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Interleukin-4 (IL-4) is an anti-inflammatory and analgesic cytokine that induces opioid receptor transcription. We investigated IL-4 knockout (ko) mice to characterize their pain behavior before and after chronic constriction injury (CCI) of the sciatic nerve as a model for neuropathic pain. We investigated opioid responsivity and measured cytokine and opioid receptor gene expression in the peripheral and central nervous system (PNS, CNS) of IL-4 ko mice in comparison with wildtype (wt) mice. Naïve IL-4 ko mice displayed tactile allodynia (wt: 0.45 g; ko: 0.18 g; p<0.001), while responses to heat and cold stimuli and to muscle pressure were not different. No compensatory changes in the gene expression of tumor necrosis factor-alpha (TNF), IL-1β, IL-10, and IL-13 were found in the PNS and CNS of naïve IL-4 ko mice. However, IL-1β gene expression was stronger in the sciatic nerve of IL-4 ko mice (p<0.001) 28 days after CCI and only IL-4 ko mice had elevated IL-10 gene expression (p = 0.014). Remarkably, CCI induced TNF (p<0.01), IL-1β (p<0.05), IL-10 (p<0.05), and IL-13 (p<0.001) gene expression exclusively in the ipsilateral spinal cord of IL-4 ko mice. The compensatory overexpression of the anti-inflammatory and analgesic cytokines IL-10 and IL-13 in the spinal cord of IL-4 ko mice may explain the lack of genotype differences for pain behavior after CCI. Additionally, CCI induced gene expression of μ, κ, and δ opioid receptors in the contralateral cortex and thalamus of IL-4 ko mice, paralleled by fast onset of morphine analgesia, but not in wt mice. We conclude that a lack of IL-4 leads to mechanical sensitivity; the compensatory hyperexpression of analgesic cytokines and opioid receptors after CCI, in turn, protects IL-4 ko mice from enhanced pain behavior after nerve lesion.
Growth factor induced signaling cascades are key regulatory elements in tissue development, maintenance and regeneration. Perturbations of these cascades have severe consequences, leading to developmental disorders and neoplastic diseases. As a major function in signal transduction, activating mutations in RAF family kinases are the cause of human tumorigenesis, where B-RAF V600E has been identified as the prevalent mutant. In order to address the oncogenic function of B-RAF V600E, we have generated transgenic mice expressing the activated oncogene specifically in lung alveolar epithelial type II cells. Constitutive expression of B-RAF V600E caused abnormalities in alveolar epithelium formation that led to airspace enlargements. These lung lesions showed signs of tissue remodeling and were often associated with chronic inflammation and low incidence of lung tumors. The inflammatory cell infiltration did not precede the formation of the lung lesions but was rather accompanied with late tumor development. These data support a model where the continuous regenerative process initiated by oncogenic B-RAF-driven alveolar disruption provides a tumor-promoting environment associated with chronic inflammation.
Metastatic tumor cells in body fluids are important targets for treatment, and critical surrogate markers for evaluating cancer prognosis and therapeutic response. Here we report, for the first time, that live metastatic tumor cells in blood samples from mice bearing human tumor xenografts and in blood and cerebrospinal fluid samples from patients with cancer were successfully detected using a tumor cell-specific recombinant vaccinia virus (VACV). In contrast to the FDA-approved CellSearch system, VACV detects circulating tumor cells (CTCs) in a cancer biomarker-independent manner, thus, free of any bias related to the use of antibodies, and can be potentially a universal system for detection of live CTCs of any tumor type, not limited to CTCs of epithelial origin. Furthermore, we demonstrate for the first time that VACV was effective in preventing and reducing circulating tumor cells in mice bearing human tumor xenografts. Importantly, a single intra-peritoneal delivery of VACV resulted in a dramatic decline in the number of tumor cells in the ascitic fluid from a patient with gastric cancer. Taken together, these results suggest VACV to be a useful tool for quantitative detection of live tumor cells in liquid biopsies as well as a potentially effective treatment for reducing or eliminating live tumor cells in body fluids of patients with metastatic disease.
Obesity is a major risk factor predisposing to the development of peripheral insulin resistance and type 2 diabetes (T2D). Elevated food intake and/or decreased energy expenditure promotes body weight gain and acquisition of adipose tissue. Number of studies implicated phospholipase D (PLD) enzymes and their product, phosphatidic acid (PA), in regulation of signaling cascades controlling energy intake, energy dissipation and metabolic homeostasis. However, the impact of PLD enzymes on regulation of metabolism has not been directly determined so far. In this study we utilized mice deficient for two major PLD isoforms, PLD1 and PLD2, to assess the impact of these enzymes on regulation of metabolic homeostasis. We showed that mice lacking PLD1 or PLD2 consume more food than corresponding control animals. Moreover, mice deficient for PLD2, but not PLD1, present reduced energy expenditure. In addition, deletion of either of the PLD enzymes resulted in development of elevated body weight and increased adipose tissue content in aged animals. Consistent with the fact that elevated content of adipose tissue predisposes to the development of hyperlipidemia and insulin resistance, characteristic for the pre-diabetic state, we observed that Pld1\(^{-/-}\) and Pld2\(^{-/-}\) mice present elevated free fatty acids (FFA) levels and are insulin as well as glucose intolerant. In conclusion, our data suggest that deficiency of PLD1 or PLD2 activity promotes development of overweight and diabetes.
