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Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by Staphylococcus aureus that has recently been associated with necrotizing pneumonia. In the present study, we report that in vitro, PVL induces polymorphonuclear cell death by necrosis or by apoptosis, depending on the PVL concentration. PVL-induced apoptosis was associated with a rapid disruption of mitochondrial homeostasis and activation of caspase-9 and caspase-3, suggesting that PVL-induced apoptosis is preferentially mediated by the mitochondrial pathway. Polymorphonuclear cell exposure to PVL leads to mitochondrial localization of the toxin, whereas Bax, 1 of the 2 essential proapoptotic members of the Bcl-2 family, was still localized in the cytosol. Addition of PVL to isolated mitochondria induced the release of the apoptogenic proteins cytochrome c and Smac/DIABLO. Therefore, we suggest that PVL, which belongs to the pore-forming toxin family, could act at the mitochondrion level by creating pores in the mitochondrial outer membrane. Furthermore, LukS-PV, 1 of the 2 components of PVL, was detected in lung sections of patients with necrotizing pneumonia together with DNA fragmentation, suggesting that PVL induces apoptosis in vivo and thereby is directly involved in the pathophysiology of necrotizing pneumonia.

Microdissected rat proximal straight tubules (PST) and inner medullary collecting ducts (IMCD) highly produce urea from L-arginine, supporting the expression of the mitochondrial arginase II. However, IMCD contain a very low density of mitochondria compared with PST. Recently, arginase II has been localized by immunohistochemistry in rat PST but not IMCD. This study was designed to verify whether rat IMCD express arginase II and to identify its subcellular localization. We developed an antibody raised against arginase II that allowed the detection of a band of 38 kDa corresponding to arginase II on immunoblots. In male and female rat kidneys, Western blot analyses revealed that arginase II was highly expressed in the inner medulla (IM), the outer stripe of the outer medulla (osOM), and the deep cortex. Immunocytochemistry demonstrated that arginase II was homogeneously expressed in IMCD. Proteins of the cytosolic and mitochondrial fractions extracted from osOM and IM and analyzed by Western blot showed that 86% of arginase II was associated with mitochondria. The molecular weight of arginase II was similar in the cytosolic and mitochondrial fractions. Immunoelectron microscopy confirmed the presence of arginase II in the mitochondria of IMCD. In conclusion, arginase II is expressed in mitochondria of male and female rat IMCD.

Arginase II (AII) has been almost exclusively studied in male mammalian kidneys. Our investigations were conducted to localize AII gene expression in the female mouse kidney, and to analyze the differential expression of AII gene at the transcriptional and translational levels in the kidneys of female and male mice. Total RNAs and soluble proteins extracted from renal zones and whole kidneys were analyzed by Northern and Western blots, respectively. Mitochondrial and cytosolic proteins were analyzed by Western blot. L-[guanidino-14C]arginine hydrolysis by AII was detected in microdissected tubules and the 14CO2 released from [14C]urea hydrolysis was quantified. The results of these experiments showed that: (1) both AII mRNA and protein were highly expressed in the deep cortex and the outer stripe of the outer medulla, (2) urea was produced mainly in the proximal straight tubules (PST), (3) the 38-kDa AII protein was more abundant in the mitochondria than the cytosol, and (4) the renal content of AII mRNA and protein was about three-fold higher in female than in male mice. In conclusion, in both genders, AII gene expression is restricted to the PST and localized into mitochondria. AII gene is differentially expressed in the kidney of female and male mice since higher levels of AII mRNA, protein and activity were observed in the kidneys of the former than those of the latter. Renal AII gene expression was gender-dependent in mice but not in rats. Finally, in the PST of females, L-arginine-derived ornithine may be a precursor for the renal production of L -glutamate and L-glutamine because high levels of AII, ornithine aminotransferase and glutamine synthetase are expressed in this nephron segment.

In the kidney, l-ornithine is reabsorbed along the proximal convoluted tubule (PCT), transported by basolateral carriers, and produced by arginase II (AII). Here, the renal metabolic fate of l-ornithine was analyzed in male and female rats. Kidneys and renal zones were dissected and used for Western blot analysis, immunofluorescence, and electron microscopic studies. Ornithine aminotransferase (OAT) and AII were localized using specific antibodies. Ornithine oxidation was determined by incubating microdissected tubules with l-[1-14C] or l-[U-14C]ornithine in the presence or absence of energy-providing substrates. Ornithine decarboxylase (ODC) mRNAs were localized by in situ hybridization. The 48-kDa OAT protein was detected in male and female kidneys, but its level was fourfold higher in the latter. OAT relative distribution increased from the superficial cortex toward the outer medulla to reach its highest level. Almost all OAT protein was localized in cortical and medullary proximal straight tubules (CPST and OSPST, respectively). In proximal straight tubule (PST), AII protein distribution overlapped that of OAT. No gender difference in AII protein level was found. OAT and AII were colocalized within PST mitochondria. l-[1-14C]ornithine decarboxylation occurred in all tubules, but predominantly in proximal tubules. l-[1-14C]ornithine decarboxylation was enhanced when l-[1-14C]ornithine was given to tubules as the sole substrate. The use of l-[U-14C]ornithine demonstrated the complete oxidation of ornithine. In conclusion, the OAT gene was expressed more in female rat proximal tubules than in male. Because OAT and AII proteins overlapped in PST mitochondria, l-arginine-derived ornithine may be preferentially converted to l-glutamate, as proven by ornithine oxidation. However, the coexpression of ODC, glutamate decarboxylase, and glutamine synthetase in PST suggests that l-ornithine can also be metabolized to putrescine, GABA, and l-glutamine. The fate of l-ornithine may depend on the cellular context.

