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Cardiovascular magnetic resonance (CMR) is a useful noninvasive technique for determining the presence of microvascular obstruction (MVO) and intramyocardial hemorrhage (IMH), frequently occurring in patients after reperfused myocardial infarction (MI). MVO, or the so-called no-reflow phenomenon, is associated with adverse ventricular remodeling and a poor prognosis during follow-up. Similarly, IMH is considered a severe damage after revascularization by percutaneous primary coronary intervention (PPCI) or fibrinolysis, which represents a worse prognosis. However, the pathophysiology of IMH is not fully understood and imaging modalities might help to better understand that phenomenon. While, during the past decade, several studies examined the distribution patterns of late gadolinium enhancement with different CMR sequences, the standardized CMR protocol for assessment of IMH is not yet well established. The aim of this review is to evaluate the available literature on this issue, with particular regard to CMR sequences. New techniques, such as positron emission tomography/magnetic resonance imaging (PET/MRI), could be useful tools to explore molecular mechanisms of the myocardial infarction healing process.

Dendritic cells (DC) sense saprophytic yeast and pathogenic, filamentous forms of Candida albicans in a specific way, resulting in disparate patterns of DC and T(h) cell activation. Using human and murine DC, such disparate patterns could be traced to the exploitation of distinct recognition receptors. Although usage of mannose receptors led to protective type 1 responses in mice, entry through Fcgamma receptors was responsible for suppression of mannose receptor-dependent reactivity, onset of type 2 responses and associated pathology. As the usage of distinct receptors selectively occurred with yeast or hyphal forms of the fungus, these findings suggest that the responsibility for pathogenicity of C. albicans is shared by the organism and DC, with implications for fungal virulence, immunity and vaccine development.

To find out whether polymorphonuclear neutrophils (PMN), abundantly recruited in disseminated Candida albicans infection, could directly affect the activation of Th cells we addressed the issues as to whether murine PMN, like their human counterparts, express costimulatory molecules and the functional consequence of this expression in terms of antifungal immune resistance. To this purpose, we assessed 1) the expression of CD80 (B7-1) and CD86 (B7-2) molecules on peripheral, splenic, and inflammatory murine Gr-1+ PMN; 2) its modulation upon interaction with C. albicans in vitro, in vivo, and in human PMN; 3) the effect of Candida exposure on the ability of murine PMN to affect CD4+ Th1 cell proliferation and cytokine production; and 4) the mechanism responsible for this effect. Murine PMN constitutively expressed CD80 molecules on both the surface and intracellularly; however, in both murine and human PMN, CD80 expression was differentially modulated upon interaction with Candida yeasts or hyphae in vitro as well as in infected mice. The expression of the CD86 molecule was neither constitutive nor inducible upon exposure to the fungus. In vitro, Gr-1+ PMN were found to inhibit the activation of IFN-γ-producing CD4+ T cells and to induce apoptosis through a CD80/CD28-dependent mechanism. A population of CD80+Gr-1+ myeloid cells was found to be expanded in conventional as well as in bone marrow-transplanted mice with disseminated candidiasis, but its depletion increased the IFN-γ-mediated antifungal resistance. These data indicate that alternatively activated PMN expressing CD80 may adversely affect Th1-dependent resistance in fungal infections.

Aspergilli are respiratory pathogens and pulmonary infections are usually acquired through the inhalation of conidia, able to reach small airways and the alveolar space where the impaired host defense mechanisms allow hyphal germination and subsequent tissue invasion. The invasive pulmonary aspergillosis is the most common manifestation of Aspergillus fumigatus infection in immunocompromised patients and is characterized by hyphal invasion and destruction of pulmonary tissue. A Th1/Th2 dysregulation and a switch to a Th2 immune response may contribute to the development and unfavorable outcome of invasive pulmonary aspergillosis. Dendritic cells (DC) have a primary role in surveillance for pathogens at the mucosal surfaces and are recognized as the initiators of immune responses to them. In the present study, we assessed the functional activity of pulmonary DC in response to A. fumigatus conidia and hyphae, both in vitro and in vivo. We analyzed mechanisms and receptors for phagocytosis by DC as well as DC migration, maturation, and Th priming in vivo upon exposure to either form of the fungus. We found a remarkable functional plasticity of DC in response to the different forms of the fungus, as pulmonary DC were able to: 1) internalize conidia and hyphae of A. fumigatus through distinct phagocytic mechanisms and recognition receptors; 2) discriminate between the different forms in terms of cytokine production; 3) undergo functional maturation upon migration to the draining lymph nodes and spleens; and 4) instruct local and peripheral Th cell reactivity to the fungus.

