Implication of chromosome 13 on hypertension and associated disorders in Lyon hypertensive rats.

BACKGROUND: Hypertension and associated disorders are major risk factors for cardiovascular disease. The Lyon hypertensive rat (LH) is a genetically hypertensive strain that exhibits spontaneous and salt-sensitive hypertension, exaggerated proteinuria, high body weight, hyperlipidemia, and elevated insulin-to-glucose ratio. Previous genetic mapping identified quantitative trait loci (QTLs) influencing blood pressure (BP) on rat chromosome 13 (RNO13) in several models of hypertension. METHODS: To study the effects of a single chromosome on the mapped traits, we generated consomic strains by substituting LH RNO13 with that of the normotensive Brown Norway (BN) strain (LH-13BN) and reciprocal consomics by substituting a BN RNO13 with that of LH (BN-13LH). These reciprocal consomic strains, as well as the two parental strains were characterized for BP, metabolic and morphological parameters. RESULTS: Compared with LH parents, LH-13BN rats showed decreased mean BP (up to -24 mmHg on 2% NaCl in the drinking water), urine proteins and lipids, and increased body weight. Differences between BN-13LH and BN rats were much smaller than those observed between LH-13BN and LH rats, demonstrating the effects of the highly resistant BN genome background. Plasma renin activity was not affected by the substitution of RNO13, despite the significant BP differences. CONCLUSION: The present work demonstrates that RNO13 is a determinant of BP, proteinuria, and plasma lipids in the LH rat. The distinct phenotypic differences between the consomic LH-13BN and the LH make it a powerful model to determine genes and pathways leading to these risk factors for cardiovascular and renal disease.

The fate and persistence of Leishmania major in mice of different genetic backgrounds: an example of exploitation of the immune system by intracellular parasites.

Leishmania spp. are intracellular protozoan parasites that are delivered within the dermis of their vertebrate hosts. Within this peripheral tissue and the draining lymph node, they find and/or rapidly create dynamic microenvironments that determine their ultimate fate, namely their more or less successful expansion, and favour their transmission to another vertebrate host though a blood-feeding vector. Depending on their genetic characteristics as well as the genetic make-up of their hosts, once within the dermis Leishmania spp. very rapidly drive and maintain sustained T cell-dependent immune responses that arbitrate their ultimate fate within their hosts. The analysis of the parasitism exerted by Leishmania major in mice of different genetic backgrounds has allowed us to recognize some of the early and late mechanisms driven by this parasite that lead to either uncontrolled or restricted parasitism. Uncontrolled parasitism by Leishmania major characterizing mice from a few inbred strains (e.g. BALB/c) is associated with the expansion of parasite reactive Th2 CD4 lymphocytes and results from their rapid and sustained activity. In contrast, restricted parasitism characteristic of mice from the majority of inbred strains results from the development of a polarized parasite-specific Th1 CD4 response. This murine model of infection has already been and will continue to be particularly instrumental in dissecting the rules controlling the pathway of differentiation of T cells in vivo. In the long run, the understanding of these rules should contribute to the rational development of novel immunotherapeutic interventions against severe infectious diseases.

Transmyocardial laser revascularisation in acutely ischaemic myocardium.

