The IFIH1-A946T risk variant promotes diabetes in a sex-dependent manner.

Type 1 diabetes (T1D) is an autoimmune disease in which pancreatic islet ß-cells are attacked by the immune system, resulting in insulin deficiency and hyperglycemia. One of the top non-synonymous single-nucleotide polymorphisms (SNP) associated with T1D is in the interferon-induced helicase C domain-containing protein 1 (IFIH1), which encodes an anti-viral cytosolic RNA sensor. This SNP results in an alanine to threonine substitution at amino acid 946 (IFIH1A946T) and confers an increased risk for several autoimmune diseases, including T1D. We hypothesized that the IFIH1A946T risk variant, (IFIH1R) would promote T1D pathogenesis by stimulating type I interferon (IFN I) signaling leading to immune cell alterations. To test this, we developed Ifih1R knock-in mice on the non-obese diabetic (NOD) mouse background, a spontaneous T1D model. Our results revealed a modest increase in diabetes incidence and insulitis in Ifih1R compared to non-risk Ifih1 (Ifih1NR) mice and a significant acceleration of diabetes onset in Ifih1R females. Ifih1R mice exhibited a significantly enhanced interferon stimulated gene (ISG) signature compared to Ifih1NR, indicative of increased IFN I signaling. Ifih1R mice exhibited an increased frequency of plasma cells as well as tissue-dependent changes in the frequency and activation of CD8+ T cells. Our results indicate that IFIH1R may contribute to T1D pathogenesis by altering the frequency and activation of immune cells. These findings advance our knowledge on the connection between the rs1990760 variant and T1D. Further, these data are the first to demonstrate effects of Ifih1R in NOD mice, which will be important to consider for the development of therapeutics for T1D.

The IFIH1-A946T risk variant promotes diabetes in a sex-dependent manner.

Type 1 diabetes (T1D) is an autoimmune disease in which pancreatic islet ß-cells are attacked by the immune system, resulting in insulin deficiency and hyperglycemia. One of the top non-synonymous single-nucleotide polymorphisms (SNP) associated with T1D is in the interferon-induced helicase C domain-containing protein 1 ( IFIH1 ), which encodes an anti-viral cytosolic RNA sensor. This SNP results in an alanine to threonine substitution at amino acid 946 (IFIH1 A946T ) and confers an increased risk for several autoimmune diseases, including T1D. We hypothesized that the IFIH1 A946T risk variant, ( IFIH1 R ) would promote T1D pathogenesis by stimulating type I interferon (IFN I) signaling leading to immune cell alterations. To test this, we developed Ifih1 R knock-in mice on the non-obese diabetic (NOD) mouse background, a spontaneous T1D model. Our results revealed a modest increase in diabetes incidence and insulitis in Ifih1 R compared to non-risk Ifih1 ( Ifih1 NR ) mice and a significant acceleration of diabetes onset in Ifih1 R females. Ifih1 R mice exhibited a significantly enhanced interferon stimulated gene (ISG) signature compared to Ifih1 NR , indicative of increased IFN I signaling. Ifih1 R mice exhibited an increased frequency of plasma cells as well as tissue-dependent changes in the frequency and activation of CD8 + T cells. Our results indicate that IFIH1 R may contribute to T1D pathogenesis by altering the frequency and activation of immune cells. These findings advance our knowledge on the connection between the rs1990760 variant and T1D. Further, these data are the first to demonstrate effects of Ifih1 R in NOD mice, which will be important to consider for the development of therapeutics for T1D.

Corrigendum: IgG2 rules: N-acetyl-ß-D-glucosamine-specific IgG2 and Th17/Th1 cooperation may promote the pathogenesis of acute rheumatic heart disease and be a biomarker of the autoimmune sequelae of Streptococcus pyogenes.

[This corrects the article DOI: 10.3389/fcvm.2022.919700.].

Effects of SPA4 peptide on lipopolysaccharide-disrupted lung epithelial barrier, injury, and function in a human cell system and mouse model of lung injury.