Blood tests are necessary, easy-to-perform and low-cost alternatives for monitoring of oncolytic virotherapy and other biological therapies in translational research. Here we assessed three candidate proteins with the potential to be used as biomarkers in biological fluids: two glucuronidases from E. coli (GusA) and Staphylococcus sp. RLH1 (GusPlus), and the luciferase from Gaussia princeps (GLuc). The three genes encoding these proteins were inserted individually into vaccinia virus GLV-1h68 genome under the control of an identical promoter. The three resulting recombinant viruses were used to infect tumor cells in cultures and human tumor xenografts in nude mice. In contrast to the actively secreted GLuc, the cytoplasmic glucuronidases GusA and GusPlus were released into the supernatants only as a result of virus-mediated oncolysis. GusPlus resulted in the most sensitive detection of enzyme activity under controlled assay conditions in samples containing as little as 1 pg/ml of GusPlus, followed by GusA (25 pg/ml) and GLuc (≥375 pg/ml). Unexpectedly, even though GusA had a lower specific activity compared to GusPlus, the substrate conversion in the serum of tumor-bearing mice injected with the GusA-encoding virus strains was substantially higher than that of GusPlus. This was attributed to a 3.2 fold and 16.2 fold longer half-life of GusA in the blood stream compared to GusPlus and GLuc respectively, thus a more sensitive monitor of virus replication than the other two enzymes. Due to the good correlation between enzymatic activity of expressed marker gene and virus titer, we conclude that the amount of the biomarker protein in the body fluid semiquantitatively represents the amount of virus in the infected tumors which was confirmed by low light imaging. We found GusA to be the most reliable biomarker for monitoring oncolytic virotherapy among the three tested markers.
Meningococci spread via respiratory droplets, whereas the closely related gonococci are transmitted sexually. Several outbreaks of invasive meningococcal disease have been reported in Europe and the United States among men who have sex with men (MSM). We recently identified an outbreak of serogroup C meningococcal disease among MSM in Germany and France. In this study, genomic and proteomic techniques were used to analyze the outbreak isolates. In addition, genetically identical urethritis isolates were recovered from France and Germany and included in the analysis. Genome sequencing revealed that the isolates from the outbreak among MSM and from urethritis cases belonged to a clade within clonal complex 11. Proteome analysis showed they expressed nitrite reductase, enabling anaerobic growth as previously described for gonococci. Invasive isolates from MSM, but not urethritis isolates, further expressed functional human factor H binding protein associated with enhanced survival in a newly developed transgenic mouse model expressing human factor H, a complement regulatory protein. In conclusion, our data suggest that urethritis and outbreak isolates followed a joint adaptation route including adaption to the urogenital tract.
Mesenteric lymph nodes (mLNs) are sentinel sites of enteral immunosurveillance and immune homeostasis. Immune cells from the gastrointestinal tract (GIT) are constantly recruited to the mLNs in steady-state and under inflammatory conditions resulting in the induction of tolerance and immune cells activation, respectively. Surgical dissection and transplantation of lymph nodes (LN) is a technique that has supported seminal work to study LN function and is useful to investigate resident stromal and endothelial cell biology and their cellular interactions in experimental disease models. Here, we provide a detailed protocol of syngeneic mLN transplantation and report assays to analyze effective mLN engraftment in congenic recipients. Transplanted mLNs allow to study T cell activation and proliferation in preclinical mouse models. Donor mLNs proved viable and functional after surgical transplantation and regenerated blood and lymphatic vessels. Immune cells from the host completely colonized the transplanted mLNs within 7-8 weeks after the surgical intervention. After allogeneic hematopoietic cell transplantation (allo-HCT), adoptively transferred allogeneic CD4+ T cells from FVB/N (H-2q) mice homed to the transplanted mLNs in C57BL/6 (H-2b) recipients during the initiation phase of acute graft-versus-host disease (aGvHD). These CD4+ T cells retained full proliferative capacity and upregulated effector and gut homing molecules comparable to those in mLNs from unmanipulated wild-type recipients. Wild type mLNs transplanted into MHCII deficient syngeneic hosts sufficed to activate alloreactive T cells upon allogeneic hematopoietic cell transplantation, even in the absence of MHCII+ CD11c+ myeloid cells. These data support that orthotopically transplanted mLNs maintain physiological functions after transplantation. The technique of LN transplantation can be applied to study migratory and resident cell compartment interactions in mLNs as well as immune reactions from and to the gut under inflammatory and non-inflammatory conditions.