The fungus Candida albicans behaves as a commensal as well as a true pathogen of areas highly enriched in dendritic cells, such as skin and mucosal surfaces. The ability of the fungus to reversibly switch between unicellular yeast to filamentous forms is thought to be important for virulence. However, whether it is the yeast or the hyphal form that is responsible for pathogenicity is still a matter of debate. Here we show the interaction, and consequences, of different forms of C. albicans with dendritic cells. Immature myeloid dendritic cells rapidly and efficiently phagocytosed both yeasts and hyphae of the fungus. Phagocytosis occurred through different phagocytic morphologies and receptors, resulting in phagosome formation. However, hyphae escaped the phagosome and were found lying free in the cytoplasm of the cells. In vitro, ingestion of yeasts activated dendritic cells for interleukin (IL)-12 production and priming of T helper type 1 (Th1) cells, whereas ingestion of hyphae inhibited IL-12 and Th1 priming, and induced IL-4 production. In vivo, generation of antifungal protective immunity was induced upon injection of dendritic cells ex vivo pulsed with Candida yeasts but not hyphae. The immunization capacity of yeast-pulsed dendritic cells was lost in the absence of IL-12, whereas that of hypha-pulsed dendritic cells was gained in the absence of IL-4. These results indicate that dendritic cells fulfill the requirement of a cell uniquely capable of sensing the two forms of C. albicans in terms of type of immune responses elicited. By the discriminative production of IL-12 and IL-4 in response to the nonvirulent and virulent forms of the fungus, dendritic cells appear to meet the challenge of Th priming and education in C. albicans saprophytism and infections.

The Ca2(+)-binding proteins of the EF-hand type, S100B and S100A1, were detected in the outer segment of bovine retina photoreceptors where they are localized to disc membranes, as investigated by immunofluorescence and immunogold cytochemistry. S100B and S100A1 stimulate a membrane-bound guanylate cyclase activity associated with photoreceptor disc membranes in dark-adapted retina in a Ca2(+)-dependent manner, although with different Ca2+ requirements, as investigated by an ultracytochemical approach. Other retinal cell types express S100B and S100A1 as well. S100B is detected in the outer limiting membrane, fine cell processes in the outer nuclear layer and the outer plexiform layer, cell bodies in the inner nuclear layer and the ganglion cell layer, and the inner limiting membrane, whereas S100A1 has a more discrete distribution. S100B and S100A1 also stimulate a membrane-bound guanylate cyclase activity in photoreceptor cell bodies and Muller cells, but their effect appears independent of the light- or dark-adapted state of the retina and is observed at relatively high Ca2+ concentrations. These data represent the ultrastructural counterpart of recent biochemical observations implicating S100B and, possibly, S100A1 in the Ca2(+)-dependent stimulation of a photoreceptor membrane-bound guanylate cyclase activity [T. Duda, R. M. Goraczniak and R. K. Sharma (1996) Molecular characterization of S100A1-S1000B protein in retina and its activation mechanism of bovine photoreceptor guanylate cyclast. Biochemistry 35, 6263-6266; A. Margulis, N. Pozdnyakov and A. Sitaramayya (1996) Activation of bovine photoreceptor guanylate cyclast by S100 proteins. Biochem. Biophys. Res. Commun. 218, 243-247]. Our data suggest that at least S100B may take part in the regulation of a membrane-bound guanylate cyclase-based signalling pathway in both photoreceptors and Muller cells.

The ultracytochemical localization of guanylate cyclases A and B activity has been studied after stimulation with atrial natriuretic peptide and C-type natriuretic peptide in light- and dark-adapted retinas and pigmented epithelium. The results showed that both peptides stimulated guanylate cyclases A and B activity in light-adapted retinas only. Guanylate cyclases A and B activity was detected on plasmamembrane of body of photoreceptors, bipolar, horizontal and ganglion cells, on plasma membranes of interneuronal connections at plexiform layers and on the plasma membrane of fibres at the nerve fibres layer. Independently of the light- or dark-adapted state, the pigmented epithelium also presented guanylate cyclases A and B activity on basal and lateral plasma membranes.