Disrupted epithelial barrier, fluid accumulation, inflammation, and compromised physiology are hallmarks of lung injury. Here we investigated the structural stability of the Toll-like receptor-4 (TLR4)-interacting SPA4 peptide, its effect on Pseudomonas aeruginosa lipopolysaccharide (LPS)-disrupted epithelial barrier in a human cell system, and lung injury markers in a mouse model of LPS-induced lung inflammation. The structural properties of SPA4 peptide were investigated using circular dichroism and UV-VIS spectroscopy. The transepithelial electrical resistance (TEER), an indicator of barrier function, was measured after the cells were challenged with 1 µg/ml LPS and treated with 10 or 100 µM SPA4 peptide. The expression and localization of tight junction proteins were studied by immunoblotting and immunocytochemistry, respectively. Mice were intratracheally challenged with 5 µg LPS per g body weight and treated with 50 µg SPA4 peptide. The lung wet/dry weight ratios or edema, surfactant protein-D (SP-D) levels in serum, lung function, tissue injury, body weights, and temperature, and survival were determined as study parameters. The spectroscopy results demonstrated that the structure was maintained among different batches of SPA4 peptide throughout the study. Treatment with 100 µM SPA4 peptide restored the LPS-disrupted epithelial barrier, which correlated with the localization pattern of Zonula Occludens (ZO)-1 and occludin proteins. Correspondingly, SPA4 peptide treatment helped suppress the lung edema and levels of serum SP-D, improved some of the lung function parameters, and reduced the mortality risk against LPS challenge. Our results suggest that the anti-inflammatory activity of the SPA4 peptide facilitates the resolution of lung pathology.

IgG2 rules: N-acetyl-ß-D-glucosamine-specific IgG2 and Th17/Th1 cooperation may promote the pathogenesis of acute rheumatic heart disease and be a biomarker of the autoimmune sequelae of Streptococcus pyogenes.

Antecedent group A streptococcal pharyngitis is a well-established cause of acute rheumatic fever (ARF) where rheumatic valvular heart disease (RHD) and Sydenham chorea (SC) are major manifestations. In ARF, crossreactive antibodies and T cells respond to streptococcal antigens, group A carbohydrate, N-acetyl-ß-D-glucosamine (GlcNAc), and M protein, respectively, and through molecular mimicry target heart and brain tissues. In this translational human study, we further address our hypothesis regarding specific pathogenic humoral and cellular immune mechanisms leading to streptococcal sequelae in a small pilot study. The aims of the study were to (1) better understand specific mechanisms of pathogenesis in ARF, (2) identify a potential early biomarker of ARF, (3) determine immunoglobulin G (IgG) subclasses directed against GlcNAc, the immunodominant epitope of the group A carbohydrate, by reaction of ARF serum IgG with GlcNAc, M protein, and human neuronal cells (SK-N-SH), and (4) determine IgG subclasses deposited on heart tissues from RHD. In 10 pediatric patients with RHD and 6 pediatric patients with SC, the serum IgG2 subclass reacted significantly with GlcNAc, and distinguished ARF from 7 pediatric patients with uncomplicated pharyngitis. Three pediatric patients who demonstrated only polymigrating arthritis, a major manifestation of ARF and part of the Jones criteria for diagnosis, lacked the elevated IgG2 subclass GlcNAc-specific reactivity. In SC, the GlcNAc-specific IgG2 subclass in cerebrospinal fluid (CSF) selectively targeted human neuronal cells as well as GlcNAc in the ELISA. In rheumatic carditis, the IgG2 subclass preferentially and strongly deposited in valve tissues (n = 4) despite elevated concentrations of IgG1 and IgG3 in RHD sera as detected by ELISA to group A streptococcal M protein. Although our human study of ARF includes a very small limited sample set, our novel research findings suggest a strong IgG2 autoantibody response against GlcNAc in RHD and SC, which targeted heart valves and neuronal cells. Cardiac IgG2 deposition was identified with an associated IL-17A/IFN-γ cooperative signature in RHD tissue which displayed both IgG2 deposition and cellular infiltrates demonstrating these cytokines simultaneously. GlcNAc-specific IgG2 may be an important autoantibody in initial stages of the pathogenesis of group A streptococcal sequelae, and future studies will determine if it can serve as a biomarker for risk of RHD and SC or early diagnosis of ARF.

