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Objectives: Nosocomial pneumonia is a common complication in critically ill patients. The goal of this study was to examine the efficacy of the Toll-like receptor 4 agonist 3-deacyl phosphorylated hexacyl disaccharide (3D PHAD), in a clinically relevant murine model of pneumonia, and assess the cellular mechanisms that mediate the protective response. Design: Mice received intrapulmonary 3D PHAD (20 μg) or vehicle for 2 consecutive days before challenge with intrapulmonary Klebsiella pneumoniae (2.3 × 10 3 colony-forming units). Mice were followed for 14-day survival, pulmonary K. pneumoniae burden, lung leukocyte profile, leukocyte phagocytic capacity, and cytokine production. Pneumonia severity and leukocyte recruitment were further assessed by histological evaluation. Setting: Research laboratory. Subjects: Wild-type, male C57BL/6 J mice. Interventions: Intrapulmonary treatment with 20 μg 3D PHAD for 2 consecutive days. Measurements and main results: Intrapulmonary treatment with 3D PHAD decreased lung K. pneumoniae colony-forming units and pneumonia severity with an associated improvement in survival compared with mice treated with vehicle. The numbers of neutrophils, monocytes, and macrophages in the lungs of 3D PHAD-treated mice were higher than those in vehicle-treated mice before infection but were not significantly different from vehicle-treated mice at 48 h after K. pneumoniae challenge. Lung innate leukocytes from 3D PHAD-treated mice had increased phagocytic capacity. Treatment with 3D PHAD alone increased cytokines in the lungs but decreased cytokines in plasma during K. pneumoniae pneumonia as compared with control. Conclusions: Intrapulmonary treatment with 3D PHAD augments innate immunity in the lung and facilitates resistance to K. pneumoniae pneumonia.

Critical limb ischemia (CLI) occurs when blood flow is restricted through the arteries, resulting in ulcers, necrosis, and chronic wounds in the downstream extremities. The development of collateral arterioles (i.e. arteriogenesis), either by remodeling of pre-existing vascular networks orde novogrowth of new vessels, can prevent or reverse ischemic damage, but it remains challenging to stimulate collateral arteriole development in a therapeutic context. Here, we show that a gelatin-based hydrogel, devoid of growth factors or encapsulated cells, promotes arteriogenesis and attenuates tissue damage in a murine CLI model. The gelatin hydrogel is functionalized with a peptide derived from the extracellular epitope of Type 1 cadherins. Mechanistically, these “GelCad” hydrogels promote arteriogenesis by recruiting smooth muscle cells to vessel structures in bothex vivoandin vivoassays. In a murine femoral artery ligation model of CLI, delivery ofin situcrosslinking GelCad hydrogels was sufficient to restore limb perfusion and maintain tissue health for 14 days, whereas mice treated with gelatin hydrogels had extensive necrosis and autoamputated within 7 days. A small cohort of mice receiving the GelCad hydrogels were aged out to 5 months and exhibited no decline in tissue quality, indicating durability of the collateral arteriole networks. Overall, given the simplicity and off-the-shelf format of the GelCad hydrogel platform, we suggest it could have utility for CLI treatment and potentially other indications that would benefit from arteriole development.

Generalized cellular ‘stress’ (i.e., oxidative, mitochondrial) within the syncytiotrophoblast (STB) layer of placenta is theorized to drive the pathogenesis of preeclampsia (PE), but the causes and consequences of this stress have yet to be clearly elucidated. Many hormones implicated in the disorder signal through the Gq pathway and have been associated with placental oxidative damage and maternal symptoms. Here, we provide novel evidence of elevated Gq stimulation (n=8-10 normalized area PLCB protein, control 1340±370, PE 3350±750, P=0.04) and an antioxidant response (n=8 normalized area SOD2 protein, control 3020±1160, PE 6700±1250, P=0.04) within STB cells of PE placenta. Thus, we hypothesized excess STB-specific Gq signaling would be sufficient to cause phenotypes of PE and that administration of a mitochondrial-targeted antioxidant (mitoquinone) would mitigate these effects. Activation of the Gq cascade in STB cells was achieved by crossing dams harboring a Cre-dependent Gq-coupled DREADD (hM3Dq) with Gcm1-Cre+/- sires and injecting clozapine N-oxide (CNO) or saline mid-gestation (GD12.5-14.5) before tissue collection at GD14.5. Gq stimulation increased mean arterial pressure (n=6-8, CNO 115±2mmHg, saline 110±2mmHg, GD 13-16.5, P=0.04), exacerbated urine protein excretion (n=9-11 CNO 43±4mg/d, saline 28±3mg/day, P=0.001), and elevated circulating pro-inflammatory factors (MCP5, LIX, TIMP1, MIP3B, GCSF, IL12p40, MDC, P NIH: HL134850, HL084207, DK133121, HL150340; AHA: 826132, 18EIA33890055 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PDF file, 707K, S1. Comparison of alpha3beta1 and alpha6beta4 integrin expression in 4T1 cell lines. S2. BLI of spontaneous and experimental metastasis (color) S3. Certain large primary mammary fat pad tumors exhibited substantial centralized zones of necrosis. S4. Silencing alpha3 integrin leads to fewer pulmonary metastases following mammary fat pad injection. S5. RNAi-mediated silencing of RhoC has no impact on 4T1 cell metastatic colonization. S6. Cox-2, matrix metalloproteinase, VEGF, and transendothelial migration assays. S7. MDA-MB-231 breast cancer cells do not secrete laminin-332 or laminin-511 or depend on alpha3 integrin in short term proliferation assays.

