Experimental peripheral arterial disease: new insights into muscle glucose uptake, macrophage, and T-cell polarization during early and late stages.

Peripheral arterial disease (PAD) is a common disease with increasing prevalence, presenting with impaired walking ability affecting patient's quality of life. PAD epidemiology is known, however, mechanisms underlying functional muscle impairment remain unclear. Using a mouse PAD model, aim of this study was to assess muscle adaptive responses during early (1 week) and late (5 weeks) disease stages. Unilateral hindlimb ischemia was induced in ApoE(-/-) mice by iliac artery ligation. Ischemic limb perfusion and oxygenation (Laser Doppler imaging, transcutaneous oxygen pressure assessments) significantly decreased during early and late stage compared to pre-ischemia, however, values were significantly higher during late versus early phase. Number of arterioles and arteriogenesis-linked gene expression increased at later stage. Walking ability, evaluated by forced and voluntary walking tests, remained significantly decreased both at early and late phase without any significant improvement. Muscle glucose uptake ([18F]fluorodeoxyglucose positron emission tomography) significantly increased during early ischemia decreasing at later stage. Gene expression analysis showed significant shift in muscle M1/M2 macrophages and Th1/Th2 T cells balance toward pro-inflammatory phenotype during early ischemia; later, inflammatory state returned to neutrality. Muscular M1/M2 shift inhibition by a statin prevented impaired walking ability in early ischemia. High-energy phosphate metabolism remained unchanged (31-Phosphorus magnetic resonance spectroscopy). Results show that rapid transient muscular inflammation contributes to impaired walking capacity while increased glucose uptake may be a compensatory mechanisms preserving immediate limb viability during early ischemia in a mouse PAD model. With time, increased ischemic limb perfusion and oxygenation assure muscle viability although not sufficiently to improve walking impairment. Subsequent decreased muscle glucose uptake may partly contribute to chronic walking impairment. Early inflammation inhibition and/or late muscle glucose impairment prevention are promising strategies for PAD management.

Macrophage migration inhibitory factor: a counter-regulator of glucocorticoid action and critical mediator of septic shock.

Recent studies have led to the discovery of a mediator that acts as an endogenous counter-regulator of glucocorticoid action within the immune system. Isolated as a product of anterior pituitary cells, this protein was found to have the sequence of macrophage migration inhibitory factor (MIF), one of the first cytokine activities to be described. Macrophages and T cells release MIF in response both to various inflammatory stimuli and upon incubation with low concentrations of glucocorticoids. The glucocorticoid-induced secretion of MIF is tightly regulated and decreases at high, anti-inflammatory steroid concentrations. Once secreted, MIF "overrides" the anti-inflammatory and immunosuppressive effects of steroids on macrophage and T-cell cytokine production. The physiological role of MIF thus appears to be to counter-balance steroid inhibition of the inflammatory response. Anti-MIF antibodies fully protect animals from experimentally induced gram-negative or gram-positive septic shock, an effect that may be the result of the increased anti-inflammatory effects of glucocorticoids after neutralization of endogenous MIF. Anti-MIF therapeutic strategies are presently under development and may prove to be a means to modulate cytokine production in septic shock as well as in other inflammatory disease states.

Papel de la Interleucina 6 (IL-6) en la regulación de la expresión de Osteopontina (OPN) y CD44 tras axotomía del nervio facial

Investigación producida a partir de una estancia en la University of Sidney, Australia, entre octubre del 2008 y enero del 2009. Se ha desarrollado el proyecto titulado "Papel de la interleucina 6 (IL6) en la regulación de la expresión de Osteopontina (OPN) y de CD44 tras axotomía del nervio facial". Tras efectuar una transección del nervio facial, se indujo una reactividad glial en el núcleo facial (NF) localizado en el tronco cerebral, utilizando ratones transgénicos que sobrexpresan IL6 bajo promotor GFAP (tg GFAP-IL6), es decir selectivamente en astrocitos. Se han utilizado técnicas histoquímicas e inmunohistoquímicas, así como también se ha completado el estudio utilizando análisis de RPA, western blotting y citometría de flujo para la identificación de poblaciones celulares. Los resultados obtenidos indican que la OPN se expresa constitutivamente en las neuronas del NF. Tras axotomía del nervio facial, la expresión de OPN y CD44 incrementa en los ratones WT, mientras que en los tg GFAP-IL6 disminuye significativamente, sugiriendo que la IL6 podría estar involucrada en la modulación de la expresión de ambas moléculas. Sin embargo, no se ha visto diferencias en otros receptores de OPN como la integrina Alpha-5. La ctometría de flujo corroboró algunos de los resultados histológicos sobre la reactividad microglial y permitió concluir que la proporción de microglía activada (CD11b+/CD45+mid) y macrófagos (CD11b+/CD45+high) que expresan CD44 incrementa en in los tg GFAP-IL6 versus WT donde la mayor parte de microglia activada mostraba un perfil CD11b+/CD45+low.

