Olivia Kershaw

IFNs Modify the Proteome of Legionella-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid

Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.

Depletion of Cultivatable Gut Microbiota by Broad-Spectrum Antibiotic Pretreatment Worsens Outcome After Murine Stroke

Background and Purpose— Antibiotics disturbing microbiota are often used in treatment of poststroke infections. A bidirectional brain–gut microbiota axis was recently suggested as a modulator of nervous system diseases. We hypothesized that gut microbiota may be an important player in the course of stroke. Methods— We investigated the outcome of focal cerebral ischemia in C57BL/6J mice after an 8-week decontamination with quintuple broad-spectrum antibiotic cocktail. These microbiota-depleted animals were subjected to 60 minutes middle cerebral artery occlusion or sham operation. Infarct volume was measured using magnetic resonance imaging, and mice were monitored clinically throughout the whole experiment. At the end point, tissues were preserved for further analysis, comprising histology and immunologic investigations using flow cytometry. Results— We found significantly decreased survival in the middle cerebral artery occlusion microbiota-depleted mice when the antibiotic cocktail was stopped 3 days before surgery (compared with middle cerebral artery occlusion specific pathogen-free and sham-operated microbiota-depleted mice). Moreover, all microbiota-depleted animals in which antibiotic treatment was terminated developed severe acute colitis. This phenotype was rescued by continuous antibiotic treatment or colonization with specific pathogen-free microbiota before surgery. Further, infarct volumes on day one did not differ between any of the experimental groups. Conclusions— Conventional microbiota ensures intestinal protection in the mouse model of experimental stroke and prevents development of acute and severe colitis in microbiota-depleted mice not given antibiotic protection after cerebral ischemia. Our experiments raise the clinically important question as to whether microbial colonization or specific microbiota are crucial for stroke outcome.

Mechanical ventilation drives pneumococcal pneumonia into lung injury and sepsis in mice: protection by adrenomedullin

IntroductionVentilator-induced lung injury (VILI) contributes to morbidity and mortality in acute respiratory distress syndrome (ARDS). Particularly pre-injured lungs are susceptible to VILI despite protective ventilation. In a previous study, the endogenous peptide adrenomedullin (AM) protected murine lungs from VILI. We hypothesized that mechanical ventilation (MV) contributes to lung injury and sepsis in pneumonia, and that AM may reduce lung injury and multiple organ failure in ventilated mice with pneumococcal pneumonia.MethodsWe analyzed in mice the impact of MV in established pneumonia on lung injury, inflammation, bacterial burden, hemodynamics and extrapulmonary organ injury, and assessed the therapeutic potential of AM by starting treatment at intubation.ResultsIn pneumococcal pneumonia, MV increased lung permeability, and worsened lung mechanics and oxygenation failure. MV dramatically increased lung and blood cytokines but not lung leukocyte counts in pneumonia. MV induced systemic leukocytopenia and liver, gut and kidney injury in mice with pneumonia. Lung and blood bacterial burden was not affected by MV pneumonia and MV increased lung AM expression, whereas receptor activity modifying protein (RAMP) 1–3 expression was increased in pneumonia and reduced by MV. Infusion of AM protected against MV-induced lung injury (66% reduction of pulmonary permeability p < 0.01; prevention of pulmonary restriction) and against VILI-induced liver and gut injury in pneumonia (91% reduction of AST levels p < 0.05, 96% reduction of alanine aminotransaminase (ALT) levels p < 0.05, abrogation of histopathological changes and parenchymal apoptosis in liver and gut).ConclusionsMV paved the way for the progression of pneumonia towards ARDS and sepsis by aggravating lung injury and systemic hyperinflammation leading to liver, kidney and gut injury. AM may be a promising therapeutic option to protect against development of lung injury, sepsis and extrapulmonary organ injury in mechanically ventilated individuals with severe pneumonia.

In vitro and in vivo trypanocidal effect of lipophilic extracts of medicinal plants from Mali and Burkina Faso

AIM OF THE STUDY To determine the in vitro and in vivo antitrypanosomal activity of extracts of traditionally used plants. MATERIALS AND METHODS 47 dichloromethane extracts were tested in vitro in the Long-term Viability Assay (LtVA) on Trypanosoma brucei brucei. The most active ones were also tested in vivo using a standardised mouse test. RESULTS 13 extracts (28%) were active in vitro with MIC-values < or = 100 microg/ml, 6 extracts showed MIC-values < or = 50 microg/ml. The root extract of Securidaca longepedunculata Fresen. (Polygalaceae) and the leaf extract of Guiera senegalensis J. F. Gmel. (Combretaceae) were able to reduce parasitaemia in mice, experimentally infected with Trypanosoma brucei brucei by 48 and 42% at the dose of 150 mg/kg b.w. intraperitoneally, two times daily for 3 days. The extract of Acacia nilotica Delile (Mimosaceae) stem bark showed immunosuppressive effect in vivo. CONCLUSION The results confirm an effect of the ethnobotanically used plants. Further investigation is needed to optimize the effectiveness of the extracts.