Significant evidence implicates α3β1 integrin in promoting breast cancer tumorigenesis and metastasis-associated cell behaviors in vitro and in vivo. However, the extent to which α3β1 is actually required for breast cancer metastasis remains to be determined. We used RNA interference to silence α3 integrin expression by approximately 70% in 4T1 murine mammary carcinoma cells, a model of aggressive, metastatic breast cancer. Loss of α3 integrin reduced adhesion, spreading, and proliferation on laminin isoforms, and modestly reduced the growth of orthotopically implanted cells. However, spontaneous metastasis to lung was strikingly curtailed. Experimental lung colonization after tail vein injection revealed a similar loss of metastatic capacity for the α3-silenced (α3si) cells, suggesting that critical, α3-dependent events at the metastatic site could account for much of α3β1′s contribution to metastasis in this model. Reexpressing α3 in the α3si cells reversed the loss of metastatic capacity, and silencing another target, the small GTPase RhoC, had no effect, supporting the specificity of the effect of silencing α3. Parental, α3si, and α3-rescued cells, all secreted abundant laminin α5 (LAMA5), an α3β1 integrin ligand, suggesting that loss of α3 integrin might disrupt an autocrine loop that could function to sustain metastatic growth. Analysis of human breast cancer cases revealed reduced survival in cases where α3 integrin and LAMA5 are both overexpressed.Implications: α3 integrin or downstream effectors may be potential therapeutic targets in disseminated breast cancers, especially when laminin α5 or other α3 integrin ligands are also over-expressed. Mol Cancer Res; 12(1); 143–54. ©2013 AACR.

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PDF file, 38K, Flow cytometry of integrin expression in 4T1 carcinoma cells.

Regulatory T (Treg) cells are one of the major immunosuppressive cell types in cancer and a potential target for immunotherapy, but targeting tumor-infiltrating (TI) Treg cells has been challenging. Here, using single-cell RNA sequencing of immune cells from renal clear cell carcinoma (ccRCC) patients, we identify two distinct transcriptional fates for TI Treg cells, Fate-1 and Fate-2. The Fate-1 signature is associated with a poorer prognosis in ccRCC and several other solid cancers. CD177, a cell surface protein normally expressed on neutrophil, is specifically expressed on Fate-1 TI Treg cells in several solid cancer types, but not on other TI or peripheral Treg cells. Mechanistically, blocking CD177 reduces the suppressive activity of Treg cells in vitro, while Treg-specific deletion of Cd177 leads to decreased tumor growth and reduced TI Treg frequency in mice. Our results thus uncover a functional CD177+ TI Treg population that may serve as a target for TI Treg-specific immunotherapy., Regulatory T (Treg) cells are important modulators of the tumor microenvironment. Here the authors perform transcriptome profiling of immune cells from patients with renal clear cell carcinoma to find a Treg signature that correlates with poorer prognosis, with CD177 being implicated as the main mediator for related alterations in Treg activity and tumor outcome.

Syncytiotrophoblast (STB) stress is theorized to drive the pathogenesis of preeclampsia (PE), and Gq-activating hormones implicated in PE have been associated with placenta oxidative damage, mitochondrial dysfunction, and maternal symptomology. As the causes and consequences of STB stress remain largely unknown, we used an in vivo model to determine whether excess STB-specific Gq signaling is sufficient to cause phenotypes of PE and if a mitochondrial-targeted antioxidant (mitoquinone) mitigates these effects. Activation of Gq signaling in STB cells was achieved by crossing dams harboring a cre-dependent Gq-coupled DREADD (hM3Dq) with Gcm1 -Cre +/- sires and injecting clozapine N-oxide (CNO) or saline mid-gestation (GD12.5-14.5) before tissue collection at GD14.5. Gq activation resulted in maternal proteinuria (n=9-11 CNO 43±4 vs saline 28±3mg/day; p=0.01), mild segmental congestion of glomerular capillaries, and elevated circulating IP-10 (p=0.03), MCP-5 (p=0.04), and IL-12p40 (p=0.01). hM3Dq stimulation reduced labyrinth vascularization (n=10 Cre + 19±1%, Cre - 24±1%, saline 23±1%; p+ vs each), spiral artery diameter (n=7-11 Cre + 110±11μm, Cre - 135±9, saline 149±12 μm; p=0.03), placenta mass (n=10-13 Cre + 0.12±0.01, Cre - 0.14±0.01, saline 0.13±0.01g; p+ 0.23±0.03, Cre - 0.30±0.02, saline 0.29±0.02g; p

The superantigen staphylococcal enterotoxin C (SEC) is critical for Staphylococcus aureus infective endocarditis (SAIE) in rabbits. Superantigenicity, its hallmark function, was proposed to be a major underlying mechanism driving SAIE but was not directly tested. With the use of S. aureus MW2 expressing SEC toxoids, we show that superantigenicity does not sufficiently account for vegetation growth, myocardial inflammation, and acute kidney injury in the rabbit model of native valve SAIE. These results highlight the critical contribution of an alternative function of superantigens to SAIE. In support of this, we provide evidence that SEC exerts antiangiogenic effects by inhibiting branching microvessel formation in an ex vivo rabbit aortic ring model and by inhibiting endothelial cell expression of one of the most potent mediators of angiogenesis, VEGF-A. SEC’s ability to interfere with tissue revascularization and remodeling after injury serves as a mechanism to promote SAIE and its life-threatening systemic pathologies.