OBJECTIVE: Although recent experience suggests that transmyocardial laser revascularisation (TMLR) relieves angina, its mechanism of action remains undefined. We examined its functional effects and analysed its morphological features in an animal model of acute ischaemia. METHODS: A total of 15 pigs were randomised to ligation of left marginal arteries (infarction group, n = 5), to TMLR of the left lateral wall using a holmium:yttrium-aluminium garnet (Ho:YAG) laser (laser group, n = 5), and to both (laser-infarction group, n = 5). All the animals were sacrificed 1 month after the procedure. Haemodynamics and echocardiography with segmental wall motion score were carried out at both time intervals (scale 0-3: 0, normal; 1, hypokinesia; 2, akinesia; 3, dyskinesia). Histology of the involved area was analysed. RESULTS: Laser group showed no change of the segmental wall motion score of the involved area 30 min after the laser channels were made (score: 0 +/- 0). Infarction and laser infarction groups both showed a persistent and definitive increase of the segmental wall motion score (at 30 min: 1.6 +/- 0.3 and 2 +/- 0, respectively; at 1 month: 1.8 +/- 0.2 and 1.8 +/- 0.4, respectively). These increases were all statistically significant in comparison with baseline values (P < 0.5), however comparison between infarction and laser-infarction groups showed no significant difference. On macroscopic examination of the endocardial surface, no channel was opened. On histology, there were signs of neovascularisation around the channels in the laser group, whereas in the laser-infarction group the channels were embedded in the infarction scar. CONCLUSIONS: In this acute pig model, TMLR did not provide improvement of contractility of the ischaemic myocardium. To the degree that the present study pertains to the clinical setting, the results suggest that mechanisms other than blood flow through the channels should be considered, such as a laser-induced triggering of neovascularisation or neural destruction.

Specific inhibition of HCN channels slows rhythm differently in atria, ventricle and outflow tract and stabilizes conduction in the anoxic-reoxygenated embryonic heart model.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in pacemaker cells very early during cardiogenesis. This work aimed at determining to what extent these channels are implicated in the electromechanical disturbances induced by a transient oxygen lack which may occur in utero. Spontaneously beating hearts or isolated ventricles and outflow tracts dissected from 4-day-old chick embryos were exposed to a selective inhibitor of HCN channels (ivabradine 0.1-10microM) to establish a dose-response relationship. The effects of ivabradine on electrocardiogram, excitation-contraction coupling and contractility of hearts submitted to anoxia (30min) and reoxygenation (60min) were also determined. The distribution of the predominant channel isoform, HCN4, was established in atria, ventricle and outflow tract by immunoblotting. Intrinsic beating rate of atria, ventricle and outflow tract was 164+/-22 (n=10), 78+/-24 (n=8) and 40+/-12bpm (n=23, mean+/-SD), respectively. In the whole heart, ivabradine (0.3microM) slowed the firing rate of atria by 16% and stabilized PR interval. These effects persisted throughout anoxia-reoxygenation, whereas the variations of QT duration, excitation-contraction coupling and contractility, as well as the types and duration of arrhythmias were not altered. Ivabradine (10microM) reduced the intrinsic rate of atria and isolated ventricle by 27% and 52%, respectively, whereas it abolished activity of the isolated outflow tract. Protein expression of HCN4 channels was higher in atria and ventricle than in the outflow tract. Thus, HCN channels are specifically distributed and control finely atrial, ventricular and outflow tract pacemakers as well as conduction in the embryonic heart under normoxia and throughout anoxia-reoxygenation.

Expression of brain-derived neurotrophic factor is not modulated by chronic mild stress in the rat hippocampus and amygdala.

Accumulating evidence supports a role for brain-derived neurotrophic factor (BDNF) in depression. However, most of these studies have been performed in animal models that have a low face validity with regard to the human disease. Here, we examined the regulation of BDNF expression in the hippocampus and amygdala of rats subjected to the chronic mild stress (CMS) model of depression, a paradigm that induces anhedonia, a core symptom of depression. We found that exposure of rats to the CMS paradigm did not modulate BDNF mRNA expression in the hippocampus and amygdala. In addition, chronic administration of imipramine, which reversed CMS-induced anhedonia, did not alter BDNF mRNA expression in these limbic structures.

Triggering of Bcl-2-related pathway is associated with apoptosis of photoreceptors in Rpe65-/- mouse model of Leber's congenital amaurosis.