Lung and general health effects of Toll-like receptor-4 (TLR4)-interacting SPA4 peptide.

BACKGROUND: A surfactant protein-A-derived peptide, which we call SPA4 peptide (amino acids: GDFRYSDGTPVNYTNWYRGE), alleviates lung infection and inflammation. This study investigated the effects of intratracheally administered SPA4 peptide on systemic, lung, and health parameters in an outbred mouse strain, and in an intratracheal lipopolysaccharide (LPS) challenge model. METHODS: The outbred CD-1 mice were intratracheally administered with incremental doses of SPA4 peptide (0.625-10 µg/g body weight) once every 24 h, for 3 days. Mice left untreated and those treated with vehicle were included as controls. Mice were euthanized after 24 h of last administration of SPA4 peptide. In order to assess the biological activity of SPA4 peptide, C57BL6 mice were intratracheally challenged with 5 µg LPS/g body weight and treated with 50 µg SPA4 peptide via intratracheal route 1 h post LPS-challenge. Mice were euthanized after 4 h of LPS challenge. Signs of sickness and body weights were regularly monitored. At the time of necropsy, blood and major organs were harvested. Blood gas and electrolytes, serum biochemical profiles and SPA4 peptide-specific immunoglobulin G (IgG) antibody levels, and common lung injury markers (levels of total protein, albumin, and lactate, lactate dehydrogenase activity, and lung wet/dry weight ratios) were determined. Lung, liver, spleen, kidney, heart, and intestine were examined histologically. Differences in measured parameters were analyzed among study groups by analysis of variance test. RESULTS: The results demonstrated no signs of sickness or changes in body weight over 3 days of treatment with various doses of SPA4 peptide. It did not induce any major toxicity or IgG antibody response to SPA4 peptide. The SPA4 peptide treatment also did not affect blood gas, electrolytes, or serum biochemistry. There was no evidence of injury to the tissues and organs. However, the SPA4 peptide suppressed the LPS-induced lung inflammation. CONCLUSIONS: These findings provide an initial toxicity profile of SPA4 peptide. Intratracheal administration of escalating doses of SPA4 peptide does not induce any significant toxicity at tissue and organ levels. However, treatment with a dose of 50 µg SPA4 peptide, comparable to 2.5 µg/g body weight, alleviates LPS-induced lung inflammation.

Vaginal Suppositories Containing SHetA2 to Treat Cervical Dysplasia: Pharmacokinetics of Daily Doses and Preliminary Safety Profile.

SHetA2 is a new drug with potential to treat cervical dysplasia, but only 0.02% of the dose is absorbed into the cervix after oral administration. By contrast, 23.9% of the dose is absorbed into the cervix after vaginal administration. This study determines the pharmacokinetic and pharmacodynamic parameters after daily vaginal doses of SHetA2 in suppositories and assesses its safety. Daily dosed mice maintained therapeutic concentrations of SHetA2 in the cervix for 65 h. The steady-state area under the curve concentration versus time (AUCcervix) after the last dose was similar to that after a single dose indicating that there was no drug accumulation in the cervix. By contrast, the maximum drug concentration (Cmax-cervix) was smaller in the daily dosed group (52.19 µg/g) than after a single dose (121.84 µg/g), whereas the half-life (t1/2-cervix) was also shorter in the daily dosed group (9.94 h) than after a single dose (23.32 h). Notably, daily vaginal doses of SHetA2 reduced the levels of cyclin D1 (the pharmacodynamic endpoint) to a larger extent (∼45%) than after the administration of a single dose (∼26%). No adverse effects were observed in the mice for the duration of the study; thus, daily vaginal doses of SHetA2 appear to be safe.

NZO/HlLtJ as a novel model for the studies on the role of metabolic syndrome in acute radiation toxicity.