In inflammatory pulmonary diseases, cigarette smoking is a major risk factor influencing the phenotype of immune cells and their functions (Arnson et al., 2010). Nicotine is a relevant constituent of cigarette smoking and, on monocytes, it binds nicotinic acetylcholine receptors inducing a quantitative increase in the levels of CD14 and a decrease in TNF-a. Altogether, these data supports an anti-inflammatory effect of nicotine on these cells.Our previous studies have shown that NGF and its receptors TrKA and p75 are involved in inflammatory pulmonary diseases. In particular, we have shown that smokers present an increase in p75 expression on monocytes, but the cause that triggers this increase is actually unknown. In addition, cigarette smoking seems not to vary the TrKA expression on these cells. The aim of our study was to investigate in vitro the biological effects of nicotine on the percentage of TrKA- and p75-positive monocytes from human healthy non smoker donors. Cytofluorimetric investigation confirms that in monocytes nicotine treatment does not influence the percentage of TrKA, but induces a relevant dose-dependent increase in the percentage of p75 that, in turn, could be an element in the general anti-inflammatory effects of nicotine. Consequently, in order to investigate the involvement of p75 in inflammatory/non-inflammatory mechanisms, we added in vitro the inflammatory stimulus MCP-1 on monocytes. Cytofluorimetric investigation have shown that, in this latest inflammatory condition, the percentage of p75-positive monocytes was greatly reduced. In conclusion, our data support the hypothesis that nicotine-induced p75 overexpression could be involved in its anti-inflammatory effect., Italian Journal of Anatomy and Embryology, Vol 117, No 2 (Supplement) 2012

We studied the activity and the ultracytochemical localization of membrane-bound guanylate cyclase (GC) after stimulation with rat atrial natriuretic peptide (rANP), porcine brain natriuretic peptide (pBNP), rat brain natriuretic peptide (rBNP), or porcine C-type natriuretic peptide (CNP) in rat C6 glioma cells during proliferation or following exposure of confluent cells to dibutyryl cyclic AMP (db-cAMP) or retinoic acid (RA). Under our experimental conditions all peptides were activators of GC as demonstrated by the accumulation of cGMP within cells. During proliferation of C6 cells, the amounts of cGMP remained approximately constant. However, at subconfluency, confluency and postconfluency, the GC reaction product was located at different sites in C6 cells. At subconfluency, GC reaction product was on membranes of protoplasmic extensions, at postconfluency, GC reaction product was in association with membranes of cell bodies, and at confluency, both localizations of GC reaction product were detected. Incubation of confluent cells in culture medium containing db-cAMP or RA induced the appearance of long and slender protoplasmic extensions. Under these conditions, the GC reaction product was localized exclusively to these processes. These data suggest that GC is differently located depending on the state of growth of glial cells, and that in differentiating glial cells GC is preferentially located in cell processes.

S-100 protein and annexin II 2 -p11 2 were reported to inhibit and to stimulate the assembly of glial fibrillary acidic protein (GFAP), respectively, in a Ca 2+ -dependent manner. Here we show by a number of experimental approaches that S-100 protein contrasts all the effects of annexin II 2 -p11 2 on GFAP assembly and, conversely, that annexin II 2 -p11 2 contrasts the inhibitory effects of S-100 protein on GFAP assembly, in a dose-dependent manner in both cases. Altogether, these data suggest that two specific Ca 2+ effecters, i.e., annexin II 2 -p11 2 and S-100 protein, might regulate the state of assembly of glial filaments in a concerted manner.