Mutations in RPE65 protein is characterized by the loss of photoreceptors, although the molecular pathways triggering retinal cell death remain largely unresolved. The role of the Bcl-2 family of proteins in retinal degeneration is still controversial. However, alteration in Bcl-2-related proteins has been observed in several models of retinal injury. In particular, Bax has been suggested to play a crucial role in apoptotic pathways in murine glaucoma model as well as in retinal detachment-associated cell death. We demonstrated that Bcl-2-related signaling pathway is involved in Rpe65-dependent apoptosis of photoreceptors during development of the disease. Pro-apoptotic Bax alpha and beta isoforms were upregulated in diseased retina. This was associated with a progressive reduction of anti-apoptotic Bcl-2, reflecting imbalanced Bcl-2/Bax ratio as the disease progresses. Moreover, specific translocation of Bax beta from cytosol to mitochondria was observed in Rpe65-deficient retina. This correlated with the initiation of photoreceptor cell loss at 4 months of age, and further increased during disease development. Altogether, these data suggest that Bcl-2-apoptotic pathway plays a crucial role in Leber's congenital amaurosis disease. They further highlight a new regulatory mechanism of Bax-dependent apoptosis based on regulated expression and activation of specific isoforms of this protein.

Protective effect of intravitreal injection of vasoactive intestinal peptide-loaded liposomes on experimental autoimmune uveoretinitis.

PURPOSE: The aim of this study was to investigate the effect of a single intravitreal (i.v.t.) injection of vasoactive intestinal peptide (VIP) loaded in rhodamine-conjugated liposomes (VIP-Rh-Lip) on experimental autoimmune uveoretinitis (EAU). METHODS: An i.v.t. injection of VIP-Rh-Lip, saline, VIP, or empty-(E)-Rh-Lip was performed simultaneously, either 6 or 12 days after footpad immunization with retinal S-antigen in Lewis rats. Clinical and histologic scores were determined. Immunohistochemistry and cytokine quantification by multiplex enzyme-linked immunosorbent assay were performed in ocular tissues. Systemic immune response was determined at day 20 postimmunization by measuring proliferation and cytokine secretion of cells from inguinal lymph nodes (ILNs) draining the immunization site, specific delayed-type hypersensitivity (DTH), and the serum concentration of cytokines. Ocular and systemic biodistribution of VIP-Rh-Lip was studied in normal and EAU rats by immunofluorescence. RESULTS: The i.v.t. injection of VIP-Rh-Lip performed during the afferent, but not the efferent, phase of the disease reduced clinical EAU and protected against retinal damage. No effect was observed after saline, E-Rh-Lip, or VIP injection. VIP-Rh-Lip and VIP were detected in intraocular macrophages and in lymphoid organs. In VIP-Rh-Lip-treated eyes, macrophages expressed transforming growth factor-beta2, low levels of major histocompatibility complex class II, and nitric oxide synthase-2. T-cells showed activated caspase-3 with the preservation of photoreceptors. Intraocular levels of interleukin (IL)-2, interferon-gamma (IFN-gamma), IL-17, IL-4, GRO/KC, and CCL5 were reduced with increased IL-13. At the systemic level, treatment reduced retinal soluble autoantigen lymphocyte proliferation, decreased IL-2, and increased IL-10 in ILN cells, and diminished specific DTH and serum concentration of IL-12 and IFN-gamma. CONCLUSIONS: An i.v.t. injection of VIP-Rh-Lip, performed during the afferent stage of immune response, reduced EAU pathology through the immunomodulation of intraocular macrophages and deviant stimulation of T-cells in ILN. Thus, the encapsulation of VIP within liposomes appears as an effective strategy to deliver VIP into the eye and is an efficient means of the prevention of EAU severity.

Selective inhibition of JNK with a peptide inhibitor attenuates pain hypersensitivity and tumor growth in a mouse skin cancer pain model.

