Nitrosative stress and corneal transplant endothelial cell death during acute graft rejection.

BACKGROUND: Nitrosative stress takes place in endothelial cells (EC) during corneal acute graft rejection. The purpose of this study was to evaluate the potential role of peroxynitrite on corneal EC death. METHODS: The effect of peroxynitrite was evaluated in vivo. Fifty, 250, and 500 microM in 1.5 microL of the natural or denatured peroxynitrite in 50 microM NaOH, 50 microM NaOH alone, or balanced salt solution were injected into the anterior chamber of rat eyes (n=3/group). Corneal toxic signs after injection were assessed by slit-lamp, in vivo confocal imaging, pachymetry, and EC count. The effect of peroxynitrite was also evaluated on nitrotyrosine and leucocyte elastase inhibitor/LDNase II immunohistochemistry. Human corneas were incubated with peroxynitrite and the effect on EC viability was evaluated. A specific inducible nitric oxide synthase inhibitor (iNOS) was administered systemically in rats undergoing allogeneic corneal graft rejection and the effect on EC was evaluated by EC count. RESULTS: Rat eyes receiving as little as 50 microM peroxynitrite showed a specific dose-dependent toxicity on EC. We observed an intense nitrotyrosine staining of human and rat EC exposed to peroxynitrite associated with leucocyte elastase inhibitor nuclear translocation, a noncaspase dependent apoptosis reaction. Specific inhibition of iNOS generation prevented EC death and enhanced EC survival of the grafted corneas. However, inhibition of iNOS did not have a significant influence on the incidence of graft rejection. CONCLUSION: Nitrosative stress during acute corneal graft rejection in rat eyes induces a noncaspase dependent apoptotic death in EC. Inhibition of nitric oxide production during the corneal graft rejection has protective effects on the corneal EC survival.

Dominant negative MyD88 proteins inhibit interleukin-1beta /interferon-gamma -mediated induction of nuclear factor kappa B-dependent nitrite production and apoptosis in beta cells.

Insulin-dependent diabetes mellitus is an autoimmune disease in which pancreatic islet beta cells are destroyed by a combination of immunological and inflammatory mechanisms. In particular, cytokine-induced production of nitric oxide has been shown to correlate with beta cell apoptosis and/or inhibition of insulin secretion. In the present study, we investigated whether the interleukin (IL)-1beta intracellular signal transduction pathway could be blocked by overexpression of dominant negative forms of the IL-1 receptor interacting protein MyD88. We show that overexpression of the Toll domain or the lpr mutant of MyD88 in betaTc-Tet cells decreased nuclear factor kappaB (NF-kappaB) activation upon IL-1beta and IL-1beta/interferon (IFN)-gamma stimulation. Inducible nitric oxide synthase mRNA accumulation and nitrite production, which required the simultaneous presence of IL-1beta and IFN-gamma, were also suppressed by approximately 70%, and these cells were more resistant to cytokine-induced apoptosis as compared with parental cells. The decrease in glucose-stimulated insulin secretion induced by IL-1beta and IFN-gamma was however not prevented. This was because these dysfunctions were induced by IFN-gamma alone, which decreased cellular insulin content and stimulated insulin exocytosis. These results demonstrate that IL-1beta is involved in inducible nitric oxide synthase gene expression and induction of apoptosis in mouse beta cells but does not contribute to impaired glucose-stimulated insulin secretion. Furthermore, our data show that IL-1beta cellular actions can be blocked by expression of MyD88 dominant negative proteins and, finally, that cytokine-induced beta cell secretory dysfunctions are due to the action of IFN-gamma.

Long-term use and tolerability of irbesartan for control of hypertension.