Elastic fibers form a complex network that contributes to the elasticity of connective tissues. Alterations in the elastic fiber network are involved in several disease affecting organs in which compliance of the connective tissue is essential: skin, main vasculature, lung, joints, muscle, and ligament. The aim of our work was to study the deposition, maturation, and organization of elastic fiber components in a dermal equivalent model consisting of collagen-GAG-chitosan seeded with fibroblasts. The influence of keratinocytes was studied in parallel, thus constituting a skin equivalent model. These models were examined by transmission electron microscopy (TEM) and by immunohistochemistry to determine the staining patterns of fibrillin-1 and elastin proteins representative of the microfibrillar framework and of the elastic fibers, respectively. After 2 mo of fibroblast culture in the dermal equivalent, elastin was undetectable, whereas fibrillin-1 staining was weak and microfibrils were infrequently observed by TEM. In the skin equivalent, fibrillin-1 and elastin were detected by immunostaining 15 d after epidermization and TEM revealed the typical structure and organization of the elastic network in the dermis, with elastin deposition on the microfibrillar scaffold. This in vitro skin equivalent model is to our knowledge the first in which elastic fibers have been detected, thus demonstrating the influence of keratinocytes on the maturation and organization of the elastic network.

Cdc14, a dual-specificity protein phosphatase, has been previously implicated in triggering exit from mitosis in the yeast Saccharomyces cerevisiae. Using immunofluorescence microscopy and immunogold labeling, we demonstrate that a functional HA-tagged version of the phosphatase Cdc14 localizes to the nucleolus. Moreover, Cdc14-HA co-localized with the nucleolar NOP2 and GAR1 proteins. By immunofluorescence, Cdc14-HA was found in the nucleolus during most of the mitotic cell cycle, except during anaphase-telophase when it redistributed along the mitotic spindle. While this work was in progress, the same pattern of Cdc14 localization was described by others (Visintin et al, Nature 398 (1999) 818). Constitutive overexpression of CDC14 was toxic and led to cell cycle arrest of cells, mainly in G1. This correlated with the appearance of abnormal nuclear structures. A genetic search for suppressors of the lethality associated with CDC14 overexpression identified YJL076W. Because overproduction of Yj1076w buffered the toxic effect of Cdc14 overproduction, this suggested that it might be a substrate of Cdc14. This has indeed been found to be the case by others who recently described Yj1076wNet1 as a nucleolar protein that physically associates with Cdc14 (Shou et al, Cell 97 (1999) 233). The present data confirm several recently uncovered aspects of the regulation of Cdc14 localization and activity and suggest that the level of expression of CDC14 influences the structural organization of the nucleolus.

Reparative process of second and third degree burns usually results in hypertrophic scar formation that can be treated by pressure. Although this method is efficient, its mechanisms of action are not known. In this work, we have studied the histological organization of hypertrophic scars submitted to pressure. Skin biopsies were performed 2 to 7 months after the onset of treatment in two adjacent regions of the scar, non-pressure- or pressure-treated and analyzed by immunohistochemistry and transmission electron microscopy for extracellular matrix organization and cellular morphology. In non-pressure-treated regions, fibrillin deposits did not present the classical candelabra-like pattern under epidermis and were reduced in dermis; in pressure-treated regions the amount was increased compared to non-pressure-treated regions but the organization was still disturbed. In non-pressure-treated regions, elastin was present in patch deposits; in pressure-treated regions elastin formed fibers, smaller than in normal dermis. Tenascin was present in the whole dermis in non-pressure-treated regions, whereas in pressure-treated regions it was observed only under epidermis and around vessels, as in normal skin. α-Smooth muscle actin-expressing myofibroblasts were absent in normal skin, present in large amounts in non-pressure-treated regions, and almost absent in pressure-treated regions. The disturbed ultrastructural organization of dermal-epidermal junction observed in non-pressure-treated regions disappeared after pressure therapy; typical features of apoptosis in fibroblastic cells and morphological aspects of collagen degradation were observed in pressure-treated regions. Our results show that, in hypertrophic scars, pressure therapy restores in part the extracellular matrix organization observed in normal scar and induces the disappearance of α-smooth muscle actin-expressing myofibroblasts, probably by apoptosis. We suggest that the pressure acts by accelerating the remission phase of the postburn reparative process.

We have isolated a mouse lysyl oxidase-like (LOXL) cDNA from a mouse embryo cDNA library and used this cDNA to measure changes in steady state levels of LOXL mRNA during the development of carbon tetrachloride-induced liver fibrosis in adult mice. These results revealed the coincident appearance of increased steady state levels of LOXL mRNA and type III procollagen mRNA early in the development of liver fibrosis. In contrast, steady state levels of lysyl oxidase mRNA increased throughout the onset of hepatic fibrosis and appeared in parallel with the increased steady state levels of pro-αI (I) collagen mRNA. These findings suggest that the LOXL protein (possibly an isoform of lysyl oxidase) is involved in the development of lysine-derived cross-links in collagenous substrates. Moreover, the substrate specificity of the LOXL protein may be different to that of lysyl oxidase and this difference may be collagen-type specific. J. Cell. Biochem. 72:181–188, 1999. © 1999 Wiley-Liss, Inc.

Cystic fibrosis is a genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator gene. Chronic inflammation and proteolysis lead to progressive damage of the bronchial wall. Extracellular matrix determines the structural organization and the mechanical properties of lung airways. It was thus examined in nine patients with cystic fibrosis (six bronchial biopsies and three lobectomies) in order to assess its level of alteration. The submucosal changes in matrix protein distribution were analyzed by immunochemistry and electron microscopy: the subepithelial basal lamina was thinned; an acellular collagen fiber layer composed of interstitial collagens (types I and III) subtended by tenascin and devoid of elastin-associated microfibrils was deposited beneath the basal lamina; this dense fibrous deposit generally formed a thick layer and could extend into the bronchial wall; the bronchial elastic framework lost arborescent distribution and appeared slender, packed, or lacunar; ultrastructural observation gave evidence for elastic and collagenic fiber lysis. Proteolytic activity is probably the major cause of matrix degradation. Fibrosis appears as a repair process rather than as an active fibrogenesis. The reversibility of extracellular matrix alterations is an important challenge and various interventions such as anti-inflammatory treatments can be targeted to halt or reverse this degradation process.

