphin for naloxone-insensitive binding sites on rat brain membranes.

The synthetic decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (termed immunorphin) corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain and its synthetic fragment VKGFY were found to compete with 125I-labeled beta-endorphin for high-affinity naloxone-insensitive binding sites on membranes isolated from the rat brain cortex (K(i)=1.18+/-0.09 and 1.58+/-0.11 nM, respectively). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but to [Met(5)]enkephalin and [Leu(5)]enkephalin as well. The K(d) values characterizing the specific binding of 125I-labeled immunorphin and its fragment Val-Lys-Gly-Phe-Tyr to these binding sites were determined to be 2.93+/-0.27 nM and 3.17+/-0.29 nM, respectively.

Beta-endorphin-like peptide SLTCLVKGFY reduces the production of 11-oxycorticosteroids by rat adrenal cortex through nonopioid beta-endorphin receptors.

Beta-endorphin-like peptide immunorphin (SLTCLVKGFY), a selective agonist of nonopioid beta-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of nonopioid beta-endorphin receptors on rat adrenal cortex membranes (Kd = 31.6 +/- 0.2 nM, Bmax = 37.4 +/- 2.2 pmol/mg protein). Immunorphin at concentrations of 10(-9) to 10(-6) M was found to inhibit the adenylate cyclase activity in adrenal cortex membranes, while intramuscular injection of immunorphin at doses of 10-100 microg/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream.

Reverse transcriptase and endonuclease activities encoded by Penelope-like retroelements.

Penelope-like elements are a class of retroelement that have now been identified in >50 species belonging to at least 10 animal phyla. The Penelope element isolated from Drosophila virilis is the only transpositionally active representative of this class isolated so far. The single ORF of Penelope and its relatives contains regions homologous to a reverse transcriptase of atypical structure and to the GIY-YIG, or Uri, an endonuclease (EN) domain not previously found in retroelements. We have expressed the single ORF of Penelope in a baculovirus expression system and have shown that it encodes a polyprotein with reverse transcriptase activity that requires divalent cations (Mn2+ and Mg2+). We have also expressed and purified the EN domain in Escherichia coli and have demonstrated that it has EN activity in vitro. Mutations in the conserved residues of the EN catalytic module abolish its nicking activity, whereas the DNA-binding properties of the mutant proteins remain unaffected. Only one strand of the target sequence is cleaved, and there is a certain degree of cleavage specificity. We propose that the Penelope EN cleaves the target DNA during transposition, generating a primer for reverse transcription. Our results show that an active Uri EN has been adopted by a retrotransposon.

The Stimulating Effect of the beta-Endorphin-Like Peptide Immunorphin on the Human T-Lymphoblastoid Cell Line Jurkat Is Mediated by a Non-Opioid Receptor for beta-Endorphin.

It was shown that beta-endorphin and the synthetic decapeptide SLTCLVKGFY that corresponds to the amino acid sequence 364-373 of the human IgG heavy chain (referred to as immunorphin) is able to stimulate growth of the human T-lymphoblastoid cell line Jurkat. The antagonist of opioid receptors naloxone did not inhibit the stimulating effect of the peptides. Studies on [(3)H]-immunorphin binding to Jurkat cell receptors have demonstrated that it binds with high affinity to naloxone-insensitive receptors (K(d) = 1.3 nM; n = 5.2 x 10(5)). Unlabeled beta-endorphin and the 6-10 fragment of immunorphin completely inhibited the labeled ligand specific binding to naloxone-insensitive receptors on T lymphocytes (K(i) = 1.4 x 10(-7) and 3.7 x 10(-5) M, respectively). Thus, beta-endorphin and immunorphin share the naloxone-insensitive receptors on human T-lymphoblastoid cell line Jurkat.