In this review, we discuss the pharmacological and clinical properties of irbesartan, a noncompetitive angiotensin II receptor type 1 antagonist, successfully used for more than a decade in the treatment of essential hypertension. Irbesartan exerts its antihypertensive effect through an inhibitory effect on the pressure response to angiotensin II. Irbesartan 150-300 mg once daily confers a lasting effect over 24 hours, and its antihypertensive efficacy is further enhanced by the coadministration of hydrochlorothiazide. Additionally and partially beyond its blood pressure-lowering effect, irbesartan reduces left ventricular hypertrophy, favors right atrial remodeling in atrial fibrillation, and increases the likelihood of maintenance of sinus rhythm after cardioversion in atrial fibrillation. In addition, the renoprotective effects of irbesartan are well documented in the early and later stages of renal disease in type 2 diabetics. Furthermore, both the therapeutic effectiveness and the placebo-like side effect profile contribute to a high adherence rate to the drug. Currently, irbesartan in monotherapy or combination therapy with hydrochlorothiazide represent a rationale pharmacologic approach for arterial hypertension and early-stage and late-stage diabetic nephropathy in hypertensive type II diabetics.

The NLRP3 inflammasome in health and disease: the good, the bad and the ugly.

While interleukin (IL)-1β plays an important role in combating the invading pathogen as part of the innate immune response, its dysregulation is responsible for a number of autoinflammatory disorders. Large IL-1β activating platforms, known as inflammasomes, can assemble in response to the detection of endogenous host and pathogen-associated danger molecules. Formation of these protein complexes results in the autocatalysis and activation of caspase-1, which processes precursor IL-1β into its secreted biologically active form. Inflammasome and IL-1β activity is required to efficiently control viral, bacterial and fungal pathogen infections. Conversely, excess IL-1β activity contributes to human disease, and its inhibition has proved therapeutically beneficial in the treatment of a spectrum of serious, yet relatively rare, heritable inflammasomopathies. Recently, inflammasome function has been implicated in more common human conditions, such as gout, type II diabetes and cancer. This raises the possibility that anti-IL-1 therapeutics may have broader applications than anticipated previously, and may be utilized across diverse disease states that are linked insidiously through unwanted or heightened inflammasome activity.

A protective cocktail vaccine against murine cutaneous leishmaniasis with DNA encoding cysteine proteinases of Leishmania major.

The protection elicited by the intramuscular injection of two plasmid DNAs encoding Leishmania major cysteine proteinase type I (CPb) and type II (CPa) was evaluated in a murine model of experimental cutaneous leishmaniasis. BALB/c mice were immunized either separately or with a cocktail of the two plasmids expressing CPa or CPb. It was only when the cpa and cpb genes were co-injected that long lasting protection against parasite challenge was achieved. Similar protection was also observed when animals were first immunized with cpa/cpb DNA followed by recombinant CPa/CPb boost. Analysis of the immune response showed that protected animals developed a specific Th1 immune response, which was associated with an increase of IFN-gamma production. This is the first report demonstrating that co-injection of two genes expressing different antigens induces a long lasting protective response, whereas the separate injection of cysteine proteases genes is not protective.

Ultrastructure of Endogenous Stages of Eimeria ninakohlyakimovae Yakimoff & Rastegaieff, 1930 Emend. Levine, 1961 in Experimentally Infected Goat

The ultrastructure of endogenous stages of Eimeria ninakohlyakimovae was observed in epithelial cells of cecum and colon crypts from a goat experimentally infected with 2.0 x 105 oocysts/kg. The secondary meronts developed above the nucleus of the host cell. The nucleus first divides and merozoites then form on the surface of multinucleated meronts. Free merozoites in the parasitophorous vacuole present a conoid, double membrane, one pair of rhoptries, micronemes, micropore, anterior and posterior polar ring, a nucleus with a nucleolus and peripheral chromatin. The microgamonts are located below the nucleus of the host cell and contain several nuclei at the periphery of the parasite. The microgametes consist of a body, a nucleus, three flagella and mitochondria. The macrogamonts develop below the nucleus of the host cell and have a large nucleus with a prominent nucleolus. The macrogametes contain a nucleus, wall-forming bodies of type I and type II. The young oocysts present a wall containing two layers and a sporont