Isolated liver perfusion was developed for the study of liver physiology and preservation. The recent development of new perfusion devices and appropriate liver preservation solutions prompted us to reconsider liver perfusion for the specific purpose of evaluating viability in terms of biochemical changes, paying special attention to modifications in the histological ultrastructure. Twenty-two isolated pig livers were perfused with autologous blood. Arterio-portal perfusions were carried out using an extracorporeal perfusion circuit with a hollow fibre membrane oxygenator. Four groups of pig livers were studied using three different liver flushing solutions [Ringer's lactate, ELOHES, and University of Wisconsin (UW)] and two different oxygenation modalities. Liver function tests and histological studies were done. Our results revealed that a high partial oxygen pressure (PO2) level was deleterious to the ultrastructural elements of hepatocytes, in particular to the mitochondria. It was also associated with deficient metabolic performance, i. e., poor bile production and lack of aerobic metabolism. Normal blood gas values could be obtained with the use of air for liver oxygenation. Flushing of the liver with Ringer's lactate or a macromolecular solution such as ELOHES was associated with severe liver cell injuries, as reflected by a marked rise in liver enzymes and histological lesions. Satisfactory results were obtained when UW solution was used for liver harvesting. We conclude that an appropriate liver preservation solution, normal blood gas values, and normal physiological arterio-portal pressure and blood flow are essential for appropriate liver function with preservation of liver architecture and of hepatocyte ultrastructures. Total bilirubin in bile and Factor V are sensitive indicators of good liver function.

We have recently shown that some squirrel monkeys (Saimiri sciureus) develop cerebral malaria when experimentally infected with asexual blood stage forms of different Plasmodium falciparum isolates. Since cerebral malaria is neither an inconsistent nor predictable event, several clones of endothelial cells isolated from the squirrel monkey brain microvasculature have been developed. Infected red blood cell (IRBC) adherence involved the knobs and direct membrane interactions through pseudopodes and microvilli on the Saimiri brain endothelial cell (SBEC) surface, similar to that observed with both brain microvascular endothelial cells from a patient who died of cerebral malaria and the rhesus monkey/P. coatneyi cerebral malaria model. The involvement of pseudopodes and microvilli increase the endothelial cell surface for the attachment of IRBCs; however, they are already present before the SBECs are exposed to IRBCs. With some SBEC phenotypes, embedding of IRBCs into the cytoplasma membrane of the endothelial cell was observed, resulting in an extremely close apposition of both SBEC and IRBC membranes during the adherence process. Once IRBCs are adherent, particularly for the embedding type, heterocellular communication-like structures between the cells become apparent. The upregulation of CD36 and intercellular adhesion molecule-1 by soluble recombinant (sr)-tumor necrosis factor-α or sr-interferon-γ did not modify the IRBC interactions with SBECs at the ultrastructural level. The study shows further that the observed differences of IRBC adherence are due to unidentified phenotypic differences of SBECs rather than to a parasite isolate or particular endothelial cell receptor-associated phenomenon. Exploring P. falciparum IRBC cytoadherence in the squirrel monkey using a homologous physiologic target cell model in vitro should be useful for the evaluation of vaccine strategies and drugs to prevent human cerebral malaria.

The adhesion of parasitized red blood cells (PRBC) to the endothelium (sequestration) may contribute to the pathogenic events in severe human malaria caused by P. falciparum . However, the factors involved in the pathophysiology, especially cerebral malaria are poorly understood. Previously, we have shown that the squirrel monkey Saimiri sciureus is a potential model for human cerebral malaria. In this paper we describe five stable clones of endothelial cell lines isolated immediately postmortem from different regions of the brain of Saimiri monkeys. The endothelial cell characteristics of these clones were confirmed by analyzing their ultrastructural aspects by transmission electron microscopy and by immunodetection of various endothelial cell markers. The Saimiri brain endothelial cell clones (SBEC) varied in their expression of different surface molecules. For example, various combinations of receptors involved in P. falciparum PRBC adherence such as CD36, ICAM-1 and E-selectin, were expressed at baseline values and could be up-regulated by human srTNF-α and human srIFN-γ. One of the SBEC clones showed a strong cytoadherence for various laboratory strains of P. falciparum despite the absence of surface expression of any of the known endothelial receptors implicated in PRBC adherence. This finding suggests the existence of a new and uncharacterized PRBC binding receptor. The use of target organ specific endothelial cell lines expressing a number of different potential P. falciparum PRBC cytoadherence receptors, will be a useful in vitro system for the evaluation of strategies for the development of vaccine and antimalarial drugs to prevent human cerebral malaria.

Vascular Ehlers–Danlos syndrome (vEDS) results from a mutation in the gene encoding alpha-1, type III pro-collagen (COL3A1) and confers fragility to skin, ligament and vascular tissue. We tested the value of skin biopsy for diagnosis of vEDS through an ultrastructure scoring procedure. Study design was a multicentric, case–control, blinded trial consisting of two phases: phase 1 was to identify an ultra-structure score providing the best discriminative value for vEDS and phase 2 was to replicate this result in a different population. We enrolled 103 patients, 66 cases defined through the revised nosology for Ehlers–Danlos syndromes and 37 control subjects selected from patients referred for other pathologies. Ultrastructure of extracellular matrix was read by three to five experienced pathologists blinded for diagnosis. We used the receiver operating curves and logistic regression analysis for ranking ultrastructure scores. We created a detailed description of lesions observed in vEDS patients with 27 items (coded 0 or 1). In the phase 1 (17 cases and 20 controls), abnormal fibroblast shape, presence of lysosomes in the fibroblast and abnormal basal lamina were found to be independent discriminative items. Addition of these three items (defining an ultrastructure score) had the best diagnosis value (area under the curve (AUC) = 0.96). In the phase 2 (49 cases, 17 controls), ultrastructure score provided odds ratio of 9.76 (95 % CI 2.91–32.78), and AUC of 0.90. The ultrastructure score of skin biopsy has predictive value for the diagnosis of vEDS. Presence of two or more signs (either abnormal fibroblast, presence of lysosomes in the fibroblast or abnormal basal lamina) is very evocative of vEDS.