Intravascular (IV) perfusion of tissue fixative is commonly used in the field of neuroscience as the central nervous system tissues are exquisitely sensitive to handling and fixation artifacts which can affect downstream microscopic analysis. Both 10% neutral-buffered formalin (NBF) and 4% paraformaldehyde (PFA) are used, although IV perfusion with PFA is most commonly referenced. The study objective was to compare the severity of handling and fixation artifacts, semiquantitative scores of inflammatory and neurodegenerative changes, and quantitative immunohistochemistry following terminal IV perfusion of mice with either 10% NBF or 4% PFA in a model of experimental autoimmune encephalitis (EAE). The study included 24 mice; 12 were control animals not immunized and an additional 12 were immunized with PLP139–151subcutaneously, harvested at day 20, and fixed in the same fashion. Equal numbers (4 per group) were perfused with 10% NBF or 4% PFA, and 4 were immersion-fixed in 10% NBF. NBF-perfused mice had less severe dark neuron artifact than PFA-perfused mice ( P < .001). Immersion-fixed animals had significantly higher scores for oligodendrocyte halos, dark neuron artifact, and perivascular clefts than perfusion-fixed animals. Histopathology scores in EAE mice for inflammation, demyelination, and necrosis did not differ among fixation methods. Also, no significant differences in quantitative immunohistochemistry for CD3 and Iba-1 were observed in immunized animals regardless of the method of fixation. These findings indicate that IV perfusion of mice with 10% NBF and 4% PFA are similar and adequate fixation techniques in this model.

Interleukin-1 alpha (IL-1α) is a cytokine involved in the acute phase immune response and its expression is upregulated in a variety of solid tumors including head and neck squamous cell carcinomas (HNSCCs). Tumor expression of IL-1α is associated with increased tumor aggressiveness in HNSCCs, but this has yet to be studied in the context of human papilloma virus (HPV) status. This study is aimed at determining differences in tumor expression and subcellular localization of IL-1α in HPV-positive (HPV+) and HPV-negative (HPV−) HNSCC tumors. Tissue microarrays (TMAs) containing HPV+ (n = 31) and HPV− (n = 47) primary and metastatic HNSCCs were analyzed for IL-1α expression using immunohistochemical (IHC) staining. HPV status was confirmed using p16 IHC staining and RNA in situ hybridization (RNA ISH). Differences in IL-1α protein expression and secretion in HPV+ and HPV− HNSCC cell lines were determined by western blot and ELISA respectively. Associations between tumor IL1A expression and survival outcomes were assessed in HPV+ and HPV− HNSCC patients from publicly available gene expression datasets. Tumor expression of IL-1α was significantly increased in HPV− tumors and cell lines (as detected by IHC and western blot respectively) compared to HPV+ tumors and cell lines. There was no difference in IL-1α release between HPV+ and HPV− cell lines. IL-1α was expressed in both nuclear and cytoplasmic compartments, with predominant expression in the nucleus. Gene expression of IL1A was significantly increased in HPV-tumors/cell lines compared to HPV+ tumors/cell lines. Lastly, increased IL1A gene expression was significantly associated with worse survival in HPV- tumors but not in HPV+ tumors. Overall IL-1α expression particularly in the nucleus may possess more prognostic significance in HPV− tumors rather than HPV+ tumors. This work warrants further investigation into the role of intracellular IL-1α ligand expression in HNSCCs and may have important implications in IL-1 pathway blockade as therapeutic strategy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12105-022-01440-x.

Nicotinamide riboside (NR) is a vitamin B3 precursor of NAD(+) that blunts diabetic and chemotherapy-induced peripheral neuropathy in preclinical models. This study examined whether NR also blunts the loss of intraepidermal nerve fibers (IENF) induced by paclitaxel, which is associated with peripheral neuropathy. The work was conducted in female rats with N-methyl-nitrosourea (MNU)-induced tumors of the mammary gland to increase its translational relevance, and to assess the interaction of NR with paclitaxel and NR’s effect on tumor growth. Once daily oral administration of 200 mg/kg NR p.o. beginning with the first of three i.v. injections of 6.6 mg/kg paclitaxel to tumor-bearing rats significantly decreased paclitaxel-induced hypersensitivity to tactile and cool stimuli, as well as place-escape avoidance behaviors. It also blunted the loss of IENF in tumor-bearing rats, as well as a separate cohort of tumor-naïve rats. Unexpectedly, concomitant administration of NR during paclitaxel treatment further decreased tumor growth; thereafter, tumor growth resumed at the same rate as vehicle-treated controls. Administration of NR also decreased the percentage of Ki67-positive tumor cells in these rats. Once daily administration of NR did not appear to alter tumor growth or the percentage of Ki67-positive tumor cells in rats that were not treated with paclitaxel and followed for three months. These results further support the ability of NR to play a protective role following nerve injury. They also suggest that NR may not only alleviate peripheral neuropathy in patients receiving taxane chemotherapy, but also offer an added benefit by possibly enhancing its tumor-suppressing effects. In tumor-bearing rats, nicotinamide riboside blunts the allodynia and loss of intraepidermal nerve fibers induced by paclitaxel, and unexpectedly enhances paclitaxel’s suppression of tumor growth.