Mycobacteriosis in the compromised host

The studies of rare genetic defects, the preliminary results of population-based studies, being validated by the experimental immunocompromised animal models and the current observations accumulated in immunocompromised patients with mycobacterial diseases provide us with insights into the importance of the macrophage activation pathway in controlling human infection with pathogenic and non pathogenic intracellular multiplying mycobacteria. Initial cytokine production by infected macrophages and/or dendritic cells could be crucial in the overall regulation of self cure, acquired protection or immunopathological sequelae expressing the disease. Knowledge of molecular and genetic cross-talks between phagocytic and specialized antigen presenting cells and different mycobacterial products associated with persistence or replication of the intracellular bacteria, could provide further informations on the global immune regulation of the early host responses to infection and the following events. It seems likely that the development of mycobacterial infections in humans will turn out to be as much dependent on the genetic make up of the host as or the virulence of the bacteria.

Apoptosis as a target for gene therapy in rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by chronic inflammation of the synovial joints resulting from hyperplasia of synovial fibroblasts and infiltration of lymphocytes, macrophages and plasma cells, all of which manifest signs of activation. All these cells proliferate abnormally, invade bone and cartilage, produce an elevated amount of pro-inflammatory cytokines, metalloproteinases and trigger osteoclast formation and activation. Some of the pathophysiological consequences of the disease may be explained by the inadequate apoptosis, which may promote the survival of autoreactive T cells, macrophages or synovial fibroblasts. Although RA does not result from single genetic mutations, elucidation of the molecular mechanisms implicated in joint destruction has revealed novel targets for gene therapy. Gene transfer strategies include inhibition of pro-inflammatory cytokines, blockade of cartilage-degrading metalloproteinases, inhibition of synovial cell activation and manipulation of the Th1-Th2 cytokine balance. Recent findings have iluminated the idea that induction of apoptosis in the rheumatoid joint can be also used to gain therapeutic advantage in the disease. In the present review we will discuss different strategies used for gene transfer in RA and chronic inflammation. Particularly, we will highlight the importance of programmed cell death as a novel target for gene therapy using endogenous biological mediators, such as galectin-1, a beta-galactoside-binding protein that induces apoptosis of activated T cells and immature thymocytes.

Characterization of glycoinositol phospholipids in the amastigote stage of the protozoan parasite Leishmania major.

The major macromolecules on the surface of the parasitic protozoan Leishmania major appear to be down-regulated during transformation of the parasite from an insect-dwelling promastigote stage to an intracellular amastigote stage that invades mammalian macrophages. In contrast, the major parasite glycolipids, the glycoinositol phospholipids (GIPLs), are shown here to be expressed at near-constant levels in both developmental stages. The structures of the GIPLs from tissue-derived amastigotes have been determined by h.p.l.c. analysis of the deaminated and reduced glycan head groups, and by chemical and enzymic sequencing. The deduced structures appear to form a complete biosynthetic series, ranging from Man alpha 1-4GlcN-phosphatidylinositol (PI) to Gal alpha 1-3Galf beta 1-3Man alpha 1-3Man alpha 1-4GlcN-PI (GIPL-2). A small proportion of GIPL-2 was further extended by addition of a Gal residue in either alpha 1-6 or beta 1-3 linkage. From g.c.-m.s. analysis and mild base treatment, all the GIPLs were shown to contain either alkylacylglycerol or lyso-alkylglycerol lipid moieties, where the alkyl chains were predominantly C18:0, with lower levels of C20:0, C22:0 and C24:0. L. major amastigotes also contained at least two PI-specific phospholipase C-resistant glycolipids which are absent from promastigotes. These neutral glycolipids were resistant to both mild acid and mild base hydrolysis, contained terminal beta-Gal residues and were not lost during extensive purification of amastigotes from host cell membranes. It is likely that these glycolipids are glycosphingolipids acquired from the mammalian host. The GIPL profile of L. major amastigotes is compared with the profiles found in L. major promastigotes and L. donovani amastigotes.