We investigated the ultracytochemical localization of particulate guanylate cyclase (GC) in the rat neurohypophysis after activation with rat atrial natriuretic factor (rANF) or porcine brain natriuretic peptide (pBNP). Under our experimental conditions, the presence of GC reaction product indicated that rANF and pBNP were strong activators of particulate GC since samples incubated in basal conditions without rANF or pBNP did not reveal any GC reaction product. The rANF-stimulated GC was localized both to pituicytes and to nerve fibers and endings whereas the pBNP-stimulated GC was present exclusively in nerve fibers and endings. Recently, two subtypes of receptors for natriuretic peptides have been identified as two isoforms of particulate GC [24,50]. Our data indicate that the receptors of the two hormones have a partially distinct distribution in the rat neurohypophysis. In pituicytes, GC reaction product was found on plasma membrane of finger-like processes and on the membranes surrounding the lipid droplets. In nerve fibers and endings, GC reaction product was associated with intracellular membranes. This finding suggests that the enzyme could mediate an internal inhibitory action of these hormones on the release of vasopressin and oxytocin.

Neurotrophins (NTs) are a family of growth/survival factors with well-established functions in the nervous system. In the last decade, novel biological actions, from oncogenicity to inflammation, have been attributed to these factors. In particular, Nerve Growth Factor (NGF) acts through two different classes of receptors: the high affinity transducing TrKA receptor, associated to proliferation/survival, and the low affinity p75 receptor that, depending on its cross talk with TrKA, induces either apoptosis or survival. Recently, NGF and its receptors have been detected in healthy pulmonary tissues and seem to be involved in pulmonary inflammatory diseases. Since the expression of these NGF receptors in circulating monocytes is controversial, our initial aim was to investigate the role of these receptors both in pulmonary tissues and in peripheral blood monocytes of patients with Chronic Obstructive Pulmonary Disease (COPD). We therefore analyzed 38 healthy control subjects divided in two groups based on their smoking status. Serendipitously, the data obtained in these two control groups may have a value of their own, with potential implications in preventive medicine. We show here that in healthy subjects, smoking induces an early increase in p75 expression in monocytes, while TrKA seems unaffected. Furthermore, our control subjects could be divided in three subsets according to the constitutive TrKA expression in monocytes: TrKA-negatives, -intermediates (up to 50%) or -high (> 50%), independently on their smoking status. Since TrKA activation promotes inflammation, we hypothesize that subjects with high-TrKA monocytes could be more prone to pulmonary inflammatory diseases, such as COPD. In fact, all 28 COPD patients in our series belonged to the high-TrKA subset, while all long term heavy smokers with no evidence of pneumologic diseases belonged to TrKA-negative subset. In conclusion, our data support the hypothesis that, since TrKA expression promotes survival, elevated levels of TrKA-positive monocytes may render subjects more prone to long term inflammatory diseases (e.g., COPD). Moreover, in patients constitutively expressing high levels of TrKA in monocytes, the smoking-dependent increase in p75 may in turn further extend monocytes survival, contributing to a chronic inflammation. Furthermore, the early increase in p75 expression in monocytes following smoking may support the hypothesis that p75 determination might represent a novel marker for passive smoking. Acknowledgments. This study was totally supported by Fondazione Cassa di Risparmio di Terni, Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

Italian Journal of Anatomy and Embryology, Vol 115, No 1/2 (Supplement) 2010

The role of IL-17 and Th17 cells in immunity vs. pathology associated with the human commensal Candida albicans remains controversial. Both positive and negative effects on immune resistance have been attributed to IL-17/Th17 in experimental candidiasis. In this study, we provide evidence that IL-22, which is also produced by Th17 cells, has a critical, first-line defense in candidiasis by controlling the growth of infecting yeasts as well as by contributing to the host's epithelial integrity in the absence of acquired Th1-type immunity. The two pathways are reciprocally regulated, and IL-22 is upregulated under Th1 deficiency conditions and vice versa. Whereas both IL-17A and F are dispensable for antifungal resistance, IL-22 mediates protection in IL-17RA-deficient mice, in which IL-17A contributes to disease susceptibility. Thus, our findings suggest that protective immunity to candidiasis is made up of a staged response involving an early, IL-22-dominated response followed by Th1/Treg reactivity that will prevent fungal dissemination and supply memory.