Angiotensin II (ANG II) Agtr1a receptor (AT1A) is expressed in cells of the arcuate nucleus of the hypothalamus that express the leptin receptor ( Lepr) and agouti-related peptide ( Agrp). Agtr1a expression in these cells is required to stimulate resting energy expenditure in response to leptin and high-fat diets (HFDs), but the mechanism activating AT1Asignaling by leptin remains unclear. To probe the role of local paracrine/autocrine ANG II generation and signaling in this mechanism, we bred mice harboring a conditional allele for angiotensinogen ( Agt, encoding AGT) with mice expressing Cre-recombinase via the Lepr or Agrp promoters to cause cell-specific deletions of Agt ( AgtLepr-KOand AgtAgrp-KOmice, respectively). AgtLepr-KOmice were phenotypically normal, arguing against a paracrine/autocrine AGT signaling mechanism for metabolic control. In contrast, AgtAgrp-KOmice exhibited reduced preweaning survival, and surviving adults exhibited altered renal structure and steroid flux, paralleling previous reports of animals with whole body Agt deficiency or Agt disruption in albumin ( Alb)-expressing cells (thought to cause liver-specific disruption). Surprisingly, adult AgtAgrp-KOmice exhibited normal circulating AGT protein and hepatic Agt mRNA expression but reduced Agt mRNA expression in adrenal glands. Reanalysis of RNA-sequencing data sets describing transcriptomes of normal adrenal glands suggests that Agrp and Alb are both expressed in this tissue, and fluorescent reporter gene expression confirms Cre activity in adrenal gland of both Agrp-Cre and Alb-Cre mice. These findings lead to the iconoclastic conclusion that extrahepatic (i.e., adrenal) expression of Agt is critically required for normal renal development and survival.

Aiming to identify immune molecules with a novel function in cancer pathogenesis, we found the cluster of differentiation 177 (CD177), a known neutrophil antigen, to be positively correlated with relapse-free (RFS), metastasis-free (MFS) or overall survival (OS) in breast cancer. Additionally, CD177 expression is correlated with good prognosis in several other solid cancers including prostate, cervical, and lung. Focusing on breast cancer, we found that CD177 is expressed in normal breast epithelial cells and is significantly reduced in invasive cancers. Loss of CD177 leads to hyperproliferative mammary epithelium and contributes to breast cancer pathogenesis. Mechanistically, we found that CD177-deficiency is associated with an increase in β-Catenin signaling. Here we identified CD177 as a novel regulator of mammary epithelial proliferation and breast cancer pathogenesis likely via the modulation of Wnt/β-Catenin signaling pathway, a key signaling pathway involved in multiple cancer types.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in the regulation of biological responses to more planar aromatic hydrocarbons, like TCDD. We previously described the sequence of events following exposure of male rats to a dioxin-like polychlorinated biphenyl (PCB) congener, 3,3’,4,4’,5-pentachlorobiphenyl (PCB126), that binds avidly to the AhR and causes various types of toxicity including metabolic syndrome, fatty liver, and disruption of energy homeostasis. The purpose of this study was, to investigate the role of AhR to mediate those toxic manifestations following sub-acute exposure to PCB126 and to examine possible sex differences in effects. For this goal, we created an AhR knockout (AhR-KO) model using CRISPR/Cas9. Comparison was made to the wild type (WT) male and female Holtzman Sprague Dawley rats. Rats were injected with a single IP dose of corn oil vehicle or 5 µmol/kg PCB126 in corn oil and necropsied after 28 days. PCB126 caused significant weight loss, reduced relative thymus weights, and increased relative liver weights in WT male and female rats, but not in AhR-KO rats. Similarly, significant pathologic changes were visible which included necrosis and regeneration in female rats, micro- and macro-vesicular hepatocellular vacuolation in males, and a paucity of glycogen in livers of both sexes in WT rats only. Hypoglycemia and lower IGF1, and reduced serum non-esterified fatty acids (NEFAs) was found in serum of both sexes of WT rats, low serum cholesterol levels only in the females, and no changes in AhR-KO rats. The expression of genes encoding enzymes related to xenobiotic metabolism (e.g. CYP1A1), gluconeogenesis, glycogenolysis, and fatty acid oxidation were unaffected in the AhR-KO rats following PCB126 exposure as opposed to WT rats where expression was significantly upregulated (PPARα, females only) or downregulated suggesting a disrupted energy homeostasis. Interestingly, Acox2, Hmgcs, G6Pase and Pc were affected in both sexes, the gluconeogenesis and glucose transporter genes Pck1, Glut2, Sds, and Crem only in male WT-PCB rats. These results show the essential role of the AhR in glycogenolysis, gluconeogenesis, and fatty acid oxidation, i.e. in the regulation of energy production and homeostasis, but also demonstrate a significant difference in the effects of PCB126 in males verses females, suggesting higher vulnerability of glucose homeostasis in males and more changes in fatty acid/lipid homeostasis in females. These differences in effects, which may apply to more/all AhR agonists, should be further analyzed to identify health risks to specific groups of highly exposed human populations.

T cells in systemic lupus erythematosus (SLE) exhibit mitochondrial abnormalities including elevated oxidative stress. Because excess iron can promote these phenotypes, we tested iron regulation of SLE T cells. A CRISPR screen identified Transferrin Receptor (CD71) as important for Th1 cells but detrimental for induced regulatory T cells (iTreg). Activated T cells induce CD71 to increase iron uptake, but this was exaggerated in T cells from SLE-prone mice which accumulated iron. Treatment of T cells from SLE-prone mice with CD71 blocking antibody reduced intracellular iron and mTORC1 signaling and restored mitochondrial physiology. While Th1 cells were inhibited, CD71 blockade enhanced iTreg. In vivo this treatment reduced pathology and increased IL-10 in SLE-prone mice. Importantly, disease severity correlated with CD71 expression on SLE patient T cells and blocking CD71 enhanced IL-10 secretion. Excess T cell iron uptake thus contributes to T cell dysfunction and can be targeted to correct SLE-associated pathology.