Signaling in Dynamic Contests: Some Impossibility Results

General signaling results in dynamic Tullock contests have been missing for long. The reason is the tractability of the problems. In this paper, an uninformed contestant with valuation vx competes against an informed opponent with valuation, either high vh or low vl. We show that; (i) When the hierarchy of valuations is vh ≥ vx ≥ vl, there is no pooling. Sandbagging is too costly for the high type. (ii) When the order of valuations is vx ≥ vh ≥ vl, there is no separation if vh and vl are close. Sandbagging is cheap due to the proximity of valuations. However, if vh and vx are close, there is no pooling. First period cost of pooling is high. (iii) For valuations satisfying vh ≥ vl ≥ vx, there is no separation if vh and vl are close. Bluffing in the first period is cheap for the low valuation type. Conversely, if vx and vl are close there is no pooling. Bluffing in the first stage is too costly. JEL: C72, C73, D44, D82. KEYWORDS: Signaling, Dynamic Contests, Non-existence, Sandbag Pooling, Bluff Pooling, Separating

Relocalization of telomeric Ku and SIR proteins in response to DNA strand breaks in yeast.

Telomeric TG-rich repeats and their associated proteins protect the termini of eukaryotic chromosomes from end-to-end fusions. Associated with the cap structure at yeast telomeres is a subtelomeric domain of heterochromatin, containing the silent information regulator (SIR) complex. The Ku70/80 heterodimer (yKu) is associated both with the chromosome end and with subtelomeric chromatin. Surprisingly, both yKu and the chromatin-associated Rap1 and SIR proteins are released from telomeres in a RAD9-dependent response to DNA damage. yKu is recruited rapidly to double-strand cuts, while low levels of SIR proteins are detected near cleavage sites at later time points. Consistently, yKu- or SIR-deficient strains are hypersensitive to DNA-damaging agents. The release of yKu from telomeric chromatin may allow efficient scanning of the genome for DNA strand breaks.

Comparative Analysis by Polymerase Chain Reaction Amplified Minicircles of Kinetoplast DNA of a Stable Strain of Trypanosoma cruzi from São Felipe, Bahia, its Clones and Subclones: Possibility of Predominance of a Principal Clone in this Area

Molecular characterization of one stable strain of Trypanosoma cruzi, the 21 SF, representative of the pattern of strains isolated from the endemic area of São Felipe, State of Bahia, Brazil, maintained for 15 years in laboratory by serial passages in mice and classified as biodeme Type II and zymodeme 2 has been investigated. The kinetoplast DNA (kDNA) of parental strain, 5 clones and 14 subclones were analyzed. Schizodeme was established by comparative study of the fragments obtained from digestion of the 330-bp fragments amplified by polymerase chain reaction (PCR) from the variable regions of the minicicles, and digested by restriction endonucleases Rsa I and Hinf I. Our results show a high percentual of similarity between the restriction fragment lenght polymorphism (RFLP) for the parental strain and its clones and among these individual clones and their subclones at a level of 80 to 100%.This homology indicates a predominance of the same "principal clone" in the 21SF strain and confirms the homogeneity previously observed at biological and isozymic analysis. These results suggest the possibility that the T. cruzi strains with similar biological and isoenzymic patterns, circulating in this endemic area, are representative of one dominant clone. The presence of "principal clones" could be responsible for a predominant tropism of the parasites for specific organs and tissues and this could contribute to the pattern of clinico-pathological manifestations of Chagas's disease in one geographical area.

Long-term follow-up of four patients with Langer-Giedion syndrome: clinical course and complications.