Experimental evaluation of an electromechanical artificial urinary sphincter in an animal model.

WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: The AMS 800 urinary control system is the gold standard for the treatment of urinary incontinence due to sphincter insufficiency. Despite excellent functional outcome and latest technological improvements, the revision rate remains significant. To overcome the shortcomings of the current device, we developed a modern electromechanical artificial urinary sphincter. The results demonstrated that this new sphincter is effective and well tolerated up to 3 months. This preliminary study represents a first step in the clinical application of novel technologies and an alternative compression mechanism to the urethra. OBJECTIVES: To evaluate the effectiveness in continence achievement of a new electromechanical artificial urinary sphincter (emAUS) in an animal model. To assess urethral response and animal general response to short-term and mid-term activation of the emAUS. MATERIALS AND METHODS: The principle of the emAUS is electromechanical induction of alternating compression of successive segments of the urethra by a series of cuffs activated by artificial muscles. Between February 2009 and May 2010 the emAUS was implanted in 17 sheep divided into three groups. The first phase aimed to measure bladder leak point pressure during the activation of the device. The second and third phases aimed to assess tissue response to the presence of the device after 2-9 weeks and after 3 months respectively. Histopathological and immunohistochemistry evaluation of the urethra was performed. RESULTS: Bladder leak point pressure was measured at levels between 1091 ± 30.6 cmH2 O and 1244.1 ± 99 cmH2 O (mean ± standard deviation) depending on the number of cuffs used. At gross examination, the explanted urethra showed no sign of infection, atrophy or stricture. On microscopic examination no significant difference in structure was found between urethral structure surrounded by a cuff and control urethra. In the peripheral tissues, the implanted material elicited a chronic foreign body reaction. Apart from one case, specimens did not show significant presence of lymphocytes, polymorphonuclear leucocytes, necrosis or cell degeneration. Immunohistochemistry confirmed the absence of macrophages in the samples. CONCLUSIONS: This animal study shows that the emAUS can provide continence. This new electronic controlled sequential alternating compression mechanism can avoid damage to urethral vascularity, at least up to 3 months after implantation. After this positive proof of concept, long-term studies are needed before clinical application could be considered.

Colitis induced in mice with dextran sulfate sodium (DSS) is mediated by the NLRP3 inflammasome.

BACKGROUND: The proinflammatory cytokines interleukin 1beta (IL-1beta) and IL-18 are central players in the pathogenesis of inflammatory bowel disease (IBD). In response to a variety of microbial components and crystalline substances, both cytokines are processed via the caspase-1-activating multiprotein complex, the NLRP3 inflammasome. Here, the role of the NLRP3 inflammasome in experimental colitis induced by dextran sodium sulfate (DSS) was examined. METHODS: IL-1beta production in response to DSS was studied in macrophages of wild-type, caspase-1(-/-), NLRP3(-/-), ASC(-/-), cathepsin B(-/-) or cathepsin L(-/-) mice. Colitis was induced in C57BL/6 and NLRP3(-/-) mice by oral DSS administration. A clinical disease activity score was evaluated daily. Histological colitis severity and expression of cytokines were determined in colonic tissue. RESULTS: Macrophages incubated with DSS in vitro secreted high levels of IL-1beta in a caspase-1-dependent manner. IL-1beta secretion was abrogated in macrophages lacking NLRP3, ASC or caspase-1, indicating that DSS activates caspase-1 via the NLRP3 inflammasome. Moreover, IL-1beta secretion was dependent on phagocytosis, lysosomal maturation, cathepsin B and L, and reactive oxygen species (ROS). After oral administration of DSS, NLRP3(-/-) mice developed a less severe colitis than wild-type mice and produced lower levels of proinflammatory cytokines in colonic tissue. Pharmacological inhibition of caspase-1 with pralnacasan achieved a level of mucosal protection comparable with NLRP3 deficiency. CONCLUSIONS: The NLRP3 inflammasome was identified as a critical mechanism of intestinal inflammation in the DSS colitis model. The NLRP3 inflammasome may serve as a potential target for the development of novel therapeutics for patients with IBD.