Purpose: Growing rates of metabolic syndrome and associated obesity warrant the development of appropriate animal models for better understanding of how those conditions may affect sensitivity to IR exposure.Materials and methods: We subjected male NZO/HlLtJ mice, a strain prone to spontaneous obesity and diabetes, to 0, 5.5, 6.37, 7.4 or 8.5 Gy (137Cs) of total body irradiation (TBI). Mice were monitored for 30 days, after which proximal jejunum and colon tissues were collected for further histological and molecular analysis.Results: Obese NZO/HlLtJ male mice are characterized by their lower sensitivity to IR at doses of 6.37 Gy and under, compared to other strains. Further escalation of the dose, however, results in a steep survival curve, reaching LD100/30 values at a dose of 8.5 Gy. Alterations in the expression of various tight junction-related proteins coupled with activation of inflammatory responses and cell death were the main contributors to the gastrointestinal syndrome.Conclusions: We demonstrate that metabolic syndrome with exhibited hyperglycemia but without alterations to the microvasculature is not a pre-requisite of the increased sensitivity to TBI at high doses. Our studies indicate the potential of NZO/HlLtJ mice for the studies on the role of metabolic syndrome in acute radiation toxicity.

TLR4-interacting SPA4 peptide improves host defense and alleviates tissue injury in a mouse model of Pseudomonas aeruginosa lung infection.

Interaction between surfactant protein-A (SP-A) and toll-like receptor (TLR)4 plays a critical role in host defense. In this work, we studied the host defense function of SPA4 peptide (amino acids GDFRYSDGTPVNYTNWYRGE), derived from the TLR4-interacting region of SP-A, against Pseudomonas aeruginosa. We determined the binding of SPA4 peptide to live bacteria, and its direct antibacterial activity against P. aeruginosa. Pro-phagocytic and anti-inflammatory effects were investigated in JAWS II dendritic cells and primary alveolar macrophages. The biological relevance of SPA4 peptide was evaluated in a mouse model of acute lung infection induced by intratracheal challenge with P. aeruginosa. Our results demonstrate that the SPA4 peptide does not interact with or kill P. aeruginosa when cultured outside the host. The SPA4 peptide treatment induces the uptake and localization of bacteria in the phagolysosomes of immune cells. At the same time, the secreted amounts of TNF-α are significantly reduced in cell-free supernatants of SPA4 peptide-treated cells. In cells overexpressing TLR4, the TLR4-induced phagocytic response is maintained, but the levels of TLR4-stimulated TNF-α are reduced. Furthermore, our results demonstrate that the therapeutic administration of SPA4 peptide reduces bacterial burden, inflammatory cytokines and chemokines, intracellular signaling, and lactate levels, and alleviates lung edema and tissue damage in P. aeruginosa-infected mice. Together, our results suggest that the treatment with SPA4 peptide can help control the bacterial burden, inflammation, and tissue injury in a P. aeruginosa lung infection model.

Histopathology of Bordetella pertussis in the Baboon Model.

Pertussis is a severe respiratory disease caused by Bordetella pertussis The classic symptoms of pertussis include paroxysmal coughing with an inspiratory whoop, posttussive vomiting, cyanosis, and persistent coryzal symptoms. Infants under 2 months of age experience more severe disease, with most deaths occurring in this age group. Most of what is known about the pathology of pertussis in humans is from the evaluation of fatal human infant cases. The baboon model of pertussis provides the opportunity to evaluate the histopathology of severe but nonfatal pertussis. The baboon model recapitulates the characteristic clinical signs of pertussis observed in humans, including leukocytosis, paroxysmal coughing, mucus production, heavy colonization of the airway, and transmission of the bacteria between hosts. As in humans, baboons demonstrate age-related differences in clinical presentation, with younger animals experiencing more severe disease. We examined the histopathology of 5- to 6-week-old baboons, with the findings being similar to those reported for fatal human infant cases. In juvenile baboons, we found that the disease is highly inflammatory and concentrated to the lungs with signs of disease that would typically be diagnosed as acute respiratory distress syndrome (ARDS) and bronchopneumonia. In contrast, no significant pathology was observed in the trachea. Histopathological changes in the trachea were limited to cellular infiltrates and mucus production. Immunohistostaining revealed that the bacteria were localized to the surface of the ciliated epithelium in the conducting airways. Our observations provide important insights into the pathology of pertussis in typical, severe but nonfatal pertussis cases in a very relevant animal model.