Lymphangiomyomatosis (LAM) consists of smooth muscle (SM) cell proliferation of unknown origin involving the lymph nodes and the lung interstitium. From morphological studies showing both SM differentiation of the proliferating cells and lymphatic hyperplasia, hypotheses were suggested concerning the origin of the proliferation. Two cases of LAM were investigated by electron microscopy and immunohistochemistry; tissues were obtained by lymph node and open lung biopsies. Cytoplasmic and matrix protein markers were used in order to clarify the pattern of differentiation of the proliferating cells and to characterize their connective tissue environment. The proliferating cells present ultrastructural characteristics of SM cells; they contain vimentin, desmin, and alpha-SM actin and are devoid of Factor VIII, favouring a parieto-arterial origin. The connective tissue matrix inside the infiltrate is composed of interstitial collagens and basement membrane components. At the late stage of the disease, remodelling of the interstitial matrix accompanies the infiltrate and remains perilesional.

We have studied the expression of the desmin gene, a muscle-specific intermediate filament protein in the granuloma cells of mouse liver infected with Schistosoma mansoni . In situ hybridization using a desmin DNA probe showed that fibroblastic cells in the granuloma strongly expressed desmin mRNAs, while in normal liver these cells did not express this mRNA to a detectable degree. The quantitative analysis of total RNAs demonstrated that the proportion of specific desmin mRNA increased from 14 to 18 weeks after infection and decreased at 20 weeks. The analysis of collagen gene expression indicated that the amount of type III collagen mRNAs was still increasing after 18 weeks from infection; in contrast, the amount of type I collagen mRNAs remained unchanged at that stage. A good correlation was observed between the detection of the specific mRNAs and the detection of both desmin and collagen molecules. Therefore, these data point to a coordinate induction of desmin and collagen gene expression during Schistosomal granuloma formation. They also suggest that the expression of the myofibroblast phenotype involves the induction of both genes.

International audience; Cutis laxa (CL) is a rare genodermatosis, which is clinically and genetically heterogeneous. It is characterized by redundant, loose, sagging, and inelastic skin. In a consanguineous family from Lebanon with autosomal-recessive transmission, we identified a homozygous missense mutation (c.649T --> C; p.C217R) in the fibulin-5 gene (FBLN5), which was, to our knowledge, previously unreported. Small skin biopsies were performed, which permitted isolation of skin fibroblasts harboring this FBLN5 mutation; they exhibited a deficit in cell growth. A CL skin equivalent (CL-SE) model compared with control SE was successfully developed to define the behavior of CL fibroblasts in a three-dimensional model. There was increased cell death and a global extracellular matrix deficiency in the dermis of this CL-SE model, and a low level of the main elastic fiber expression. There was no basement membrane evident at the ultrastructural level, and type-VII collagen could not be detected at the histological level. This model reproduced some defects of the extracellular matrix and highlighted other defects, which occurred at the time of the basement membrane formation, which were not evident in skin from patients. This CL-SE model could be adapted to screen for therapeutically active molecules.Cutis laxa (CL) is a rare genodermatosis, which is clinically and genetically heterogeneous. It is characterized by redundant, loose, sagging, and inelastic skin. In a consanguineous family from Lebanon with autosomal-recessive transmission, we identified a homozygous missense mutation (c.649T --> C; p.C217R) in the fibulin-5 gene (FBLN5), which was, to our knowledge, previously unreported. Small skin biopsies were performed, which permitted isolation of skin fibroblasts harboring this FBLN5 mutation; they exhibited a deficit in cell growth. A CL skin equivalent (CL-SE) model compared with control SE was successfully developed to define the behavior of CL fibroblasts in a three-dimensional model. There was increased cell death and a global extracellular matrix deficiency in the dermis of this CL-SE model, and a low level of the main elastic fiber expression. There was no basement membrane evident at the ultrastructural level, and type-VII collagen could not be detected at the histological level. This model reproduced some defects of the extracellular matrix and highlighted other defects, which occurred at the time of the basement membrane formation, which were not evident in skin from patients. This CL-SE model could be adapted to screen for therapeutically active molecules.

International audience; Mitochondrial dysfunction in skeletal muscle has been implicated in the development of type 2 diabetes. However, whether these changes are a cause or a consequence of insulin resistance is not clear. We investigated the structure and function of muscle mitochondria during the development of insulin resistance and progression to diabetes in mice fed a high-fat, high-sucrose diet. Although 1 month of high-fat, high-sucrose diet feeding was sufficient to induce glucose intolerance, mice showed no evidence of mitochondrial dysfunction at this stage. However, an extended diet intervention induced a diabetic state in which we observed altered mitochondrial biogenesis, structure, and function in muscle tissue. We assessed the role of oxidative stress in the development of these mitochondrial abnormalities and found that diet-induced diabetic mice had an increase in ROS production in skeletal muscle. In addition, ROS production was associated with mitochondrial alterations in the muscle of hyperglycemic streptozotocin-treated mice, and normalization of glycemia or antioxidant treatment decreased muscle ROS production and restored mitochondrial integrity. Glucose- or lipid-induced ROS production resulted in mitochondrial alterations in muscle cells in vitro, and these effects were blocked by antioxidant treatment. These data suggest that mitochondrial alterations do not precede the onset of insulin resistance and result from increased ROS production in muscle in diet-induced diabetic mice.