Italian Journal of Anatomy and Embryology, Vol 115, No 1/2 (Supplement) 2010

We investigated the subcellular distribution of S-100b protein in primary cultures of Schwann cells. The subcellular localization of the protein in cells fixed and then permeabilized is similar, if not identical, to that seen in Schwann cells in peripheral nerves, i.e., S-100b protein is found in the cytoplasm and associated with membranes and filamentous structures. In cells either fixed in the presence of Triton X-100 or exposed to Triton X-100 for a short time before fixation (Triton cytoskeletons), the immune reaction product is considerably less intense, and the protein is associated with filaments running parallel to the long axis of the cell as well as in a submembranous position. Including CaCl2 in the buffer during fixation in the presence of Triton X-100 does not result in any increase in the intensity of the immune reaction product in Triton cytoskeletons, suggesting that, within the limits of the technique employed, no binding of additional S-100b protein to the Triton X-100-resistant material can be induced. On the other hand, including EGTA results in a substantial decrease in the intensity of the immune reaction product in Triton cytoskeletons. Altogether, these findings suggest that a remarkable fraction of S-100b protein in cultured Schwann cells is associated with elements of the cytoskeleton and that Ca2+ exerts some regulatory role in the association of S-100b protein with the cytoskeleton.

The ultracytochemical localization of adenylate cyclase (AC) and guanylate cyclase B (GC-B) and C (GC-C) activity was studied after stimulation with pituitary adenylate cyclase activating peptide, C-type natriuretic peptide and guanylin, respectively, in normal human respiratory nasal mucosa and mucosa of nasal polyps. To demonstrate these enzymatic activities, we employed enzyme-ultracytochemical methods for electron microscopy. Both normal and pathologic nasal mucosa contained AC, GC-B and GC-C activity. In the upper portion of respiratory epithelium, the enzymes were detected on ciliary and microvillar membranes. In ciliary membranes, GC-B was the predominant form expressed. In goblet cells and in glands of the lamina propria, enzymatic activities were localized mainly on plasma membranes and on membranes lining secretory granules. The results did not reveal any evident differences between the enzymatic activities in normal and pathological nasal mucosa and suggest complementary activities for these enzymes and their stimulators in the regulation of mucociliary transport and glandular secretion.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that, expressed by different cell types, has regulatory effects on T cells resulting from tryptophan depletion in specific local tissue microenvironments. The discovery that inhibition of IDO activity reduces the survival of MHC-mismatched fetuses in mice and that the risk of fetal allograft rejection correlates with the degree of parental tissue incompatibility has led to the hypothesis that IDO activity protects fetal allografts from maternal T cell-mediated immunity. Different mechanisms, however, might contribute to IDO-dependent immune regulation. We have found that tryptophan metabolites in the kynurenine pathway, such as 3-hydroxyanthranilic and quinolinic acids, will induce the selective apoptosis in vitro of murine thymocytes and Th1 but not Th2 cells. T cell apoptosis was observed at relatively low concentrations of kynurenines, did not require Fas/Fas ligand interactions and was associated with the activation of casapase-8 and the release of cytochrome c from mitochondria. In vivo, the two kynurenines caused depletion of specific thymocyte subsets in a fashion qualitatively similar to dexamethasone. These data may represent the first experimental evidence for the involvement of tryptophan catabolism in the regulation of T cell apoptosis and maintenance of peripheral T cell tolerance.