Long-term observations of individuals with the so-called Langer-Giedion (LGS) or tricho-rhino-phalangeal type II (TRPS2) are scarce. We report here a on follow-up of four LGS individuals, including one first described by Andres Giedion in 1969, and review the sparse publications on adults with this syndrome which comprises ectodermal dysplasia, multiple cone-shaped epiphyses prior to puberty, multiple cartilaginous exostoses, and mostly mild intellectual impairment. LGS is caused by deletion of the chromosomal segment 8q24.11-q24.13 containing among others the genes EXT1 and TRPS1. Most patients with TRPS2 are only borderline or mildly cognitively delayed, and few are of normal intelligence. Their practical skills are better than their intellectual capability, and, for this reason and because of their low self-esteem, they are often underestimated. Some patients develop seizures at variable age. Osteomas on processes of cervical vertebrae may cause pressure on cervical nerves or dissection of cerebral arteries. Joint stiffness is observed during childhood and changes later to joint laxity causing instability and proneness to trauma. Perthes disease is not rare. Almost all males become bald at or soon after puberty, and some develop (pseudo) gynecomastia. Growth hormone deficiency was found in a few patients, TSH deficiency so far only in one. Puberty and fertility are diminished, and no instance of transmission of the deletion from a non-mosaic parent to a child has been observed so far. Several affected females had vaginal atresia with consequent hydrometrocolpos.

Down-regulation of NOSII gene expression by iontophoresis of anti-sense oligonucleotide in endotoxin-induced uveitis.

Transcorneoscleral iontophoresis was used to enhance ocular penetration of a 21-bp NH(2) protected anti-NOSII oligonucleotides (ODNs) (fluorescein or infrared-41 labeled) in Lewis rats. Both histochemical localization and acrylamide sequencing gels were used. To evaluate the potential to down-regulate NOSII expression in the rat model of endotoxin-induced uveitis (EIU), anti-sense NOSII ODN, scrambled ODN or saline were iontophorezed into these animals' eyes. Iontophoresis facilitated the penetration of intact ODNs into the intraocular tissues of the rat eye and only the eyes receiving ODNs and electrical current demonstrated intact ODNs within the ocular tissues of both segments of the eye. Iontophoresis of anti-NOSII ODN significantly down-regulated the expression of NOSII expression in iris/ciliary body compared to the saline or scrambled ODN treated eyes. Nitrite production was also significantly reduced in the anti-NOSII applied eyes compared to those treated with saline. Using this system, intraocular delivery of ODNs can be significantly enhanced increasing the potential for successful gene therapy for human eye diseases.

Functional analysis of pyochelin-/enantiopyochelin-related genes from a pathogenicity island of Pseudomonas aeruginosa strain PA14.

Genomic islands are foreign DNA blocks inserted in so-called regions of genomic plasticity (RGP). Depending on their gene content, they are classified as pathogenicity, symbiosis, metabolic, fitness or resistance islands, although a detailed functional analysis is often lacking. Here we focused on a 34-kb pathogenicity island of Pseudomonas aeruginosa PA14 (PA14GI-6), which is inserted at RGP5 and carries genes related to those for pyochelin/enantiopyochelin biosynthesis. These enantiomeric siderophores of P. aeruginosa and certain strains of Pseudomonas protegens are assembled by a thiotemplate mechanism from salicylate and two molecules of cysteine. The biochemical function of several proteins encoded by PA14GI-6 was investigated by a series of complementation analyses using mutants affected in potential homologs. We found that PA14_54940 codes for a bifunctional salicylate synthase/salicyl-AMP ligase (for generation and activation of salicylate), that PA14_54930 specifies a dihydroaeruginoic acid (Dha) synthetase (for coupling salicylate with a cysteine-derived thiazoline ring), that PA14_54910 produces a type II thioesterase (for quality control), and that PA14_54880 encodes a serine O-acetyltransferase (for increased cysteine availability). The structure of the PA14GI-6-specified metabolite was determined by mass spectrometry, thin-layer chromatography, and HPLC as (R)-Dha, an iron chelator with antibacterial, antifungal and antitumor activity. The conservation of this genomic island in many clinical and environmental P. aeruginosa isolates of different geographical origin suggests that the ability for Dha production may confer a selective advantage to its host.

Hypoxic contraction of small pulmonary arteries from normal and endotoxemic rats: fundamental role of NO.