Abstract
In the murine model of Leishmania major infection, resistance or susceptibility to the parasite has been associated with the development of a Th1 or Th2 type of immune response. Recently, however, the immunosuppressive effects of IL-10 have been ascribed a crucial role in the development of the different clinical correlates of Leishmania infection in humans. Since T cells and professional APC are important cellular sources of IL-10, we compared leishmaniasis disease progression in T cell-specific, macrophage/neutrophil-specific and complete IL-10-deficient C57BL/6 as well as T cell-specific and complete IL-10-deficient BALB/c mice. As early as two weeks after infection of these mice with L. major, T cell-specific and complete IL-10-deficient animals showed significantly increased lesion development accompanied by a markedly elevated secretion of IFN-γ or IFN-γ and IL-4 in the lymph nodes draining the lesions of the C57BL/6 or BALB/c mutants, respectively. In contrast, macrophage/neutrophil-specific IL-10-deficient C57BL/6 mice did not show any altered phenotype. During the further course of disease, the T cell-specific as well as the complete IL-10-deficient BALB/c mice were able to control the infection. Furthermore, a dendritic cell-based vaccination against leishmaniasis efficiently suppresses the early secretion of IL-10, thus contributing to the control of parasite spread. Taken together, IL-10 secretion by T cells has an influence on immune activation early after infection and is sufficient to render BALB/c mice susceptible to an uncontrolled Leishmania major infection.
Author Summary
The clinical symptoms caused by infections with Leishmania parasites range from self-healing cutaneous to uncontrolled visceral disease and depend not only on the parasite species but also on the type of the host's immune response. It is estimated that 350 million people worldwide are at risk, with a global incidence of 1–1.5 million cases of cutaneous and 500,000 cases of visceral leishmaniasis. Murine leishmaniasis is the best-characterized model to elucidate the mechanisms underlying resistance or susceptibility to Leishmania major parasites in vivo. Using T cell-specific and macrophage-specific mutant mice, we demonstrate that abrogating the secretion of the immunosuppressive cytokine IL-10 by T cells is sufficient to render otherwise susceptible mice resistant to an infection with the pathogen. The healing phenotype is accompanied by an elevated specific inflammatory immune response very early after infection. We further show that dendritic cell-based vaccination against leishmaniasis suppresses the early secretion of IL-10 following challenge infection. Thus, our study unravels a molecular mechanism critical for host immune defense, aiding in the development of an effective vaccine against leishmaniasis.
Introduction: The aim of our study was to develop a reproducible murine model of elastase-induced aneurysm formation combined with aortic transplantation.
Methods: Adult male mice (n = 6-9 per group) underwent infrarenal, orthotopic transplantation of the aorta treated with elastase or left untreated. Subsequently, both groups of mice were monitored by ultrasound until 7 weeks after grafting.
Results: Mice receiving an elastase-pretreated aorta developed aneurysms and exhibited a significantly increased diastolic vessel diameter compared to control grafted mice at 7 week after surgery (1.11 +/- 0.10 mm vs. 0.75 +/- 0.03 mm; p <= 0.001). Histopathological examination revealed disruption of medial elastin, an increase in collagen content and smooth muscle cells, and neointima formation in aneurysm grafts.
Conclusions: We developed a reproducible murine model of elastase-induced aneurysm combined with aortic transplantation. This model may be suitable to investigate aneurysm-specific inflammatory processes and for use in gene-targeted animals.
Background
Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by inflammation, demyelination and axonal pathology. Myelin basic protein/proteolipid protein (MBP-PLP) fusion protein MP4 is capable of inducing chronic experimental autoimmune encephalomyelitis (EAE) in susceptible mouse strains mirroring diverse histopathological and immunological hallmarks of MS. Limited availability of human tissue underscores the importance of animal models to study the pathology of MS.
Methods
Twenty-two female C57BL/6 (B6) mice were immunized with MP4 and the clinical development of experimental autoimmune encephalomyelitis (EAE) was observed. Methylene blue-stained semi-thin and ultra-thin sections of the lumbar spinal cord were assessed at the peak of acute EAE, three months (chronic EAE) and six months after onset of EAE (long-term EAE). The extent of lesional area and inflammation were analyzed in semi-thin sections on a light microscopic level. The magnitude of demyelination and axonal damage were determined using electron microscopy. Emphasis was put on the ventrolateral tract (VLT) of the spinal cord.
Results
B6 mice demonstrated increasing demyelination and severe axonal pathology in the course of MP4-induced EAE. In addition, mitochondrial swelling and a decrease in the nearest neighbor neurofilament distance (NNND) as early signs of axonal damage were evident with the onset of EAE. In semi-thin sections we observed the maximum of lesional area in the chronic state of EAE while inflammation was found to a similar extent in acute and chronic EAE. In contrast to the well-established myelin oligodendrocyte glycoprotein (MOG) model, disease stages of MP4-induced EAE could not be distinguished by assessing the extent of parenchymal edema or the grade of inflammation.
Conclusions
Our results complement our previous ultrastructural studies of B6 EAE models and suggest that B6 mice immunized with different antigens constitute useful instruments to study the diverse histopathological aspects of MS.