Background Dogs are the primary reservoir for human visceral leishmaniasis due to Leishmania infantum. Phlebotomine sand flies maintain zoonotic transmission of parasites between dogs and humans. A subset of dogs is infected transplacentally during gestation, but at what stage of the clinical spectrum vertically infected dogs contribute to the infected sand fly pool is unknown. Methodology/Principal findings We examined infectiousness of dogs vertically infected with L. infantum from multiple clinical states to the vector Lutzomyia longipalpis using xenodiagnosis and found that vertically infected dogs were infectious to sand flies at differing rates. Dogs with mild to moderate disease showed significantly higher transmission to the vector than dogs with subclinical or severe disease. We documented a substantial parasite burden in the skin of vertically infected dogs by RT-qPCR, despite these dogs not having received intradermal parasites via sand flies. There was a highly significant correlation between skin parasite burden at the feeding site and sand fly parasite uptake. This suggests dogs with high skin parasite burden contribute the most to the infected sand fly pool. Although skin parasite load and parasitemia correlated with one another, the average parasite number detected in skin was significantly higher compared to blood in matched subjects. Thus, dermal resident parasites were infectious to sand flies from dogs without detectable parasitemia. Conclusions/Significance Together, our data implicate skin parasite burden and earlier clinical status as stronger indicators of outward transmission potential than blood parasite burden. Our studies of a population of dogs without vector transmission highlights the need to consider canine vertical transmission in surveillance and prevention strategies., Author summary Sand flies transmit Leishmania parasites between infected dogs and humans leading to the life-threatening tropical disease Visceral Leishmaniasis (VL). Identifying which dogs transmit parasites well to sand flies is important to curb disease spread. The offspring of both dogs and humans can also be infected vertically while in utero. Despite this, the infectiousness of dogs that receive parasites in utero to sand flies has not been thoroughly investigated. Thus, we allowed sand flies to feed on a group of vertically infected dogs at varying stages of VL disease severity and measured sand fly parasite uptake. We found vertically infected dogs were readily able to transmit parasites to the sand flies. Dogs that were most infectious had mild to moderate clinical disease and relatively high levels of parasite infection in their blood and skin. However, the level of skin infection was significantly higher than that observed in the blood, and the skin parasite load had the strongest correlation with sand fly parasite uptake. This implicates the skin may be an underappreciated driver of canine infectiousness to the sand fly vector. In addition, this work highlights that vertically infected dogs are very important parts of the transmission cycle and must be considered in all public health efforts addressing VL.

Isoflavone-metabolizing gut bacteria ameliorate CNS autoimmunity in mice., The gut microbiota is a potential environmental factor that influences the development of multiple sclerosis (MS). We and others have demonstrated that patients with MS and healthy individuals have distinct gut microbiomes. However, the pathogenic relevance of these differences remains unclear. Previously, we showed that bacteria that metabolize isoflavones are less abundant in patients with MS, suggesting that isoflavone-metabolizing bacteria might provide protection against MS. Here, using a mouse model of MS, we report that an isoflavone diet provides protection against disease, which is dependent on the presence of isoflavone-metabolizing bacteria and their metabolite equol. Notably, the composition of the gut microbiome in mice fed an isoflavone diet exhibited parallels to healthy human donors, whereas the composition in those fed an isoflavone-free diet exhibited parallels to patients with MS. Collectively, our study provides evidence that dietary-induced gut microbial changes alleviate disease severity and may contribute to MS pathogenesis.

By combining next generation whole exome sequencing and induced pluripotent stem cell (iPSC) technology we found that an Alu repeat inserted in exon 9 of the MAK gene results in a loss of normal MAK transcript and development of human autosomal recessive retinitis pigmentosa (RP). Although a relatively rare cause of disease in the general population, the MAK variant is enriched in individuals of Jewish ancestry. In this population, 1 in 55 individuals are carriers and one third of all cases of recessive RP is caused by this gene. The purpose of this study was to determine if a viral gene augmentation strategy could be used to safely restore functional MAK protein as a step toward a treatment for early stage MAK-associated RP. Patient iPSC-derived photoreceptor precursor cells were generated and transduced with viral vectors containing the MAK transcript. One week after transduction, transcript and protein could be detected via rt-PCR and western blotting respectively. Using patient-derived fibroblast cells and mak knockdown zebra fish we demonstrate that over-expression of the retinal MAK transgene restored the cells ability to regulate primary cilia length. In addition, the visual defect in mak knockdown zebrafish was mitigated via treatment with the retinal MAK transgene. There was no evidence of local or systemic toxicity at 1-month or 3-months following subretinal delivery of clinical grade vector into wild type rats. The findings reported here will help pave the way for initiation of a phase 1 clinical trial for the treatment of patients with MAK-associated RP.

Melanocortin 1 receptor (MC1R) is under investigation as a target for drug delivery for metastatic melanoma therapy and imaging. The purpose of this study was to determine the potential of using BRAF inhibitors (BRAF(i)) and histone deacetylase inhibitors (HDAC(i)) to enhance the delivery of MC1R-targeted radiolabeled peptide ([(212)Pb]DOTA-MC1L) by pharmacologically upregulating the MC1R expression in metastatic melanoma cells and tumors. MC1R expression was analyzed in de-identified melanoma biopsies by immunohistochemical staining. Upregulation of MC1R expression was determined in BRAF(V600E) cells (A2058) and BRAF wild-type melanoma cells (MEWO) by quantitative real-time polymerase chain reaction, flow cytometry, and receptor-ligand binding assays. The role of microphthalmia-associated transcription factor (MITF) in the upregulation of MC1R was also examined in A2058 and MEWO cells. The effectiveness of [(212)Pb]DOTA-MC1L α-particle radiotherapy in combination with BRAF(i) and/or HDAC(i) was determined in athymic nu/nu mice bearing A2058 and MEWO human melanoma xenografts. High expression of MC1R was observed in situ in clinical melanoma biopsies. BRAF(i) and HDAC(i) significantly increased the MC1R expression (up to 10-fold in mRNA and 4-fold in protein levels) via MITF-dependent pathways, and this increase led to enhanced ligand binding on the cell surface. Inhibition of MITF expression antagonized the upregulation of MC1R in both BRAF(V600E) and BRAF(WT) cells. Combining [(212)Pb]DOTA-MC1L with BRAF(i) and/or HDAC(i) improved the tumor response by increasing the delivery of (212)Pb α-particle emissions to melanoma tumors via augmented MC1R expression. These data suggest that FDA-approved HDAC(i) and BRAF(i) could improve the effectiveness of MC1R-targeted therapies by enhancing drug delivery via upregulated MC1R.