Cancer pain significantly affects the quality of cancer patients, and current treatments for this pain are limited. C-Jun N-terminal kinase (JNK) has been implicated in tumor growth and neuropathic pain sensitization. We investigated the role of JNK in cancer pain and tumor growth in a skin cancer pain model. Injection of luciferase-transfected B16-Fluc melanoma cells into a hindpaw of mouse induced robust tumor growth, as indicated by increase in paw volume and fluorescence intensity. Pain hypersensitivity in this model developed rapidly (<5 days) and reached a peak in 2 weeks, and was characterized by mechanical allodynia and heat hyperalgesia. Tumor growth was associated with JNK activation in tumor mass, dorsal root ganglion (DRG), and spinal cord and a peripheral neuropathy, such as loss of nerve fibers in the hindpaw skin and induction of ATF-3 expression in DRG neurons. Repeated systemic injections of D-JNKI-1 (6 mg/kg, i.p.), a selective and cell-permeable peptide inhibitor of JNK, produced an accumulative inhibition of mechanical allodynia and heat hyperalgesia. A bolus spinal injection of D-JNKI-1 also inhibited mechanical allodynia. Further, JNK inhibition suppressed tumor growth in vivo and melanoma cell proliferation in vitro. In contrast, repeated injections of morphine (5 mg/kg), a commonly used analgesic for terminal cancer, produced analgesic tolerance after 1 day and did not inhibit tumor growth. Our data reveal a marked peripheral neuropathy in this skin cancer model and important roles of the JNK pathway in cancer pain development and tumor growth. JNK inhibitors such as D-JNKI-1 may be used to treat cancer pain.

Measurement of plasma endothelin-1 in experimental hypertension and in healthy subjects.

BACKGROUND: Endothelin-1 is an endothelium-derived potent vasoconstrictor peptide of 21 amino acids. To establish reference values in different models of hypertension and in human subjects an assay for plasma immunoreactive endothelin-1 (ET-1) was optimized. METHODS: ET-1 is extracted by acetone from 1 mL of plasma and subjected to a sensitive enzyme-linked immunosorbent assay. RESULTS: The detection limit for plasma ET-1 is 0.05 fmol/mL. Mean recoveries of the 1, 2, 5, and 10 fmol of ET-1 added to 1 mL of plasma were 66%, 75%, 85%, and 92%, respectively. Within- and between-assay coefficients of variation were < or =12% and < or =10%, respectively. Assay accuracy was demonstrated by consistent recoveries of added ET-1 over the entire physiologic range of plasma concentrations and by the linearity of ET-1 concentrations measured in serially diluted plasma extracts (r = 0.99). No ET-1 was detected when albumin buffer was extracted instead of plasma. Using this method, we found increased ET-1 levels in plasma of three experimental rat models of hypertension: stroke prone spontaneously hypertensive rats (SP-SHR), deoxycorticosterone acetate-salt hypertensive rats, and one kidney-one clip hypertensive rats. In contrast, plasma ET-1 levels of SHR were half those of normotensive Wistar rats. In two kidney-one clip hypertensive rats, plasma ET-1 concentrations were not different from those found in sham-operated control rats. Plasma ET-1 concentrations of 37 healthy men were 0.85 +/- 0.26 fmol/ml (mean +/- SD). CONCLUSIONS: The present assay reliably measures ET-1 levels in rat and human plasma. It allows to discriminate between different forms of hypertension with high or low circulating levels of ET-1.

CD4+CD25+ T cell depletion impairs tolerance induction in a murine model of asthma.

BACKGROUND: Regulatory T cells (Tregs) are key players in controlling the development of airway inflammation. However, their role in the mechanisms leading to tolerance in established allergic asthma is unclear. OBJECTIVE: To examine the role of Tregs in tolerance induction in a murine model of asthma. METHODS: Ovalbumin (OVA) sensitized asthmatic mice were depleted or not of CD25(+) T cells by anti-CD25 PC61 monoclonal antibody (mAb) before intranasal treatment (INT) with OVA, then challenged with OVA aerosol. To further evaluate the respective regulatory activity of CD4(+)CD25(+) and CD4(+)CD25(-) T cells, both T cell subsets were transferred from tolerized or non-tolerized animals to asthmatic recipients. Bronchoalveolar lavage fluid (BALF), T cell proliferation and cytokine secretion were examined. RESULTS: Intranasal treatment with OVA led to increased levels of IL-10, TGF-beta and IL-17 in lung homogenates, inhibition of eosinophil recruitment into the BALF and antigen specific T cell hyporesponsiveness. CD4(+)CD25(+)Foxp3(+) T cells were markedly upregulated in lungs and suppressed in vitro and in vivo OVA-specific T cell responses. Depletion of CD25(+) cells before OVA INT severely hampered tolerance induction as indicated by a strong recruitment of eosinophils into BALF and a vigorous T cell response to OVA upon challenge. However, the transfer of CD4(+)CD25(-) T cells not only suppressed antigen specific T cell responsiveness but also significantly reduced eosinophil recruitment as opposed to CD4(+)CD25(+) T cells. As compared with control mice, a significantly higher proportion of CD4(+)CD25(-) T cells from OVA treated mice expressed mTGF-beta. CONCLUSION: Both CD4(+)CD25(+) and CD4(+)CD25(-) T cells appear to be essential to tolerance induction. The relationship between both subsets and the mechanisms of their regulatory activity will have to be further analyzed.