Influence of the estrus cycle of the mouse on the disposition of SHetA2 after vaginal administration.

SHetA2 is a novel compound with the potential to treat cervical dysplasia, but has poor water solubility. A vaginal suppository formulation was able to achieve therapeutic concentrations in the cervix of mice, but these concentrations were variable. Histological analysis indicated that mice in the same group were in different stages of their estrous cycle, which is known to induce anatomical changes in their gynecological tissues. We investigated the effects of these changes on the pharmacokinetics and pharmacodynamics of SHetA2 when administered vaginally. Mice were synchronized to be either in estrous or diestrus stage for administration of the SHetA2 suppository. Pharmacokinetic parameters were calculated from the SHetA2 concentrations vs. time data. The reduction in the expression of cyclin D1 protein in the cervix was used as pharmacodynamic endpoint. Mice dosed during diestrus had a larger AUCcervix (335 µg mL h-1), higher Cmax (121.8 ±â€¯38.7 µg/g) and longer t1/2-cervix (30.3 h) compared to mice dosed during estrus (120 µg mL h-1, 44.6 ±â€¯29.5 µg/g and 3.6 h respectively). Therapeutic concentrations of SHetA2 were maintained for 48 h in the cervix of mice dosed during diestrus and for only 12 h in the estrus group. The treatment reduced the expression of cyclin D1 protein in the cervix of mice in the estrus to a larger extent. These results indicate that the estrous cycle of mice influences significantly the disposition of SHetA2 after vaginal administration and may also influence its efficacy.

DMBA promotes ErbB2­mediated carcinogenesis via ErbB2 and estrogen receptor pathway activation and genomic instability.

Environmental factors, including 7,12­dimethylbenz[a]anthracene (DMBA) exposure, and genetic predisposition, including ErbB2 overexpression/amplification, have been demonstrated to increase breast cancer susceptibility. Although DMBA­ and ErbB2­mediated breast cancers are well­studied in their respective models, key interactions between environmental and genetic factors on breast cancer risk remain unclear. Therefore, the present study aimed to investigate the effect of DMBA exposure on ErbB2­mediated mammary tumorigenesis. MMTV­ErbB2 transgenic mice exposed to DMBA (1 mg) via weekly oral gavage for 6 weeks exhibited significantly enhanced mammary tumor development, as indicated by reduced tumor latency and increased tumor multiplicity compared with control mice. Whole mount analysis of premalignant mammary tissues from 15­week­old mice revealed increased ductal elongation and proliferative index in DMBA­exposed mice. Molecular analyses of premalignant mammary tissues further indicated that DMBA exposure enhanced epidermal growth factor receptor (EGFR)/ErbB2 and estrogen receptor (ER) signaling, which was associated with increased mRNA levels of EGFR/ErbB2 family members and ER­targeted genes. Furthermore, analysis of tumor karyotypes revealed that DMBA­exposed tumors displayed more chromosomal alterations compared with control tumors, implicating DMBA­induced chromosomal instability in tumor promotion in this model. Together, the data suggested that DMBA­induced deregulation of EGFR/ErbB2­ER pathways plays a critical role in the enhanced chromosomal instability and promotion of ErbB2­mediated mammary tumorigenesis. The study highlighted gene­environment interactions that may increase risk of breast cancer, which is a critical clinical issue.

Safety assessment of the dietary supplement OxyELITE™ Pro (New Formula) in inbred and outbred mouse strains.

Herbal dietary supplements have gained wide acceptance as alternatives to conventional therapeutic agents despite concerns regarding their efficacy and safety. In 2013, a spate of severe liver injuries across the United States was linked to the dietary supplement OxyELITE Pro-New Formula (OEP-NF), a multi-ingredient product marketed for weight loss and exercise performance enhancement. The principal goal of this study was to assess the hepatotoxic potential of OEP-NF in outbred and inbred mouse models. In an acute toxicity study, significant mortality was observed after administering 10X and 3X mouse-equivalent doses (MED) of OEP-NF, respectively. Increases in liver/body weight ratio, ALT and AST were observed in female B6C3F1 mice after gavaging 2X and 1.5X MED of OEP-NF. Similar findings were observed in a 90-day feeding study. These alterations were paralleled by altered expression of gene- and microRNA-signatures of hepatotoxicity, including Cd36, Nqo1, Aldoa, Txnrd1, Scd1 and Ccng1, as well as miR-192, miR-193a and miR-125b and were most pronounced in female B6C3F1 mice. Body weight loss, observed at week 1, was followed by weight gain throughout the feeding studies. These findings bolster safety and efficacy concerns for OEP-NF, and argue strongly for implementation of pre-market toxicity studies within the dietary supplement industry.