Light microscope histochemistry and immunohistochemistry, and routine electron microscopy techniques were performed to analyse elastin distribution and structure in the human liver compared with that in baboon and mouse. In man and baboon, elastic fibers stained by iron hematoxylin or orcinolnew fuchsin seemed to be solitary and were few in number; in the mouse they were thinner but abundant, both in the portal tract and in hepatic veins. Orcein or resorcin-fuchsin stains, employed after oxidation of tissue sections, revealed a network comprising elastic, elaunin, and oxytalan fibers, which was also demonstrated by immunofluorescence with anti-elastin antibody in man and baboon. At the ultrastructural level, the elastic fibers of the human portal tract corresponded to discontinuous patches of amorphous material intermingled with few microfibrils. These contrasted with the thinner elastic fibers of baboon and mouse liver which had a core of amorphous material. In man and baboon, these fibers meshed into slender bundles of microfibrils often exhibiting small spots of amorphous material (elaunin fibers) and terminated as isolated microfibrils (oxytalan fibers). Immunoelectron microscopy of elastin carried out on baboon liver tissue labelled the amorphous material and also its microfibrillar component. Immunoperoxidase deposits were also associated with isolated bundles of microfibrils in the baboon portal stroma. Immunolabelling and elastic stains disclosed an important elastin portal network located around vascular, biliary structures and interspaced with collagen bundles. The structural polymorphism of elastin, assembling different relative amounts of amorphous material and microfibrils, might have a relationship with the required elasticity in a given species.

Purine nucleoside phosphorylase (PNP, EC 2.4.2.1) activity was revealed by enzyme histochemistry in Toxoplasma gondii ME49 strain isolated from murine cerebral cysts and from in vitro cultivation. The activity of the enzyme was revealed by an insoluble electron-opaque precipitate of lead phosphate at the site of the reaction. In bradyzoites and tachyzoites of T. gondii, the enzyme activity could be observed only in the cytoplasm. In bradyzoites, one or two foci of important PNP activity were detected near the nucleus. In tachyzoites, an important PNP activity underlined the plasma membrane. For both bradyzoites and tachyzoites, localization neither in the nucleus nor in cytoplasmic organelles could be detected.

HCV particles were isolated from the plasma of chronically infected patients. The virus was analysed by sucrose density gradient centrifugation. The fractions were tested for viral RNA, core antigen and envelope proteins by using a monoclonal antibody directed against the natural E1E2 complex (D32.10). Two populations of particles containing RNA plus core antigen were separated: the first with a density of 1.06–1.08 g/ml did not contain the envelope proteins; the second with a density between 1.17 and 1.21 g/ml expressed both E1 and E2 glycoproteins. Electron microscopy of the enveloped population after immunoprecipitation with D32.10 showed spherical particles with a rather featureless surface and with a diameter around 40 nm. Immuno-gold staining gave evidence that the E1E2 complex was indeed positioned at the surface of these particles.

An antiserum was generated from synthetic peptides highly conserved between different mammalian species to immunolocalise the small leucine-rich proteoglycan osteoadherin (OSAD) in murine teeth. In 19-day-old embryos of rats and mice, a positive staining was found in incisor predentin and alveolar bone surrounding developing incisors and molars. In newborns, OSAD was detected at the tip of the first molar cusp where it accumulated in predentin concomitantly with odontoblast differentiation. In 2-day-old rats and mice, in the first molar, immunostaining revealed positive predentin, enamel matrix close to the apical pole of ameloblasts and a strong signal in dentin. At this stage, OSAD was detected in predentin in the second molar. Ultrastructural immunocytochemistry showed gold particles associated with collagen fibres in predentin and in foci at the dentin mineralisation front. Gold particles were also detected near the secretory pole of ameloblasts where enamel crystallites elongate. No staining was detected in pulp tissue and dental follicle. Restriction of OSAD expression to the extracellular matrix of bone, dentin and enamel suggests a role of this proteoglycan in the organisation of mineralised tissues.

As autophagic inclusions accumulate in senescent fibroblasts, we wondered whether an increase in cellular fragility during in vitro lymphocyte aging may be related to an autolysosome accumulation. We established that, during long-term cultures, repeatedly stimulated T-lymphocytes acquired characteristics of replicative senescence and became progressively intolerant to activation. Cell death following stimulations: (i) corresponded to apoptosis, associated with necrosis at the end of the culture; (ii) was not, for its main part, mediated through CD95/CD178 or TNFRII/TNF alpha interactions; and (iii) occurred in spite of bcl-2 increased expression. After 14 weeks of culture, the percentage of lymphocytes containing at least one autophagic inclusion (p

Replicative senescence appears after a finite number of cell divisions. After proliferation has ceased, senescent cells remain viable for long periods and metabolic modifications are observed such as lipofuscin accumulation. In order to understand this phenomenon, we examined the emergence of subcellular modifications corresponding to autophagy in MRC5 normal human fibroblasts. An increase of monodansylcadaverine fluorescence, a specific marker of autophagy, in aging compared to young fibroblasts was observed (p

Panton Valentine leukocidin (PVL) may be responsible for pulmonary necrosis in necrotizing Staphylococcus aureus pneumonia, a highly lethal infection. Commercial intravenous immunoglobulin (IVIg) preparations containing antibodies against PVL might have therapeutic value in this setting, as an adjunct to antimicrobial chemotherapy. To test this possibility, we determined anti-PVL antibody titers in commercial IVIg and the capacity of IVIg to prevent the cytopathic effects of PVL in vitro. Specific enzyme-linked immunosorbent assays based on purified recombinant PVL (rPVL) showed that IVIg contained specific anti-PVL antibodies. The cytotoxicity of rPVL and of crude culture supernatants of PVL-producing S. aureus strains were investigated by measuring ethidium-bromide incorporation by polymorphonuclear neutrophils (PMNs) in flow cytometric assays, as well as PMN ultrastructural changes by transmission electron microscopy. IVIg was found to neutralize pore formation and the cytopathic effect of both rPVL and S. aureus culture supernatants.