Streptococcus pneumoniae-induced regulation of cyclooxygenase-2 in human lung tissue

The majority of cases of community-acquired pneumonia are caused by Streptococcus pneumoniae and most studies on pneumococcal host interaction are based on cell culture or animal experiments. Thus, little is known about infections in human lung tissue. Cyclooxygenase-2 and its metabolites play an important regulatory role in lung inflammation. Therefore, we established a pneumococcal infection model on human lung tissue demonstrating mitogen-activated protein kinase (MAPK)-dependent induction of cyclooxygenase-2 and its related metabolites. In addition to alveolar macrophages and the vascular endothelium, cyclooxygenase-2 was upregulated in alveolar type II but not type I epithelial cells, which was confirmed in lungs of patients suffering from acute pneumonia. Moreover, we demonstrated the expression profile of all four E prostanoid receptors at the mRNA level and showed functionality of the E prostanoid4 receptor by cyclic adenosine monophosphate production. Additionally, in comparison to previous studies, cyclooxygenase-2/prostaglandin E2 related pro- and anti-inflammatory mediator regulation was partly confirmed in human lung tissue after pneumococcal infection. Overall, cell type-specific and MAPK-dependent cyclooxygenase-2 expression and prostaglandin E2 formation in human lung tissue may play an important role in the early phase of pneumococcal infections.

Does a Feline Leukemia Virus Infection Pave the Way for Bartonella henselae Infection in Cats

ABSTRACT Domestic cats serve as the reservoir hosts of Bartonella henselae and may develop mild clinical symptoms or none after experimental infection. In humans, B. henselae infection can result in self-limiting cat scratch disease. However, immunocompromised patients may suffer from more-severe courses of infection or may even develop the potentially lethal disease bacillary angiomatosis. It was reasoned that cats with immunocompromising viral infections may react similarly to B. henselae infection. The aim of our study was to investigate the influence of the most important viruses known to cause immunosuppression in cats—Feline leukemia virus (FeLV), Feline immunodeficiency virus (FIV), and Feline panleukopenia virus (FPV)—on natural B. henselae infection in cats. Accordingly, 142 cats from animal shelters were necropsied and tested for B. henselae and concurrent infections with FeLV, FIV, or FPV by PCR and immunohistochemistry. A significant association was found between B. henselae and FeLV infections (P = 0.00028), but not between B. henselae and FIV (P = 1.0) or FPV (P = 0.756) infection, age (P = 0.392), or gender (P = 0.126). The results suggest that susceptibility to B. henselae infection is higher in cats with concurrent FeLV infections, regardless of whether the infection is latent or progressive. Histopathology and immunohistochemistry for B. henselae failed to identify lesions that could be attributed specifically to B. henselae infection. We conclude that the course of natural B. henselae infection in cats does not seem to be influenced by immunosuppressive viral infections in general but that latent FeLV infection may predispose cats to B. henselae infection or persistence.

Nf1 haploinsufficiency and Icsbp deficiency synergize in the development of leukemias

Loss of neurofibromin or interferon consensus sequence binding protein (Icsbp) leads to a myeloproliferative disorder. Transcription of NF1 is directly controlled by ICSBP. It has been postulated that loss of NF1 expression resulting from loss of transcriptional activation by ICSBP contributes to human hematologic malignancies. To investigate the functional cooperation of these 2 proteins, we have established Icsbp-deficient mice with Nf1 haploinsufficiency. We here demonstrate that loss of Icsbp and Nf1 haploinsufficiency synergize to induce a forced myeloproliferation in Icsbp-deficient mice because of an expansion of a mature myeloid progenitor cell. Furthermore, Nf1 haploinsufficiency and loss of Icsbp contribute synergistically to progression of the myeloproliferative disorder toward transplantable leukemias. Leukemias are characterized by distinct phenotypes, which correlate with progressive genetic abnormalities. Loss of Nf1 heterozygosity is not mandatory for disease progression, but its occurrence with other genetic abnormalities indicates progressive genetic alterations in a defined subset of leukemias. These data show that loss of the 2 tumor suppressor genes Nf1 and Icsbp synergize in the induction of leukemias.

Does the taking of biopsies affect the metastatic potential of tumours? A systematic review of reports on veterinary and human cases and animal models.