Cytokines in parasitic diseases: the example of cutaneous leishmaniasis.

The essential role of cytokines in parasitic diseases has been emphasised since the in vivo description of the importance of T helper 1 (Th1) and T helper 2 (Th2) CD4+ T cell responses in resistance and susceptibility to infection with L. major in mice. Th1 cells produced IL-2, IFN-gamma and Lymphotoxin T (LT) and Th2 cells produce IL-4, IL-5 and IL-13. In this model of infection the correlation between on the one hand resistance to infection and the development of a Th1 response and on the other hand susceptibility and Th2 cell development allowed the identification of the mechanisms directing the differentiation of CD4+ T cell precursors towards either Th1 type or Th2 type responses. Cytokines are the crucial inducer of functional CD4+ T cell subset differentiation during infection with L. major. IL-12 and IFN-gamma direct the differentiation of Th1 response and IL-4 of a Th2 response. In susceptible mice, careful analysis of IL-4 production during the first days of infection has shown that the IL-4 produced as a result of a very early burst of IL-4 mRNA expression (16 hours) plays a essential role in the maturation of a Th2 CD4+ T cell response by rendering the CD4+ T cell precursors unresponsive to IL-12. Activation of a restricted population of CD4+ T cells expressing the V beta 4 V alpha 8 TCR heterodimer after recognition of a single antigen, the LACK (Leishmania Activated c Kinase) antigen, resulted in this rapid production of IL-4 required for the subsequent CD4+ T cell differentiation. Thus, tolerization of these cells might contribute a strategy for preventing infection with L. major.

Infectivity of Lactobacillus rhamnosus and Lactobacillus paracasei isolates in a rat model of experimental endocarditis.

The potential pathogenicity of selected (potentially) probiotic and clinical isolates of Lactobacillus rhamnosus and Lactobacillus paracasei was investigated in a rat model of experimental endocarditis. In addition, adhesion properties of the lactobacilli for fibrinogen, fibronectin, collagen and laminin, as well as the killing activity of the platelet-microbicidal proteins fibrinopeptide A (FP-A) and connective tissue activating peptide 3 (CTAP-3), were assessed. The 90 % infective dose (ID(90)) of the L. rhamnosus endocarditis isolates varied between 10(6) and 10(7) c.f.u., whereas four of the six (potentially) probiotic L. rhamnosus isolates showed an ID(90) that was at least 10-fold higher (10(8) c.f.u.) (P<0.001). In contrast, the two other probiotic L. rhamnosus isolates exhibited an ID(90) (10(6) and 10(7) c.f.u.) comparable to the ID(90) of the clinical isolates of this species investigated (P>0.05). Importantly, these two probiotic isolates shared the same fluorescent amplified fragment length polymorphism cluster type as the clinical isolate showing the lowest ID(90) (10(6) c.f.u.). L. paracasei tended to have a lower infectivity than L. rhamnosus (ID(90) of 10(7) to > or =10(8) c.f.u.). All isolates had comparable bacterial counts in cardiac vegetations (P>0.05). Except for one L. paracasei strain adhering to all substrates, all tested lactobacilli adhered only weakly or not at all. The platelet peptide FP-A did not show any microbicidal activity against the tested lactobacilli, whereas CTAP-3 killed the majority of the isolates. In general, these results indicate that probiotic lactobacilli display a lower infectivity in experimental endocarditis compared with true endocarditis pathogens. However, the difference in infectivity between L. rhamnosus endocarditis and (potentially) probiotic isolates could not be explained by differences in adherence or platelet microbicidal protein susceptibility. Other disease-promoting factors may exist in these organisms and warrant further investigation.