Heterogeneity of human monocytes: an optimized four-color flow cytometry protocol for analysis of monocyte subsets.

Monocytes are central mediators in the development of atherosclerotic plaques. They circulate in blood and eventually migrate into tissue including the vessel wall where they give rise to macrophages and dendritic cells. The existence of monocyte subsets with distinct roles in homeostasis and inflammation suggests specialization of function. These subsets are identified based on expression of the CD14 and CD16 markers. Routinely applicable protocols remain elusive, however. Here, we present an optimized four-color flow cytometry protocol for analysis of human blood monocyte subsets using a specific PE-Cy5-conjugated monoclonal antibody (mAb) to HLA-DR, a PE-Cy7-conjugated mAb to CD14, a FITC-conjugated mAb to CD16, and PE-conjugated mAbs to additional markers relevant to monocyte function. Classical CD14(+)CD16(-) monocytes (here termed "Mo1" subset) expressed high CCR2, CD36, CD64, and CD62L, but low CX(3)CR1, whereas "nonclassical" CD14(lo)CD16(+) monocytes (Mo3) essentially showed the inverse expression pattern. CD14(+)CD16(+) monocytes (Mo2) expressed high HLA-DR, CD36, and CD64. In patients with stable coronary artery disease (n = 13), classical monocytes were decreased, whereas "nonclassical" monocytes were increased 90% compared with healthy subjects with angiographically normal coronary arteries (n = 14). Classical monocytes from CAD patients expressed higher CX(3)CR1 and CCR2 than controls. Thus, stable CAD is associated with expansion of the nonclassical monocyte subset and increased expression of inflammatory markers on monocytes. Flow cytometric analysis of monocyte subsets and marker expression may provide valuable information on vascular inflammation. This may translate into the identification of monocyte subsets as selective therapeutic targets, thus avoiding adverse events associated with indiscriminate monocyte inhibition.

Histone deacetylase inhibitors impair innate immune responses

SUMMARYThe innate immune system plays a central role in host defenses against invading pathogens. Innate immune cells sense the presence of pathogens through pattern recognition receptors that trigger intracellular signaling, leading to the production of pro-inflammatory mediators like cytokines, which shape innate and adaptive immune responses. Both by excess and by default inflammation may be detrimental to the host. Indeed, severe sepsis and septic shock are lethal complications of infections characterized by a dysregulated inflammatory response.In recent years, members of the superfamily of histone deacetylases have been the focus of great interest. In mammals, histone deacetylases are broadly classified into two main subfamilies comprising histone deacetylases 1-11 (HDAC1-11) and sirtuins 1-7 (SIRT1-7). These enzymes influence gene expression by deacetylating histones and numerous non-histone proteins. Histone deacetylases have been involved in the development of oncologic, metabolic, cardiovascular, neurodegenerative and autoimmune diseases. Pharmacological modulators of histone deacetylase activity, principally inhibitors, have been developed for the treatment of cancer and metabolic diseases. When we initiated this project, several studies suggested that inhibitors of HDAC 1-11 have anti-inflammatory activity. Yet, their influence on innate immune responses was largely uncharacterized. The present study was initiated to fill in this gap.In the first part of this work, we report the first comprehensive study of the effects of HDAC 1- 11 inhibitors on innate immune responses in vitro and in vivo. Strikingly, expression studies revealed that HDAC1-11 inhibitors act essentially as negative regulators of basal and microbial product- induced expression of critical immune receptors and antimicrobial products by mouse and human innate immune cells like macrophages and dendritic cells. Furthermore, we describe a new molecular mechanism whereby HDAC1-11 inhibitors repress pro-inflammatory cytokine expression through the induction of the expression and the activity of the transcriptional repressor Μί-2β. HDAC1-11 inhibitors also impair the potential of macrophages to engulf and kill bacteria. Finally, mice treated with an HDAC inhibitor are more susceptible to non-severe bacterial and fungal infection, but are protected against toxic and septic shock. Altogether these data support the concept that HDAC 1-11 inhibitors have potent anti-inflammatory and immunomodulatory activities in vitro and in vivo.Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that plays a central role in innate immune responses, cell proliferation and oncogenesis. In the second part of this manuscript, we demonstrate that HDAC1-11 inhibitors inhibit MIF expression in vitro and in vivo and describe a novel molecular mechanism accounting for these effects. We propose that inhibition of MIF expression by HDAC 1-11 inhibitors may contribute to the antitumorigenic and anti-inflammatory effects of these drugs.NAD+ is an essential cofactor of sirtuins activity and one of the major sources of energy within the cells. Therefore, sirtuins link deacetylation to NAD+ metabolism and energy status. In the last part of this thesis, we report preliminary results indicating that a pharmacological inhibitor of SIRT1-2 drastically decreases pro-inflammatory cytokine production (RNA and protein) and interferes with MAP kinase intracellular signal transduction pathway in macrophages. Moreover, administration of the SIRT1-2 inhibitor protects mice from lethal endotoxic shock and septic shock.Overall, our studies demonstrate that inhibitors of HDAC1-11 and sirtuins are powerful anti-inflammatory molecules. Given their profound negative impact on the host antimicrobial defence response, these inhibitors might increase the susceptibility to opportunistic infections, especially in immunocompromised cancer patients. Yet, these inhibitors might be useful to control the inflammatory response in severely ill septic patients or in patients suffering from chronic inflammatory diseases.