Short-term early exposure to lapatinib confers lifelong protection from mammary tumor development in MMTV-erbB-2 transgenic mice.

BACKGROUND: Although chemopreventative agents targeting the estrogen/estrogen receptor (ER) pathway have been effective for ER+ breast cancers, prevention of hormone receptor-negative breast cancers, such as Her2/erbB-2+ breast cancers, remains a significant issue. Previous studies have demonstrated that administration of EGFR/erbB-2-targeting lapatinib to MMTV-erbB-2 transgenic mice inhibited mammary tumor development. The prevention, however, was achieved by prolonged high dose exposure. The tolerance to high dose/long-term drug administration may hinder its potential in clinical settings. Therefore, we aimed to test a novel, short-term chemopreventative strategy using lapatinib during the premalignant risk window in MMTV-erbB-2 mice. METHODS: We initially treated cultured cells with lapatinib to explore the anti-proliferative effects of lapatinib in vitro. We used a syngeneic tumor graft model to begin exploring the in vivo anti-tumorigenic effects of lapatinib in MMTV-erbB-2 mice. Then, we tested the efficacy of brief exposure to lapatinib (100 mg/kg/day for 8 weeks), beginning at 16 weeks of age, in the prevention of mammary tumor development in MMTV-erbB-2 mice. RESULTS: In the syngeneic tumor transplant model, we determined that lapatinib significantly inhibited tumor cell proliferation. Furthermore, we demonstrated that short-term lapatinib exposure resulted in life-long protective effects, as supported by increased tumor latency in lapatinib-treated mice compared to the control mice. We further established that delayed tumor development in the treated mice was preceded by decreased BrdU nuclear incorporation and inhibited mammary morphogenesis. Molecular analysis indicated that lapatinib inhibited phosphorylation and expression of EGFR, erbB-3, erbB-2, Akt1, and Erk1/2 in premalignant mammary tissues. Also, lapatinib drastically inhibited the phosphorylation and expression of ERα and the transcription of ER target genes in premalignant mammary tissues. We also determined that lapatinib suppressed the stemness of breast cancer cell lines, as evidenced by decreased tumorsphere formation and ALDH+ cell populations. CONCLUSIONS: Taken together, these data demonstrate that brief treatment with EGFR/erbB-2-targeting agents before the onset of tumors may provide lifelong protection from mammary tumors, through the concurrent inhibition of erbB-2 and ER signaling pathways and consequential reprogramming. Our findings support further clinical testing to explore the benefit of shorter lapatinib exposure in the prevention of erbB-2-mediated carcinogenesis.

An oral HemokineTM, α-methylhydrocinnamate, enhances myeloid and neutrophil recovery following irradiation in vivo.

An oral therapeutic which reduces duration of cytopenias and is active following accidental radiation exposures is an unmet need in radiation countermeasures. Alpha methylhydrocinnamate (ST7) prolongs STAT-5 phosphorylation, reduces growth-factor dependency of multi-lineage cell lines, and stimulates erythropoiesis. Here, ST7 and its isomers were studied for their effects on myeloid progenitors and hematopoietic stem cells (HSCs) following radiation, in nonhuman primates, and murine irradiation models. Addition of ST7 or ST7-S increased CFU-GM production by 1.7-fold (p<0.001), reduced neutrophil apoptosis comparable to G-CSF, and enhanced HSC survival post-radiation by 2-fold, (p=0.028). ST7 and ST7-S administered in normal baboons increased ANC and platelet counts by 50-400%. In sub-lethally-irradiated mice, ANC nadir remained >200/mm3 and neutropenia recovered in 6days with ST7 treatment and 18days in controls (p<0.05). In lethally-irradiated mice, marrow pathology at 15days was hypocellular (10% cellularity) in controls, but normal (55-75% cellularity) with complete neutrophil maturation with ST7-S treatment. Following lethal irradiation, ST7, given orally for 4days, reduced mortality, with 30% survival in ST7-animals vs 8% in controls, (p<0.05). Collectively, the studies indicate that ST7 and ST7-S enhance myeloid recovery post-radiation and merit further evaluation to accelerate hematologic recovery in conditions of radiation-related and other marrow hypoplasias.