The process leading to irreversible fibrotic constriction of the bronchioles was studied in two cases of bronchiolitis obliterans (BO) after bone marrow transplantation. Because lysyl oxidase (LOX) is the main collagen cross-linking enzyme that might account for irreversible fibrosis, its expression was studied together with expression of extracellular matrix (ECM) proteins. Characteristic types of lesions could be distinguished on the basis of histological and immunohistological criteria. An inflammatory stage was characterised by infiltration restricted to the bronchioles by lymphocytes and dendritic cells. A fibro-inflammatory stage was characterised by the coexistence of a persistent immune cellular lesion pattern with further focal modelling of a sub-epithelial neo-synthesised connective matrix. LOX expression was observed at the tips of intra-luminal fibrotic protrusions, together with tenascin and cellular fibronectin. A fibrotic stage was characterised by dense ECM deposits spreading throughout the peri-bronchiolar connective tissue, resulting in bronchiole obliteration and final disappearance. In contrast to reversible cases of fibrosis, persistence of long-term LOX expression reflecting continuing fibrosing activity might account for the irreversible status of BO. Our two cases illustrated that, at inflammatory and fibro-inflammatory stages, BO may be stabilised by immunosuppressive treatment, while the persistence of LOX expression in the fibrotic stage might correspond to a disease that becomes irreversible and fatal.The process leading to irreversible fibrotic constriction of the bronchioles was studied in two cases of bronchiolitis obliterans (BO) after bone marrow transplantation. Because lysyl oxidase (LOX) is the main collagen cross-linking enzyme that might account for irreversible fibrosis, its expression was studied together with expression of extracellular matrix (ECM) proteins. Characteristic types of lesions could be distinguished on the basis of histological and immunohistological criteria. An inflammatory stage was characterised by infiltration restricted to the bronchioles by lymphocytes and dendritic cells. A fibro-inflammatory stage was characterised by the coexistence of a persistent immune cellular lesion pattern with further focal modelling of a sub-epithelial neo-synthesised connective matrix. LOX expression was observed at the tips of intra-luminal fibrotic protrusions, together with tenascin and cellular fibronectin. A fibrotic stage was characterised by dense ECM deposits spreading throughout the peri-bronchiolar connective tissue, resulting in bronchiole obliteration and final disappearance. In contrast to reversible cases of fibrosis, persistence of long-term LOX expression reflecting continuing fibrosing activity might account for the irreversible status of BO. Our two cases illustrated that, at inflammatory and fibro-inflammatory stages, BO may be stabilised by immunosuppressive treatment, while the persistence of LOX expression in the fibrotic stage might correspond to a disease that becomes irreversible and fatal.

Williams syndrome (WS) is a complex developmental disorder with multisystem involvement known to be the result of a microdeletion in the q11.23 region of chromosome 7. This deletion involves several genes, including the elastin gene. Although elastic fibers are important constituents of skin, little is known about the skin phenotype in WS patients. We have therefore studied the skin of four WS patients in which we've shown the deletion of one copy of the elastin gene. Physical examination and indirect immunofluorescent microscopy of elastin did not detect any major phenotypic or morphologic changes in the skin. We were able, however, to show subtle textural changes in skin and, by electron microscopy, that the amorphous component of elastic fibers in WS patients was consistently reduced when compared to normal controls. These findings indicate that deletion of one copy of the elastin gene results in reduced deposition of elastin in dermal elastic fibers, an altered elastic fiber ultrastructure, and a subclinical dermal phenotype in the children and young adult patients analyzed in this study.

International audience; Cdc14, a dual-specificity protein phosphatase, has been previously implicated in triggering exit from mitosis in the yeast Saccharomyces cerevisiae. Using immunofluorescence microscopy and immunogold labeling, we demonstrate that a functional HA-tagged version of the phosphatase Cdc14 localizes to the nucleolus. Moreover, Cdc14-HA co-localized with the nucleolar NOP2 and GAR1 proteins. By immunofluorescence, Cdc14-HA was found in the nucleolus during most of the mitotic cell cycle, except during anaphase-telophase when it redistributed along the mitotic spindle. While this work was in progress, the same pattern of Cdc14 localization was described by others (Visintin et al, Nature 398 (1999) 818). Constitutive overexpression of CDC14 was toxic and led to cell cycle arrest of cells, mainly in G1. This correlated with the appearance of abnormal nuclear structures. A genetic search for suppressors of the lethality associated with CDC14 overexpression identified YJL076W. Because overproduction of Yj1076w buffered the toxic effect of Cdc14 overproduction, this suggested that it might be a substrate of Cdc14. This has indeed been found to be the case by others who recently described Yj1076w/Netl as a nucleolar protein that physically associates with Cdc14 (Shou et al, Cell 97 (1999) 233). The present data confirm several recently uncovered aspects of the regulation of Cdc14 localization and activity and suggest that the level of expression of CDC14 influences the structural organization of the nucleolus.

Schistosomiasis is a debilitating tropical disease for which an effective vaccine is needed. A 28-kDa glutathione S-transferase from Schistosoma mansoni (Sm28GST) has been shown to induce protective immunity. Sm28GST possesses significant sequence identity to mammalian GST isoforms. In order to study self-reactivity in mice immunized with Sm28GST and the concomitant phenomena of immune tolerance and epitope suppression, as well as their consequences for the protective immunity induced by this vaccination, we developed transgenic (Tg) mice that express Sm28GST under the control of a part of the mouse transferrin gene promoter. A study of (P28)Tg mice showed that the expression of Sm28GST was strictly localized in pericentrolobular hepatocytes. No histological change, inflammatory infiltrates, or modification of seric L-aspartate: 2-oxoglutarate aminotransferase concentration was observed over an 18-month period, despite a cross-reactivity between Sm28GST and a mouse molecule of 30 kDa. The immunoglobulin G anti-Sm28GST response of (P28)Tg mice immunized with recombinant Sm28GST was lower (P < 0.001) than that observed in non-(P28)Tg littermates and inversely proportional of Sm28GST liver expression. The response of non-(P28)Tg mouse spleen cells to Sm28GST stimulation was greater (P < 0.01) than that observed with (P28)Tg mouse spleen cells. (P28)Tg mice infected with 40 S. mansoni furcocercariae harbored more worms (P < 0.05) than did non-(P28)Tg control mice. The increase in the level of infection in (P28)Tg mice was reflected in concomitant increases in the numbers of adult worms and schistosome eggs found in livers and intestines after whole-body perfusion at 56 days postinfection, but no relative increase in the fertility of individual female worms was observed. The results obtained argue for the involvement of Sm28GST in reducing levels of infection and support the view that this enzyme has a central role in the maintenance of parasite viability, at least during its migration through host tissues.