The present study was aimed at examining the role of nitric oxide (NO) in the hypoxic contraction of isolated small pulmonary arteries (SPA) in the rat. Animals were treated with either saline (sham experiments) or Escherichia coli lipolysaccharide [LPS, to obtain expression of the inducible NO synthase (iNOS) in the lung] and killed 4 h later. SPA (300- to 600-micrometer outer diameter) were mounted as rings in organ chambers for the recording of isometric tension, precontracted with PGF2alpha, and exposed to either severe (bath PO2 8 +/- 3 mmHg) or milder (21 +/- 3 mmHg) hypoxia. In SPA from sham-treated rats, contractions elicited by severe hypoxia were completely suppressed by either endothelium removal or preincubation with an NOS inhibitor [NG-nitro-L-arginine methyl ester (L-NAME), 10(-3) M]. In SPA from LPS-treated rats, contractions elicited by severe hypoxia occurred irrespective of the presence or absence of endothelium and were largely suppressed by L-NAME. The milder hypoxia elicited no increase in vascular tone. These results indicate an essential role of NO in the hypoxic contractions of precontracted rat SPA. The endothelium independence of HPV in arteries from LPS-treated animals appears related to the extraendothelial expression of iNOS. The severe degree of hypoxia required to elicit any contraction is consistent with a mechanism of reduced NO production caused by a limited availability of O2 as a substrate for NOS.

Effects of ciliary muscle plasmid electrotransfer of TNF-alpha soluble receptor variants in experimental uveitis.

Intraocular inflammation has been recognized as a major factor leading to blindness. Because tumor necrosis factor-alpha (TNF-alpha) enhances intraocular cytotoxic events, systemic anti-TNF therapies have been introduced in the treatment of severe intraocular inflammation, but frequent re-injections are needed and are associated with severe side effects. We have devised a local intraocular nonviral gene therapy to deliver effective and sustained anti-TNF therapy in inflamed eyes. In this study, we show that transfection of the ciliary muscle by plasmids encoding for three different variants of the p55 TNF-alpha soluble receptor, using electrotransfer, resulted in sustained intraocular secretion of the encoded proteins, without any detection in the serum. In the eye, even the shorter monomeric variant resulted in efficient neutralization of TNF-alpha in a rat experimental model of endotoxin-induced uveitis, as long as 3 months after transfection. A subsequent downregulation of interleukin (IL)-6 and iNOS and upregulation of IL-10 expression was observed together with a decreased rolling of inflammatory cells in anterior segment vessels and reduced infiltration within the ocular tissues. Our results indicate that using a nonviral gene therapy strategy, the local self-production of monomeric TNF-alpha soluble receptors induces a local immunomodulation enabling the control of intraocular inflammation.

Leishmania cysteine proteinases: from gene to subunit vaccine.

Whole genome sequences of microbial pathogens present new opportunities for clinical application. Presently, genome sequencing of the human protozoan parasite Leishmania major is in progress. The driving forces behind the genome project are to identify genes with key cellular functions and new drug targets, to increase knowledge on mechanisms of drug resistance and to favor technology transfer to scientists from endemic countries. Sequencing of the genome is also aimed at the identification of genes that are expressed in the infectious stages of the parasite and in particular in the intracellular form of the parasite. Several protective antigens of Leishmania have been identified. In addition to these antigens, lysosomal cysteine proteinases (CPs) have been characterized in different strains of Leishmania and Trypanosoma, as new target molecules. Recently, we have isolated and characterized Type I (CPB) and Type II (CPA) cysteine proteinase encoding genes from L. major. The exact function of cysteine proteinases of Leishmania is not completely understood, although there are a few reports describing their role as virulence factors. One specific feature of CPB in Leishmania and other trypanosomatids, is the presence of a Cterminal extension (CTE) which is possibly indicative of conserved structure and function. Recently, we demonstrated that DNA immunization of genetically susceptible BALB / c mice, using a cocktail of CPB and CPA genes, induced long lasting protection against L. major infection. This review intends to give an overview of the current knowledge on genetic vaccination used against leishmaniasis and the importance of CP genes for such an approach.