Cardiac myosin-Th17 responses promote heart failure in human myocarditis.

In human myocarditis and its sequela dilated cardiomyopathy (DCM), the mechanisms and immune phenotype governing disease and subsequent heart failure are not known. Here, we identified a Th17 cell immunophenotype of human myocarditis/DCM with elevated CD4+IL17+ T cells and Th17-promoting cytokines IL-6, TGF-ß, and IL-23 as well as GM-CSF-secreting CD4+ T cells. The Th17 phenotype was linked with the effects of cardiac myosin on CD14+ monocytes, TLR2, and heart failure. Persistent heart failure was associated with high percentages of IL-17-producing T cells and IL-17-promoting cytokines, and the myocarditis/DCM phenotype included significantly low percentages of FOXP3+ Tregs, which may contribute to disease severity. We demonstrate a potentially novel mechanism in human myocarditis/DCM in which TLR2 peptide ligands from human cardiac myosin stimulated exaggerated Th17-related cytokines including TGF-ß, IL-6, and IL-23 from myocarditic CD14+ monocytes in vitro, and an anti-TLR2 antibody abrogated the cytokine response. Our translational study explains how an immune phenotype may be initiated by cardiac myosin TLR ligand stimulation of monocytes to generate Th17-promoting cytokines and development of pathogenic Th17 cells in human myocarditis and heart failure, and provides a rationale for targeting IL-17A as a therapeutic option.

Analysis of TMEFF2 allografts and transgenic mouse models reveals roles in prostate regeneration and cancer.

BACKGROUND: Previous results from our lab indicate a tumor suppressor role for the transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) in prostate cancer (PCa). Here, we further characterize this role and uncover new functions for TMEFF2 in cancer and adult prostate regeneration. METHODS: The role of TMEFF2 was examined in PCa cells using Matrigel(TM) cultures and allograft models of PCa cells. In addition, we developed a transgenic mouse model that expresses TMEFF2 from a prostate specific promoter. Anatomical, histological, and metabolic characterizations of the transgenic mouse prostate were conducted. The effect of TMEFF2 in prostate regeneration was studied by analyzing branching morphogenesis in the TMEFF2-expressing mouse lobes and alterations in branching morphogenesis were correlated with the metabolomic profiles of the mouse lobes. The role of TMEFF2 in prostate tumorigenesis in whole animals was investigated by crossing the TMEFF2 transgenic mice with the TRAMP mouse model of PCa and analyzing the histopathological changes in the progeny. RESULTS: Ectopic expression of TMEFF2 impairs growth of PCa cells in Matrigel or allograft models. Surprisingly, while TMEFF2 expression in the TRAMP mouse did not have a significant effect on the glandular prostate epithelial lesions, the double TRAMP/TMEFF2 transgenic mice displayed an increased incidence of neuroendocrine type tumors. In addition, TMEFF2 promoted increased branching specifically in the dorsal lobe of the prostate suggesting a potential role in developmental processes. These results correlated with data indicating an alteration in the metabolic profile of the dorsal lobe of the transgenic TMEFF2 mice. CONCLUSIONS: Collectively, our results confirm the tumor suppressor role of TMEFF2 and suggest that ectopic expression of TMEFF2 in mouse prostate leads to additional lobe-specific effects in prostate regeneration and tumorigenesis. This points to a complex and multifunctional role for TMEFF2 during PCa progression.

The salutary effects of diphenyldifluoroketone EF24 in liver of a rat hemorrhagic shock model.