BACKGROUND/AIMS: Interferon-gamma (IFNgamma) elicits antiproliferative and antifibrogenic activity in a variety of mesenchymal cells, including hepatic stellate cells (Ito cells), and therefore represents a possible drug for liver fibrosis. However, IFNgamma binds to heparan sulfate, and is localized by these molecules in a restricted area within the tissue. For example, in rat liver, it has been shown that following injection, IFNgamma was concentrated in a restricted area by heparan sulfate. The aim of this study was to analyze, at the tissular level in the liver, the antifibrogenic activity of IFNgamma. METHODS: Chronic inflammation due to Schistosoma infection induces hepatic fibrogenesis around the parasite eggs (portal fibrosis) and in the parenchyma (lobular fibrosis). Infected mice were treated with recombinant IFNgamma, and the collagen content of the liver was evaluated by means of biochemical dosages, histologic and morphometric examination of liver tissue, and electron microscopic analysis. RESULTS: IFNgamma reduced the whole liver collagen content by 28% compared to control mice. In control mice, collagen was found around eggs and infiltrating the parenchyma, associated with a diffuse array of inflammatory cells, while in treated mice the collagen was present only around eggs and surrounded by a dense layer of inflammatory cells. Therefore, collagen was measured in isolated granulomas and in the remaining parenchyma. We found that IFNgamma strongly reduced the parenchymal collagen (74%), but had no effect on the granuloma collagen content. CONCLUSIONS: Together these data demonstrate that IFNgamma did not act in a homogeneous manner in the liver. Since granulomas are almost completely devoid of heparan sulfate, these data could suggest, among others hypotheses, that heparan sulfate which binds IFNgamma either localizes or mediates the cytokine activity outside the granulomas.

We compared the effects of IL-10 and IL-4 on the functions of B lymphocytes triggered through their CD40. During the initial phase, IL-10 was as potent as IL-4 in inducing the expansion of viable B cells. Then, cellular expansion slowed down and after approximately 3 weeks the number of B cells started to decline. While the combination of IL-10 and IL-4 was synergistic during the first 2 weeks of culture, B cell recovery declined after 3 weeks, indicating that IL-10 prevails over IL-4. Those effects were not restricted to a specific B cell subset as both sIgD+ B cells and sIgD- B cells behaved in a similar way, though the latter population responded with a slightly accelerated kinetic. Inverted microscope examination and scanning electron microscopy showed that in response to IL-10, CD40-activated B cell cultures were heterogeneous with loose aggregates of cells as well as free floating large ovoid cells. In contrast, in the presence of IL-4, CD40-activated B cell cultures were essentially composed of tight cell clumps. IL-10 progressively induced all B cells to differentiate into non-replicating cells with intracytoplasmic Ig that secreted Ig at a high rate. Cytologic analysis indicated that IL-10 cultured cells display a basophilic cytoplasm with an arcoplasm and a low nucleus/cytoplasm ratio. Transmission electron microscopy demonstrated that when IL-10 was added to the culture, B cells displayed structures for excretion with extended endoplasmic reticulum and dilated cisternae containing paracrystalline structures, typical of plasmablasts cells. Taken together, these results indicate that IL-10 acts as a plasma cell differentiation factor for CD40-activated B cells.

International audience; This study of two cases of pulmonary Wegener's granulomatosis (WG) focuses on the ultrastructural aspects of the vascular wall injury and on the immunohistochemical characterization of the perivascular connective matrix. The iterative waves of endothelial cell necrosis and regeneration are demonstrated by the multilamellar appearance of the basal lamina. Neutrophils infiltrate the vessel wall and myofibroblasts are recruited to injured vessels. The perivascular connective matrix associates basement-membrane like and fibrillar material with fibrin deposits. The initiation of the fibrosing process was assessed by the visualization of matrix molecules involved in targeting (p-fibronectin), organizing (cellular fibronectin and tenascin) and stabilizing (lysyl-oxidase) the fibrogenic activity. These elementary lesions affect different levels of the vascular tree, and capillaritis is involved in the extension of the pathological process. Lysyl-oxidase labelling reveals the fibrosing front which is located on the border of dense fibrosis. The markers of fibrosing activity disappear in the areas of fibrosis following vasculitis and/or ischaemic necrosis and/or granulomatosis. Vasculitis plays a major role in both the genesis and progression of the fibrosis observed in the late stage of WG.This study of two cases of pulmonary Wegener's granulomatosis (WG) focuses on the ultrastructural aspects of the vascular wall injury and on the immunohistochemical characterization of the perivascular connective matrix. The iterative waves of endothelial cell necrosis and regeneration are demonstrated by the multilamellar appearance of the basal lamina. Neutrophils infiltrate the vessel wall and myofibroblasts are recruited to injured vessels. The perivascular connective matrix associates basement-membrane like and fibrillar material with fibrin deposits. The initiation of the fibrosing process was assessed by the visualization of matrix molecules involved in targeting (p-fibronectin), organizing (cellular fibronectin and tenascin) and stabilizing (lysyl-oxidase) the fibrogenic activity. These elementary lesions affect different levels of the vascular tree, and capillaritis is involved in the extension of the pathological process. Lysyl-oxidase labelling reveals the fibrosing front which is located on the border of dense fibrosis. The markers of fibrosing activity disappear in the areas of fibrosis following vasculitis and/or ischaemic necrosis and/or granulomatosis. Vasculitis plays a major role in both the genesis and progression of the fibrosis observed in the late stage of WG.

