Diagnosis of the feline upper respiratory disease complex (FURDC): questions faced by clinicians

Feline Upper Respiratory Disease Complex is a common clinical entity. Two viruses (herpesvirus, calicivirus) and two bacteria (Chlamydia, Bordetella) are the main pathogens involved. In certain infections, such as neonatal ophthalmia, viral rhinotracheitis, herpetic keratitis, symblepharon, corneal necrosis and corneal sequestrum, clinical signs are sometimes sufficient to make a diagnosis, especially if supported by a significant history. However, the etiological diagnosis of conjunctivitis and keratoconjunctivitis needs further tests. Conjunctival cytology is not specific enough, and serological testing is of little interest due to the wide variability of post-infectious antibody levels. Viral culture is too expensive for a regular use. PCR testing is now the gold standard and is essential to monitor the cat population. However, due to the specific biology of the herpes virus, PCR results in this instance are not easy to interpret, and can produce false positives and false negatives. An etiological diagnosis requires a joint study of biological results and clinical observations.Les infections respiratoires supérieures et oculaires félines constituent un complexe clinique très fréquemment observé. Des virus (herpès et calicivirus) et des bactéries (Chlamydia et Bordetella) sont les agents pathogènes responsables dominants. Les signes cliniques, surtout s'ils sont appuyés sur des commémoratifs concordants, sont significatifs dans certaines infections telles que l'ophtalmie néonatale, le coryza, les kératites herpétiques, les symblépharon, la nécrose cornéenne et le séquestre cornéen. Cependant, le diagnostic étiologique des conjonctivites et des kératoconjonctivites nécessite des examens complémentaires. La cytologie conjonctivale n'est pas spécifique et la sérologie présente peu d'intérêt en raison de la variabilité des titres post-infectieux. Les techniques de culture virale ont un coût prohibitif. Les examens par PCR ont pris un essor considérable et sont essentiels dans la surveillance des effectifs. Cependant, la biologie du virus herpès rend l'interprétation difficile avec des réponses faussement positives et faussement négatives. Seule l'étude conjointe des observations cliniques et des examens biologiques permet le diagnostic étiologique

0232: A new role of the brain natriuretic peptide in the heart: Modulation of cardiac precursor cell proliferation and differentiation

The actual role of the brain natriuretic peptide (BNP) in the heart remains elusive despite its reported protective effect in ischemic animal hearts. Because recently BNP was shown to control the proliferation and differentiation of murine embryonic stem cells, we asked in this study whether BNP could influence the proliferation and differentiation of cardiac progenitor cells (CPC) in vitro and in vivo. We first identified a c-kit+ Sca-1+ cell population present in neonatal and adult hearts which expressed the NPR-A and NPR-B receptors. In vitro, these cells proliferated and in presence of BNP differentiated into CPCs (c-kit+ Sca-1+ Nkx2.5+) and into mature cardiomyocytes. In parallel, BNP was injected to newborn and adult healthy mice (n=6 mice per group). In the hearts of both neonatal and adult mice, BNP injection increased the number of newly formed cardiomyocytes (neonatal: + 23%, p= 0.009 and adult: +68%, p= 0.005) and the number of CPCs (neonatal: + 142%, p= 0.002 and adult: +134%, p= 0.04). BrdU injection to neonatal BNP treated mice demonstrated that BNP stimulated CPC proliferation. In anticipation that BNP might be used as a therapeutic agent, we injected BNP into mice undergoing myocardial infarction (n=6-7 mice per group). Higher numbers of Nkx2.5+ cells were detected in both the infarcted (+38%, p=0.03) and non infarcted areas (+69%, p=0.02) of BNP treated hearts one week after surgery. Finally, by isolating neonatal cardiac cells from the hearts of NPR-A or NPR-B deficient mice, we demonstrated that BNP modulates the fate of CPCs via NPRB binding and that long term BNP treatment is correlated in vitro and in vivo with decreased Protein Kinase G activity. Our results highlight a new key role for BNP in the control of CPC proliferation and/or differentiation. This new function of BNP should be evaluated in therapies aimed to induce cardiac cell regeneration and should reopen the debate about the therapeutic use of BNP for patients suffering from heart diseases

Peroxynitrite induces HMGB1 release by cardiac cells in vitro and HMGB1 upregulation in the infarcted myocardium in vivo