Background The epidermal growth factor receptor (EGFR) monoclonal IgG1 antibody cetuximab is approved for first-line treatment of recurrent and metastatic (R/M) HNSCC as a part of the standard of care EXTREME regimen (platinum/5-fluorouracil/cetuximab). This regimen has relatively high response and disease control rates but is generally not curative and many patients will experience recurrent disease and/or metastasis. Therefore, there is a great need to identify predictive biomarkers for recurrence and disease progression in cetuximab-treated HNSCC patients to facilitate patient management and allow for treatment modification. The goal of this work is to assess the potential of activating interleukin-1 (IL-1) ligands (IL-1 alpha [IL-1α], IL-1 beta [IL-1β]) as predictive biomarkers of survival outcomes in HNSCC patients treated with cetuximab-based chemotherapy. Methods Baseline gene, serum and tumor expression of interleukin-1 (IL-1) ligands were analyzed from The Cancer Genome Atlas (TCGA) database or clinical trials of cetuximab-based therapies and interrogated for associations with clinical outcome data. Results High tumor gene expression of IL-1β was associated with a more favorable overall survival in cetuximab-treated HNSCC patients but not in non-cetuximab-treated patients. In HNSCC patients treated with cetuximab-based chemotherapy, higher gene and circulating levels of IL-1α and IL-1β were correlated with a more favorable progression free survival compared to patients with low or undetectable levels of IL-1 ligands. Conclusions These findings suggest that IL-1 ligands may function as predictive biomarkers for tumor response to cetuximab-based chemotherapy in HNSCC patients and warrants further investigation and validation in larger clinical studies.

Background Insulin secretion is insufficient in cystic fibrosis (CF), even before diabetes is present, though the mechanisms involved remain unclear. Acyl-ghrelin (AG) can diminish insulin secretion and is elevated in humans with CF. Methods We tested the hypothesis that elevated AG contributes to reduced insulin secretion and hyperglycemia in CF ferrets. Results Fasting AG was elevated in CF versus non-CF ferrets. Similar to its effects in other species, AG administration in non-CF ferrets acutely reduced insulin, increased growth hormone, and induced hyperglycemia. During oral glucose tolerance testing, non-CF ferrets had responsive insulin, glucagon like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) levels and maintained normal glucose levels, whereas CF ferrets had insufficient responses and became hyperglycemic. Interestingly in wild-type ferrets, the acyl-ghrelin receptor antagonist [D-Lys3]-GHRP-6 impaired glucose tolerance, and abolished insulin, GLP-1, and GIP responses during glucose tolerance testing. By contrast, in CF ferrets [D-Lys3]-GHRP-6 improved glucose tolerance, enhanced the insulin-to-glucose ratio, but did not impact the already low GLP-1 and GIP levels. Conclusions These results suggest a mechanism by which elevated AG contributes to CF hyperglycemia through inhibition of insulin secretion, an effect magnified by low GLP-1 and GIP. Interventions that lower ghrelin, ghrelin action, and/or raise GLP-1 or GIP might improve glycemia in CF.

Obesity is a risk factor for breast cancer and also predicts poor clinical outcomes regardless of menopausal status. Contributing to the poor clinical outcomes is the suboptimal efficacy of standard therapies due to dose limiting toxicities and obesity-related complications, highlighting the need to develop novel therapeutic approaches for treating obese patients. We recently found that obesity leads to an increase in tumor-infiltrating macrophages with activated NLRC4 inflammasome and increased interleukin (IL)−1β production. IL-1β, in turn, leads to increased angiogenesis and cancer progression. Using Next Generation RNA sequencing, we identified an NLRC4/IL-1β-dependent upregulation of angiopoietin-like 4 (ANGPTL4), a known angiogenic factor in cancer, in tumors from obese mice. ANGPTL4-deficiency by genetic knockout or treatment with a neutralizing antibody led to a significant reduction in obesity-induced angiogenesis and tumor growth. At a mechanistic level, ANGPTL4 expression is induced by IL-1β from primary adipocytes in a manner dependent on NF-κB- and MAP kinase-activation, which is further enhanced by hypoxia. This report shows that adipocyte-derived ANGPTL4 drives disease progression under obese conditions and is a potential therapeutic target for treating obese breast cancer patients. Ministry of Education (MOE) Accepted version RK: NIH T32 AI007260; WZ: NIH R01 grants CA200673 and CA203834, Oberley Award (National Cancer Institute Award P30CA086862) from Holden Comprehensive Cancer Center at the University of Iowa; NB: NIH F30 CA206255; AK: Mark Stinski Developmental Grant from the Department of Microbiology, University of Iowa; BD: NIH R01HL130146; NST: grant from Ministry of Education, Singapore (MOE2014-T2-1-012); FSS: NIH R01AI118719.