Leishmanial antigens in the diagnosis of active lesions and ancient scars of American tegumentary leishmaniasis patients

Cutaneous biopsies (n = 94) obtained from 88 patients with American tegumentary leishmaniasis were studied by conventional and immunohistochemical techniques. Specimens were distributed as active lesions of cutaneous leishmaniasis (n = 53) (Group I), cicatricial lesions of cutaneous leishmaniasis (n = 35) (Group II) and suggestive scars of healed mucosal leishmaniasis patients (n = 6) (Group III). In addition, active cutaneous lesions of other etiology (n = 24) (Group C1) and cutaneous scars not related to leishmaniasis (n = 10) (Group C2) were also included in the protocol. Amastigotes in Group I biopsies were detected by routine histopathological exam (30.2%), imprint (28.2%), culture (43.4%), immunofluorescence (41.4%) and immunoperoxidase (58.5%) techniques; and by the five methods together (79.3%). In Group II, 5.7% of cultures were positive. Leishmanial antigen was also seen in the cytoplasm of macrophages and giant cells (cellular pattern), vessel walls (vascular pattern) and dermal nerves (neural pattern). Positive reaction was detected in 49 (92.5%), 20 (57%) and 4 (67%) biopsies of Groups I, II and III, respectively. Antigen persistency in cicatricial tissue may be related to immunoprotection or, on the contrary, to the development of late lesions. We suggest that the cellular, vascular and neural patterns could be applied in the immunodiagnosis of active and cicatricial lesions in which leishmaniasis is suspected.

Role of glutathione in macrophage activation: effect of cellular glutathione depletion on nitrite production and leishmanicidal activity.

We have examined the effects of two agents depleting the intracellular pool of glutathione (GSH) on macrophage activation induced by IFN-gamma + LPS, as measured by nitrite production and leishmanicidal activity. Diethylmaleate (DEM), which depletes intracellular GSH by conjugation via a reaction catalyzed by the GSH-S-transferase, strongly inhibited nitrite secretion and leishmanicidal activity when added before or at the time of addition of IFN-gamma + LPS; this inhibition was progressively lost when addition of DEM was delayed up to 10 hr. A close correlation was observed between levels of intracellular soluble GSH during activation and nitrite secretion. Inhibition was partially reversed by the addition of glutathione ethyl ester (GSH-Et). Buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, also inhibited macrophage activation, although to a lesser extent than DEM despite a more pronounced soluble GSH depletion. This inhibition was completely reversed by the addition of GSH-Et. DEM and BSO did not alter cell viability or PMA-triggered O2- production by activated macrophages, suggesting that the inhibitory effects observed on nitrite secretion and leishmanicidal activity were not related to a general impairment of macrophage function. DEM and BSO treatment reduced iNOS specific activity and iNOS protein in cytosolic extracts. DEM also decreased iNOS mRNA expression while BSO had no effect. Although commonly used as a GSH-depleting agent, DEM may have additional effects because it can also act as a sulhydryl reagent; BSO, on the other hand, which depletes GSH by enzymatic inhibition, has no effect on protein-bound GSH. Our results suggest that both soluble and protein-bound GSH may be important for the induction of NO synthase in IFN-gamma + LPS-activated macrophages.