HIV-1 vaccines and adaptive trial designs.

Developing a vaccine against the human immunodeficiency virus (HIV) poses an exceptional challenge. There are no documented cases of immune-mediated clearance of HIV from an infected individual, and no known correlates of immune protection. Although nonhuman primate models of lentivirus infection have provided valuable data about HIV pathogenesis, such models do not predict HIV vaccine efficacy in humans. The combined lack of a predictive animal model and undefined biomarkers of immune protection against HIV necessitate that vaccines to this pathogen be tested directly in clinical trials. Adaptive clinical trial designs can accelerate vaccine development by rapidly screening out poor vaccines while extending the evaluation of efficacious ones, improving the characterization of promising vaccine candidates and the identification of correlates of immune protection.

Photodynamic therapy as an adjunct to surgery for malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is increasingly observed in industrial countries. Despite concerted efforts and combined treatments including surgery, chemotherapy and irradiation patients eventually succumb from relentless local progression of the disease. Recent publications have demonstrated an improved response rate with the cytostatic agent pemetrexed which will be tested in a neoadjuvant setting followed by surgery. However, effective tumor control requires new loco-regional treatment modalities, eventually in combination with neoadjuvant chemotherapy. Intraoperative photodynamic therapy (PDT) of the chest cavity has been proposed as an attractive treatment concept for MPM since a selective treatment of the tumor bed following resection has the potential to improve local tumor control. It has been shown to afford tumor destruction in patients with mesothelioma but efficiency and selectivity is not yet sufficient for routine clinical application. Experimental work on MPM has shown that tumor selectivity of PDT depend on treatment conditions and can be improved by structural modification and improved targeting of the sensitizers. Refinements of PDT for mesothelioma will depend on a more detailed understanding of the pathways for preferential sensitizer accumulation within the tumor as well as on synergistic effects between PDT and chemotherapeutic agents.

A peptide inhibitor of c-Jun N-terminal kinase protects against both aminoglycoside and acoustic trauma-induced auditory hair cell death and hearing loss.

Hearing loss can be caused by a variety of insults, including acoustic trauma and exposure to ototoxins, that principally effect the viability of sensory hair cells via the MAP kinase (MAPK) cell death signaling pathway that incorporates c-Jun N-terminal kinase (JNK). We evaluated the otoprotective efficacy of D-JNKI-1, a cell permeable peptide that blocks the MAPK-JNK signal pathway. The experimental studies included organ cultures of neonatal mouse cochlea exposed to an ototoxic drug and cochleae of adult guinea pigs that were exposed to either an ototoxic drug or acoustic trauma. Results obtained from the organ of Corti explants demonstrated that the MAPK-JNK signal pathway is associated with injury and that blocking of this signal pathway prevented apoptosis in areas of aminoglycoside damage. Treatment of the neomycin-exposed organ of Corti explants with D-JNKI-1 completely prevented hair cell death initiated by this ototoxin. Results from in vivo studies showed that direct application of D-JNKI-1 into the scala tympani of the guinea pig cochlea prevented nearly all hair cell death and permanent hearing loss induced by neomycin ototoxicity. Local delivery of D-JNKI-1 also prevented acoustic trauma-induced permanent hearing loss in a dose-dependent manner. These results indicate that the MAPK-JNK signal pathway is involved in both ototoxicity and acoustic trauma-induced hair cell loss and permanent hearing loss. Blocking this signal pathway with D-JNKI-1 is of potential therapeutic value for long-term protection of both the morphological integrity and physiological function of the organ of Corti during times of oxidative stress.