Background /Objectives: The pathogenesis of hyperglycemia during acute pancreatitis (AP) remains unknown due to inaccessibility of human tissues and lack of animal models. We aimed to develop an animal model to study the mechanisms of hyperglycemia and impaired glucose tolerance in AP. Methods We injected ferrets with intraperitoneal cerulein (50 μg/kg, 9 hourly injections) or saline. Blood samples were collected for glucose (0, 4, 8, 12, 24h); TNF-α, IL-6 (6h); amylase, lipase, insulin, glucagon, pancreatic polypeptide (PP), glucagon-like peptide-1 (GLP-1), and gastric inhibitory polypeptide (GIP) (24h). Animals underwent oral glucose tolerance test (OGTT), mixed meal tolerance test (MMTT) at 24h or 3 months, followed by harvesting pancreas for histopathology and immunostaining. Results Cerulein-injected ferrets exhibited mild pancreatic edema, neutrophil infiltration, and elevations in serum amylase, lipase, TNF-α, IL-6, consistent with AP. Plasma glucose was significantly higher in ferrets with AP at all time points. Plasma glucagon, GLP-1 and PP were significantly higher in cerulein-injected animals, while plasma insulin was significantly lower compared to controls. OGTT and MMTT showed abnormal glycemic responses with higher area under the curve. The hypoglycemic response to insulin injection was completely lost, suggestive of insulin resistance. OGTT showed low plasma insulin; MMTT confirmed low insulin and GIP; abnormal OGTT and MMTT responses returned to normal 3 months after cerulein injection. Conclusions Acute cerulein injection causes mild acute pancreatitis in ferrets and hyperglycemia related to transient islet cell dysfunction and insulin resistance. The ferret cerulein model may contribute to the understanding of hyperglycemia in acute pancreatitis.

The Toll-like receptor 8 (TLR8) agonist VTX-2337 (motolimod) is an anti-cancer immunotherapeutic agent that is believed to augment natural killer (NK) and dendritic cell (DC) activity. The goal of this work is to examine the role of TLR8 expression/activity in head and neck squamous cell carcinoma (HNSCC) to facilitate the prediction of responders to VTX-2337-based therapy. The prognostic role of TLR8 expression in HNSCC patients was assessed by TCGA and tissue microarray analyses. The anti-tumor effect of VTX-2337 was determined in SCCVII/C3H, mEERL/C57Bl/6 and TUBO-human EGFR/BALB/c syngeneic mouse models. The effect of combined VTX-2337 and cetuximab treatment on tumor growth, survival and immune cell recruitment was assessed. TLR8 expression was associated with CD8+ T cell infiltration and favorable survival outcomes. VTX-2337 delayed tumor growth in all 3 syngeneic mouse models and significantly increased the survival of cetuximab-treated mice. The anti-tumor effects of VTX-2337+ cetuximab were accompanied by increased splenic lymphoid DCs and IFNγ+ CD4+ and tumor-specific CD8+ T cells. Depletion of CD4+ T cells, CD8+ T cells and NK cells were all able to abolish the anti-tumor effect of VTX-2337+ cetuximab. Altogether, VTX-2337 remains promising as an adjuvant for cetuximab-based therapy however patients with high TLR8 expression may be more likely to derive benefit from this drug combination compared to patients with low TLR8 expression.

Evaluation of sepsis-induced immunoparalysis has highlighted how decreased lymphocyte number/function contribute to worsened infection/cancer. Yet, an interesting contrast exists with autoimmune disease development, wherein diminishing pathogenic effectors may benefit the post-septic host. Within this framework, the impact of cecal ligation and puncture (CLP)-induced sepsis on the development of experimental autoimmune encephalomyelitis (EAE) was explored. Notably, CLP mice have delayed onset and reduced disease severity, relative to sham mice. Reduction in disease severity was associated with reduced number, but not function, of autoantigen (MOG)-specific pathogenic CD4 T cells in the CNS during disease and draining lymph node during priming. Numerical deficits of CD4 T cell effectors are associated with the loss of MOG-specific naive precursors. Critically, transfer of MOG-TCR transgenic (2D2) CD4 T cells after, but not before, CLP led to EAE disease equivalent to sham mice. Thus, broad impairment of antigenic responses, including autoantigens, is a hallmark of sepsis-induced immunoparalysis., eLife digest Sepsis is a life-threatening condition that can happen when the immune system overreacts to an infection and begins to damage tissues and organs in the body. It causes an extreme immune reaction called a cytokine storm, where the body releases uncontrolled levels of cytokines, proteins that are involved in coordinating the body’s response to infections. This in turn activates more immune cells, resulting in hyperinflammation. People who survive sepsis may have long-lasing impairments in their immune system that may leave them more vulnerable to infections or cancer. But scientists do not know exactly what causes these lasting immune problems or how to treat them. The fact that people are susceptible to cancer and infection after sepsis may offer a clue. It may suggest that the immune system is not able to attack bacteria or cancer cells. One way to explore this clue would be to test the effects of sepsis on autoimmune diseases, which cause the immune system to attack the body’s own cells. For example, in the autoimmune disease multiple sclerosis, the immune system attacks and destroys cells in the nervous system. If autoimmune disease is reduced after sepsis, it would suggest the cell-destroying abilities of the immune system are lessened. Using this approach, Jensen, Jensen et al. show that sepsis reduces the number of certain immune cells, called CD4 T cells, which are are responsible for an autoimmune attack of the central nervous system. In the experiments, mice that survived sepsis were evaluated for their ability to develop a multiple sclerosis-like disease. Mice that survived sepsis developed less severe or no autoimmune disease. After sepsis, these animals also had fewer CD4 T cells. However, when these immune cells were reinstated, the autoimmune disease emerged. The experiments help explain some of the immune system changes that occur after sepsis. Jensen, Jensen et al. suggest that rather than being completely detrimental, these changes may help to block harmful autoimmune responses. The experiments may also hint at new ways to combat autoimmune diseases by trying to replicate some of the immune-suppressing effects of sepsis. Studying the effect of sepsis on other autoimmune diseases in mice might provide more clues.