Aims High-mobility group box 1 (HMGB1) is a nuclear protein actively secreted by immune cells and passively released by necrotic cells that initiates pro-inflammatory signalling through binding to the receptor for advance glycation end-products. HMGB1 has been established as a key inflammatory mediator during myocardial infarction, but the proximal mechanisms responsible for myocardial HMGB1 expression and release in this setting remain unclear. Here, we investigated the possible involvement of peroxynitrite, a potent cytotoxic oxidant formed during myocardial infarction, on these processes. Methods and results The ability of peroxynitrite to induce necrosis and HMGB1 release in vitro was evaluated in H9c2 cardiomyoblasts and in primary murine cardiac cells (myocytes and non-myocytes). In vivo, myocardial HMGB1 expression and nitrotyrosine content (a marker of peroxynitrite generation) were determined following myocardial ischaemia and reperfusion in rats, whereas peroxynitrite formation was inhibited by two different peroxynitrite decomposition catalysts: 5,10,15,20-tetrakis(4-sulphonatophenyl) porphyrinato iron (III) (FeTPPS) or Mn(III)-tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP). In all types of cells studied, peroxynitrite (100 μM) elicited significant necrosis, the loss of intracellular HMGB1, and its passive release into the medium. In vivo, myocardial ischaemia-reperfusion induced significant myocardial necrosis, cardiac nitrotyrosine formation, and marked overexpression of myocardial HMGB1. FeTPPS reduced nitrotyrosine, decreased infarct size, and suppressed HMGB1 overexpression, an effect that was similarly obtained with MnTBAP. Conclusion These findings indicate that peroxynitrite represents a key mediator of HMGB1 overexpression and release by cardiac cells and provide a novel mechanism linking myocardial oxidative/nitrosative stress with post-infarction myocardial inflammatio

Bacterial flagellin elicits widespread innate immune defense mechanisms, apoptotic signaling, and a sepsis-like systemic inflammatory response in mice

Introduction: Systemic inflammation in sepsis is initiated by interactions between pathogen molecular motifs and specific host receptors, especially toll-like receptors (TLRs). Flagellin is the main flagellar protein of motile microorganisms and is the ligand of TLR5. The distribution of TLR5 and the actions of flagellin at the systemic level have not been established. Therefore, we determined TLR5 expression and the ability of flagellin to trigger prototypical innate immune responses and apoptosis in major organs from mice. Methods: Male Balb/C mice (n = 80) were injected intravenously with 1-5 mu g recombinant Salmonella flagellin. Plasma and organ samples were obtained after 0.5 to 6 h, for molecular investigations. The expression of TLR5, the activation state of nuclear factor kappa B (NF kappa B) and mitogen-activated protein kinases (MAPKs) [extracellular related kinase (ERK) and c-jun-NH2 terminal kinase (JNK)], the production of cytokines [tumor necrosis alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), macrophage inhibitory protein-2 (MIP-2) and soluble triggering receptor expressed on myeloid cells (TREM-1)], and the apoptotic cleavage of caspase-3 and its substrate Poly(ADP-ribose) polymerase (PARP) were determined in lung, liver, gut and kidney at different time-points. The time-course of plasma cytokines was evaluated up to 6 h after flagellin. Results: TLR5 mRNA and protein were constitutively expressed in all organs. In these organs, flagellin elicited a robust activation of NF kappa B and MAPKs, and induced significant production of the different cytokines evaluated, with slight interorgan variations. Plasma TNF alpha, IL-6 and MIP-2 disclosed a transient peak, whereas IL-1 beta and soluble TREM-1 steadily increased over 6 h. Flagellin also triggered a marked cleavage of caspase-3 and PARP in the intestine, pointing to its ability to promote significant apoptosis in this organ. Conclusions: Bacterial flagellin elicits prototypical innate immune responses in mice, leading to the release of multiple pro-inflammatory cytokines in the lung, small intestine, liver and kidney, and also activates apoptotic signalling in the gut. Therefore, this bacterial protein may represent a critical mediator of systemic inflammation and intestinal barrier failure in sepsis due to flagellated micro-organism

Targeted Magnetic Intra-Lysosomal Hyperthermia produces lysosomal reactive oxygen species and causes Caspase-1 dependent cell death

Therapeutic strategies using drugs which cause Lysosomal Cell Death have been proposed for eradication of resistant cancer cells. In this context, nanotherapy based on Magnetic Intra-Lysosomal Hyperthermia (MILH) generated by magnetic nanoparticles (MNPs) that are grafted with ligands of receptors overexpressed in tumors appears to be a very promising therapeutic option. However, mechanisms whereby MILH induces cell death are still elusive. Herein, using Gastrin-grafted MNPs specifically delivered to lysosomes of tumor cells from different cancers, we provide evidences that MILH causes cell death through a non-apoptotic signaling pathway. The mechanism of cell death involves a local temperature elevation at the nanoparticle periphery which enhances the production of reactive oxygen species through the lysosomal Fenton reaction. Subsequently, MILH induces lipid peroxidation, lysosomal membrane permeabilization and leakage of lysosomal enzymes into the cytosol, including Cathepsin-B which activates Caspase-1 but not apoptotic Caspase-3. These data highlight the clear potential of MILH for the eradication of tumors overexpressing receptors