In Vitro and in Vivo Assays of 3,5-Disubstituted-Tetrahydro-2H-1,3,5-Thiadiazin-2-Thione Derivatives against Trypanosoma cruzi

Cytotoxicity assays of 24 new 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazin-2-thione derivatives were performed. The 17 compounds with higher anti-epimastigote activity and lower cytotoxicity were, thereafter, screened against amastigote of Trypanosoma cruzi. Out of these 17 derivatives S-2d was selected to be assayed in vivo, because of its remarkable trypanocidal properties. To determine toxicity against J774 macrophages, a method based on quantification of cell damage, after 24 h, was used. Cell respiration, an indicator of cell viability, was assessed by the reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to formazan. Anti-amastigote activity was estimated after 48 h by microscopic counts of May Grünwald-Giemsa-stained monolayers. Nifurtimox and benznidazole were used as reference drugs. For the in vivo experiences, mice were infected with 10(4) blood trypomastigotes and then treated during 15 days with S-2d or nifurtimox by oral route. All of the compounds were highly toxic at 100 µg/ml for macrophages and a few of them maintained this cytotoxicity even at 10 µg/ml. Of the derivatives assayed against amastigotes 3k and S-2d showed an interesting activity, that was held even at 1µg/ml. It is demonstrated that the high anti-epimastigote activity previously reported is mainly due to the non-specific toxicity of these compounds. In vivo assays assessed a reduction of parasitemia after administration of S-2d to infected mice.

In Vitro and in Vivo Anti-Trypanosoma cruzi Activity of a Novel Nitro-derivative

Nitroarylidenemalononitriles and their cyanoacetamide derivatives with remarkable anti-epimastigote properties, were synthesized attempting to obtain new 3,5-diamino-4-(5'-nitroarylidene)-4H-thiadiazine 1,1-dioxide derivatives, which in previous reports had shown anti-Trypanosoma cruzi activity. Tests to evaluate the cytotoxicity of compounds were performed on J774 macrophages. 5-nitro-2-thienyl-malononitrile (5NO2TM), was the only product which maintained a high anti-epimastigote activity at concentrations in which it was no longer cytotoxic, thus it was assayed against intracellular amastigotes. Its anti-amastigote activity was similar to that of nifurtimox. Afterwards in vivo toxicity and anti-chagasic activity were determined. A reduction in parasitemia was observed.

B-cell infiltration and frequency of cytokine producing cells differ between localized and disseminated human cutaneous leishmaniases

Biopsies from human localized cutaneous lesions (LCL n = 7) or disseminated lesions (DL n = 8) cases were characterized according to cellular infiltration,frequency of cytokine (IFN-g, TNF-alpha) or iNOS enzyme producing cells. LCL, the most usual form of the disease with usually one or two lesions, exhibits extensive tissue damage. DL is a rare form with widespread lesions throughout the body; exhibiting poor parasite containment but less tissue damage. We demonstrated that LCL lesions exhibit higher frequency of B lymphocytes and a higher intensity of IFN-gamma expression. In both forms of the disease CD8+ were found in higher frequency than CD4+ T cells. Frequency of TNF-alpha and iNOS producing cells, as well as the frequency of CD68+ macrophages, did not differ between LCL and DL. Our findings reinforce the link between an efficient control of parasite and tissue damage, implicating higher frequency of IFN-gamma producing cells, as well as its possible counteraction by infiltrated B cells and hence possible humoral immune response in situ.