Clinicians and pathologists are sporadically asked by owners whether the taking of tumour biopsies may affect the behaviour of the tumour, including its potential to metastasise. Unfortunately, systematic studies on this subject are unavailable in veterinary medicine, and the aim of this study was to estimate the risk of adverse effects of biopsy taking on tumour progression in animals. A systematic review of veterinary and human case reports and clinical studies as well as experimental animal models of biopsy-induced tumour metastasis was undertaken. There were only two veterinary case reports of needle tract metastases (NTM) following the taking of needle biopsies from urogenital and pulmonary tumours. Seventeen experimental studies found a high incidence of NTM but only a rat osteosarcoma and a hamster squamous carcinoma model showed an increased incidence of distant or regional metastases after incision or excision biopsy. In human medicine, the occurrence of NTM has been reported after the taking of biopsies from mesotheliomas (15%), melanomas (11%) and gall bladder tumours (11%), liver metastases of colon carcinomas (4%) and mammary carcinomas (4%) but an incidence of only <1% for all other tumours. Circulating tumour cells increased immediately after the taking of biopsies from human squamous cell, prostate, breast and hepatocellular carcinomas. Although no increased risk of biopsy-induced distant metastasis has been reported for any type of tumour, this is inconclusive due to a lack of non-biopsied control groups in human studies. Reports of biopsy-induced metastasis in animal tumours indicate that the taking of transcutaneous biopsies from urogenital tumours may be associated with a risk of NTM. However, there is no evidence of a general increase in risk of distant metastases in any tumour type in people or animals. The overall risk therefore appears to be negligible when compared to the valuable information obtained from biopsies in veterinary practice.

Skin TLR7 Triggering Promotes Accumulation of Respiratory Dendritic Cells and Natural Killer Cells

The TLR7 agonist imiquimod has been used successfully as adjuvant for skin treatment of virus-associated warts and basal cell carcinoma. The effects of skin TLR7 triggering on respiratory leukocyte populations are unknown. In a placebo-controlled experimental animal study we have used multicolour flow cytometry to systematically analyze the modulation of respiratory leukocyte subsets after skin administration of imiquimod. Compared to placebo, skin administration of imiquimod significantly increased respiratory dendritic cells (DC) and natural killer cells, whereas total respiratory leukocyte, alveolar macrophages, classical CD4+ T helper and CD8+ T killer cell numbers were not or only moderately affected. DC subpopulation analyses revealed that elevation of respiratory DC was caused by an increase of respiratory monocytic DC and CD11bhi DC subsets. Lymphocyte subpopulation analyses indicated a marked elevation of respiratory natural killer cells and a significant reduction of B lymphocytes. Analysis of cytokine responses of respiratory leukocytes after stimulation with Klebsiella pneumonia indicated reduced IFN-γ and TNF-α expression and increased IL-10 and IL-12p70 production after 7 day low dose skin TLR7 triggering. Additionally, respiratory NK cytotoxic activity was increased after 7d skin TLR7 triggering. In contrast, lung histology and bronchoalveolar cell counts were not affected suggesting that skin TLR7 stimulation modulated respiratory leukocyte composition without inducing overt pulmonary inflammation. These data suggest the possibility to modulate respiratory leukocyte composition and respiratory cytokine responses against pathogens like Klebsiella pneumonia through skin administration of a clinically approved TLR7 ligand. Skin administration of synthetic TLR7 ligands may represent a novel, noninvasive means to modulate respiratory immunity.

Diagnostic Value of Morphometry in Feline Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common form of feline heart disease. To date, reliable morphometric reference data for anatomical or histological changes are unavailable. The aim of this study was to identify diagnostically relevant morphometric criteria that clearly distinguish feline HCM from normal hearts. Hearts from 15 cats with HCM had increased weights (g per distance between the first and eighth vertebral bodies) when compared with hearts from 15 matched control cats. Several anatomically defined and digitally scanned areas of standardized cross sections were significantly increased in HCM when compared with controls, including the area across the entire heart half-way between the coronary sulcus and apex, the right and left ventricular walls and the ventricular septum. Differences were similar when the papillary muscles were included in the measurements of the right and left ventricular walls and the ventricular septum. Histological morphometric analyses failed to identify any significant differences, including the diameter and cross-sectional area of cardiomyocytes and the length, width or areas of cross-sectioned nuclei. In addition, morphometric analyses failed to identify any differences in the amount of cardiomyocyte fibre branching or myocardial fibrosis. Thus, only the relative weight and macroscopical analyses proved useful in distinguishing feline hearts with HCM from normal hearts. The results do not uphold the hypothesis that increased cardiomyocyte diameter is a principal change in feline HCM.