Immunohistochemistry (IHC) is a fundamental molecular technique that provides information on protein expression in the context of spatial localization and tissue morphology. IHC is used in all facets of pathology from identifying infectious agents or characterizing tumors in diagnostics, to characterizing cellular and molecular processes in investigative and experimental studies. Confidence in an IHC assay is primarily driven by the degree to which it is validated. There are many approaches to validate an IHC assay’s specificity including bioinformatics approaches using published protein sequences, careful design of positive and negative tissue controls, use of cell pellets with known target protein expression, corroboration of IHC findings with western blots and other analytical methods, and replacement of the primary antibody with an appropriate negative control reagent. Each approach has inherent strengths and weaknesses, and the thoughtful use of these approaches provides cumulative evidence, or a weight of evidence, to support the IHC assay’s specificity and build confidence in a study’s conclusions. Although it is difficult to be 100% confident in the specificity of any IHC assay, it is important to consider how validation approaches provide evidence to support or to question the specificity of labeling, and how that evidence affects the overall interpretation of a study’s results. In this review, we discuss different approaches for IHC antibody validation, with an emphasis on the characterization of antibody specificity in investigative studies. While this review is not prescriptive, it is hoped that it will be thought provoking when considering the interpretation of IHC results.

Alpha-1 antitrypsin deficiency (AATD) is the most common genetic cause and risk factor for chronic obstructive pulmonary disease, but the field lacks a large-animal model that allows for longitudinal assessment of pulmonary function. We hypothesized that ferrets would model human AATD-related lung and hepatic disease. AAT-knockout (AAT-KO) and PiZZ (E342K, the most common mutation in humans) ferrets were generated and compared with matched controls using custom-designed flexiVent modules to perform pulmonary function tests, quantitative computed tomography (QCT), bronchoalveolar lavage (BAL) proteomics, and alveolar morphometry. Complete loss of AAT (AAT-KO) led to increased pulmonary compliance and expiratory airflow limitation, consistent with obstructive lung disease. QCT and morphometry confirmed emphysema and airspace enlargement, respectively. Pathway analysis of BAL proteomics data revealed inflammatory lung disease and impaired cellular migration. The PiZ mutation resulted in altered AAT protein folding in the liver, hepatic injury, and reduced plasma concentrations of AAT, and PiZZ ferrets developed obstructive lung disease. In summary, AAT-KO and PiZZ ferrets model the progressive obstructive pulmonary disease seen in AAT-deficient patients and may serve as a platform for preclinical testing of therapeutics including gene therapy.

Cascade-specific termination of G protein signaling is catalyzed by the RGS (regulator of G protein signaling) family members, including RGS2 . Angiotensin, vasopressin, and endothelin are implicated in preeclampsia, and RGS2 is known to inhibit G protein cascades activated by these hormones. Mutations in RGS2 are associated with human hypertension and increased risk of developing preeclampsia and its sequelae. RGS family members are known to influence maternal vascular function, but the role of RGS2 within the placenta has not been explored. Here, we hypothesized that reduced expression of RGS2 within the placenta represents a risk factor for the development of preeclampsia. Although cAMP/CREB signaling was enriched in placentas from human pregnancies affected by preeclampsia compared with clinically matched controls and RGS2 is known to be a CREB-responsive gene, RGS2 mRNA was reduced in placentas from pregnancies affected by preeclampsia. Experimentally reducing Rgs2 expression within the feto-placental unit was sufficient to induce preeclampsia-like phenotypes in pregnant wild-type C57BL/6J mice. Stimulation of RGS2 transcription within immortalized human HTR8/SVneo trophoblasts by cAMP/CREB signaling was discovered to be dependent on the activity of histone deacetylase activity, and more specifically, HDAC9 (histone deacetylase-9), and HDAC9 expression was reduced in placentas from human pregnancies affected by preeclampsia. We conclude that reduced expression of RGS2 within the placenta may mechanistically contribute to preeclampsia. More generally, this work identifies RGS2 as an HDAC9 -dependent CREB-responsive gene, which may contribute to reduced RGS2 expression in placenta during preeclampsia.

The superantigen staphylococcal enterotoxin C (SEC) is critical for Staphylococcus aureus infective endocarditis (SAIE) in rabbits. Superantigenicity, its hallmark function, was proposed to be a major underlying mechanism driving SAIE but was not directly tested. With the use of S. aureus MW2 expressing SEC toxoids, we show that superantigenicity does not sufficiently account for vegetation growth, myocardial inflammation, and acute kidney injury in the rabbit model of native valve SAIE. These results highlight the critical contribution of an alternative function of superantigens to SAIE. In support of this, we provide evidence that SEC exerts anti-angiogenic effects by inhibiting branching microvessel formation in an ex vivo rabbit aortic ring model and by inhibiting endothelial cell expression of one of the most potent mediators of angiogenesis, VEGF-A. SEC’s ability to interfere with tissue re-vascularization and remodeling after injury serves as a mechanism to promote SAIE and its life-threatening systemic pathologies.