The ultracytochemical localization of adenylate cyclase (AC) and guanylate cyclase B (GC-B) and C (GC-C) activity was studied after stimulation with pituitary adenylate cyclase activating peptide, C-type natriuretic peptide and guanylin, respectively, in normal human respiratory nasal mucosa and mucosa of nasal polyps. To demonstrate these enzymatic activities, we employed enzyme-ultracytochemical methods for electron microscopy. Both normal and pathologic nasal mucosa contained AC, GC-B and GC-C activity. In the upper portion of respiratory epithelium, the enzymes were detected on ciliary and microvillar membranes. In ciliary membranes, GC-B was the predominant form expressed. In goblet cells and in glands of the lamina propria, enzymatic activities were localized mainly on plasma membranes and on membranes lining secretory granules. The results did not reveal any evident differences between the enzymatic activities in normal and pathological nasal mucosa and suggest complementary activities for these enzymes and their stimulators in the regulation of mucociliary transport and glandular secretion.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that, expressed by different cell types, has regulatory effects on T cells resulting from tryptophan depletion in specific local tissue microenvironments. The discovery that inhibition of IDO activity reduces the survival of MHC-mismatched fetuses in mice and that the risk of fetal allograft rejection correlates with the degree of parental tissue incompatibility has led to the hypothesis that IDO activity protects fetal allografts from maternal T cell-mediated immunity. Different mechanisms, however, might contribute to IDO-dependent immune regulation. We have found that tryptophan metabolites in the kynurenine pathway, such as 3-hydroxyanthranilic and quinolinic acids, will induce the selective apoptosis in vitro of murine thymocytes and Thl but not Th2 cells. T cell apoptosis was observed at relatively low concentrations of kynurenines, did not require Fas/Fas ligand interactions and was associated with the activation of casapase-8 and the release of cytochromecfrom mitochondria. In vivo, the two kynurenines caused depletion of specific thymocyte subsets in a fashion qualitatively similar to dexamethasone. These data may represent the first experimental evidence for the involvement of tryptophan catabolism in the regulation of T cell apoptosis and maintenance of peripheral T cell tolerance.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that, expressed by different cell types, has regulatory effects on T cells resulting from tryptophan depletion in specific local tissue microenvironments. Different mechanisms, however, might contribute to IDO-dependent immune regulation. We show here that tryptophan metabolites in the kynurenine pathway, such as 3-hydroxyanthranilic and quinolinic acids, will induce the selective apoptosis in vitro of murine thymocytes and of Th1 but not Th2 cells. T cell apoptosis was observed at relatively low concentrations of kynurenines, did not require Fas/Fas ligand interactions, and was associated with the activation of caspase-8 and the release of cytochrome c from mitochondria. When administered in vivo, the two kynurenines caused depletion of specific thymocyte subsets in a fashion qualitatively similar to dexamethasone. These data suggest that the selective deletion of T lymphocytes may be a major mechanism whereby tryptophan metabolism affects immunity under physiopathologic conditions.

Human beings are continuously exposed to fungi, yet they rarely get fungal diseases. The delicate balance between the host and these otherwise harmless pathogens may turn into a parasitic relationship, resulting in the development of severe infections. The ability to reversibly switch between unicellular and filamentous forms, all of which can be found in infected tissues, is thought to be important for virulence. Efficient responses to the different forms of fungi require different mechanisms of immunity. Dendritic cells (DC) are uniquely able at decoding the fungus-associated information and translating it in qualitatively different T helper (Th) immune responses, in vitro and in vivo. Myeloid DC phagocytosed yeasts and hyphae of Candida albicans and conidia and hyphae of Aspergillus fumigatus, both in vitro and in vivo. Phagocytosis occurred through distinct phagocytic morphologies, involving the engagement and cooperativity of distinct recognition receptors. However, receptor engagement and cooperativity were greatly modified by opsonization. The engagement of distinct receptors translated into disparate downstream signaling events, ultimately affecting cytokine production and costimulation. In vivo studies confirmed that the choice of receptor and mode of entry of fungi into DC was responsible for Th polarization and patterns of susceptibility or resistance to infection. Adoptive transfer of different types of DC activated protective, nonprotective and regulatory T cells, ultimately affecting the outcome of infection. The conclusions are that the selective exploitation of receptors and mode of entry into DC may determine the full range of host's immune relationships with fungi and have important implications in the design of vaccine-based strategies.

Antiidiotypic monoclonal antibodies (MAbs) representing the internal image of a yeast killer toxin (KT) have therapeutic potential against several fungal infections. The efficacy of KT MAbs againstAspergillus fumigatuswas investigated in a mouse model of T-cell-depleted allogeneic bone marrow transplantation (BMT) with invasive pulmonary aspergillosis. Mice were highly susceptible to infection at 3 days post-BMT, when profound neutropenia was observed both in the periphery and in the lungs. Treatment with KT MAbs protected the mice from infection, as judged by the long-term survival and decreased pathology associated with inhibition of fungal growth and hyphal development in the lungs. In vitro, similar to polymorphonuclear neutrophils, KT MAbs significantly inhibited the hyphal development and metabolic activity of germinatedAspergillusconidia. These results indicate that mimicking the action of neutrophils could be a strategy through which KT MAbs exert therapeutic efficacy inA. fumigatusinfections.