BACKGROUND: Liver is a target for injury in low flow states and it plays a central role in the progression of systemic failure associated with hemorrhagic shock. Pharmacologic support can help recover liver function even after it has suffered extensive damage during ischemia and reperfusion phases. In this work we assessed the efficacy of a diphenyldifluoroketone EF24, an IKKß inhibitor, in controlling hepatic inflammatory signaling caused by hemorrhagic shock in a rat model. METHODS: Sprague Dawley rats were bled to about 50% of blood volume. The hemorrhaged rats were treated with vehicle control or EF24 (0.4 mg/kg) after 1 h of hemorrhage without any accompanying resuscitation. The study was terminated after additional 5 h to excise liver tissue for biochemical analyses and histology. RESULTS: EF24 treatment alleviated hemorrhagic shock-induced histologic injury in the liver and restored serum transaminases to normal levels. Hemorrhagic shock induced the circulating levels of CD163 (a marker for macrophage activation) and CINC (an IL-8 analog), as well as myeloperoxidase activity in liver tissue. These markers of inflammatory injury were reduced by EF24 treatment. EF24 treatment also suppressed the expression of the Toll-like receptor 4, phospho-p65/Rel A, and cyclooxygenase-2 in liver tissues, indicating that it suppressed inflammatory pathway. Moreover, it reduced the hemorrhagic shock-induced increase in the expression of high mobility group box-1 protein. The evidence for apoptosis after hemorrhagic shock was inconclusive. CONCLUSION: Even in the absence of volume support, EF24 treatment suppresses pro-inflammatory signaling in liver tissue and improves liver functional markers in hemorrhagic shock.

Defective selection of thymic regulatory T cells accompanies autoimmunity and pulmonary infiltrates in Tcra-deficient mice double transgenic for human La/Sjögren's syndrome-B and human La-specific TCR.

A human La/Sjögren's syndrome-B (hLa)-specific TCR/hLa neo-self-Ag double-transgenic (Tg) mouse model was developed and used to investigate cellular tolerance and autoimmunity to the ubiquitous RNA-binding La Ag often targeted in systemic lupus erythematosus and Sjögren's syndrome. Extensive thymic clonal deletion of CD4(+) T cells occurred in H-2(k/k) double-Tg mice presenting high levels of the I-E(k)-restricted hLa T cell epitope. In contrast, deletion was less extensive in H-2(k/b) double-Tg mice presenting lower levels of the epitope, and some surviving thymocytes were positively selected as thymic regulatory T cells (tTreg). These mice remained serologically tolerant to hLa and healthy. H-2(k/b) double-Tg mice deficient of all endogenous Tcra genes, a deficiency known to impair Treg development and function, produced IgG anti-hLa autoantibodies and displayed defective tTreg development. These autoimmune mice had interstitial lung disease characterized by lymphocytic aggregates containing Tg T cells with an activated, effector memory phenotype. Salivary gland infiltrates were notably absent. Thus, expression of nuclear hLa Ag induces thymic clonal deletion and tTreg selection, and lymphocytic infiltration of the lung is a consequence of La-specific CD4(+) T cell autoimmunity.

Identification of streptococcal m-protein cardiopathogenic epitopes in experimental autoimmune valvulitis.

The M protein of rheumatogenic group A streptococci induces carditis and valvulitis in Lewis rats and may play a role in pathogenesis of rheumatic heart disease. To identify the epitopes of M5 protein that produce valvulitis, synthetic peptides spanning A, B, and C repeat regions contained within the extracellular domain of the streptococcal M5 protein were investigated. A repeat region peptides NT4, NT5/6, and NT7 induced valvulitis similar to the intact pepsin fragment of M5 protein. T cell lines from rats with valvulitis recognized M5 peptides NT5/6 and NT6. Passive transfer of an NT5/6-specific T cell line into naïve rats produced valvulitis characterized by infiltration of CD4+ cells and upregulation of VCAM-1, while an NT6-specific T cell line did not target the valve. Our new data suggests that M protein-specific T cells may be important mediators of valvulitis in the Lewis rat model of rheumatic carditis.