Multiple Myeloma (MM) is a plasma cell (PC) malignancy producing large amounts of monoclonal Igs requiring a highly developed endoplasmic reticulum (ER). A coordinated adaptative program called the unfolded protein response (UPR) allows cells to survive the stress consecutive to a transient protein overload of the ER. This process is referred to as "physiological UPR". If the stress is excessive or sustained, the UPR leads to cell death ("terminal UPR"). We have recently documented that the spontaneous death of non transformed human PC is initiated by an ER stress. Moreover the group of R. Sitia has established that the proteasomal activity decreases in the late phases of PC differentiation. These findings suggest that the imbalance between the high-rate of Igs secretion and the impaired proteolytic machinery predisposes PC to apoptosis. We have postulated that a dysregulated adaptative UPR contributes to the increased survival capacity of MM cells. We have undertaken a study aimed at promoting death of MM cells by disruption of ER homeostasis. Our approach has consisted in using RNA interference to knock-down the UPR sensors PERK, ATF6 and IRE1, in two MM cell lines (U 266 and NCI-H929). We show that the anti-UPR siRNAs induce a 70 to 80% reduction of the levels of expression of PERK, ATF6 and IRE1 and promote cell death of these two human MM cell lines. The PERK-targeted siRNA was consistently found to be the best apoptosis inducer. We found that invalidation of PERK leads to induction of a terminal UPR as revealed by the upregulation of GADD153/CHOP and of the spliced form of XBP-1. The death elicited by invalidation of the UPR sensors was accompanied by phosphatidylserine (PS) exposure and early loss of the mitochondrial transmembrane potential. By contrast, we found no evidence of DNA fragmentation and electronic microscopy revealed only a partial nuclear chromatin condensation. Cell death induced by UPR-targeted siRNAs operated in a caspase -independent fashion since it was not inhibited by the pan-caspase inhibitor z-VAD-fmk. Accordingly, the activated forms of caspase-3 and-4 were undetectable. However, calpain inhibitors and calcium chelators prevented the loss of the mitochondrial transmembrane potential and partially inhibited cell death. This suggests that the death signal initiated at the level of the ER is transmitted to the mitochondria via the calcium cysteine protease calpain. Transmission electron microscopy revealed that the PERK siRNA induces formation of numerous autophagic vacuoles, a result which was confirmed by the positive staining with monodansylcadaverine (MDC). Appearance of mature autophagic vacuoles preceeded acquisition of death morphology which was characterized by partial nuclear condensation and protein accumulation. The anti-UPR siRNAs induced the presence of immature and mature autophagic vacuoles but, in contrast, when acquiring death morphology, cells were devoid of autophagic vacuoles although they showed partial nuclear condensation. The autophagic specific inhibitor 3 methyladenine reduced both PS exposure and MDC staining induced by PERK siRNA. This suggests that UPR-targeted siRNAs induce death of MM cells by autophagy. Altogether, our data demonstrate that disruption of ER homeostasis by extinction of UPR sensors induce death of MM cells. They also reveal that a connection exists between ER stress and autophagy in MM cells.

A gel-like reconstituted basement membrane matrix containing type IV collagen, laminin, entactin, nidogen, and heparan sulfate proteoglycan was used to examine the interactions between normal calf skin fibroblasts and basement membranes. Within 6 h after seeding, fibroblasts initiated a migration that resulted in the formation of a cellular network after 1 day of culture on top of the gel. Electron microscopy revealed that fibroblasts were able to remodel the basement membrane matrix by penetrating into the gel (from day 3), depositing fibronectin and collagen fibers, and retracting this extracellular matrix. Fibroblasts cultured on the Engelbreth-Holm-Swarm reconstituted basement membrane matrix displayed ultrastructural features characterized by a poor synthetic apparatus (rough endoplasmic reticulum and Golgi vesicles), a large cytoskeleton, and intracytoplasmic vesicles containing laminin. Thus the reconstituted basement membrane matrix is remodeled by skin fibroblasts, and reciprocally their ultrastructural morphologic features are affected by this matrix.

International audience; Elastic fiber formation involves the secretion of tropoelastin which is converted to insoluble elastin by cross-linking, initiated by the oxidative deamination of lysine residues by lysyl oxidase. Five lysyl oxidase genes have been discovered. This study deals with the expression of two isoforms, LOX and LOX-like (LOXL), in human foreskin and in a human skin-equivalent (SE) model that allows the formation of elastic fibers. In this model, keratinocytes are added to a dermal equivalent made of fibroblasts grown on a chitosan-cross-linked collagen-GAG matrix. LOX and LOXL were detected by immunohistochemistry in the dermis and the epidermis of both normal skin and in a SE. This expression was confirmed by in situ hybridization on the SE. LOX and LOXL expression patterns were confirmed in human skin. The ultrastructural localization of LOXL was indicative of its association with elastin-positive materials within the SE and human skin, though interaction with collagen could not be discarded. LOX was found on collagen fibers and could be associated with elastin-positive materials in the SE and human skin. LOXL and LOX were detected in keratinocytes where LOX was mainly expressed by differentiating keratinocytes, in contrast to LOXL that can be found in both proliferating and differentiating fibroblasts. These data favor a role for LOXL in elastic fiber formation, together with LOX, and within the epidermis where both enzymes should play a role in post-translational modification of yet unknown substrates.Elastic fiber formation involves the secretion of tropoelastin which is converted to insoluble elastin by cross-linking, initiated by the oxidative deamination of lysine residues by lysyl oxidase. Five lysyl oxidase genes have been discovered. This study deals with the expression of two isoforms, LOX and LOX-like (LOXL), in human foreskin and in a human skin-equivalent (SE) model that allows the formation of elastic fibers. In this model, keratinocytes are added to a dermal equivalent made of fibroblasts grown on a chitosan-cross-linked collagen-GAG matrix. LOX and LOXL were detected by immunohistochemistry in the dermis and the epidermis of both normal skin and in a SE. This expression was confirmed by in situ hybridization on the SE. LOX and LOXL expression patterns were confirmed in human skin. The ultrastructural localization of LOXL was indicative of its association with elastin-positive materials within the SE and human skin, though interaction with collagen could not be discarded. LOX was found on collagen fibers and could be associated with elastin-positive materials in the SE and human skin. LOXL and LOX were detected in keratinocytes where LOX was mainly expressed by differentiating keratinocytes, in contrast to LOXL that can be found in both proliferating and differentiating fibroblasts. These data favor a role for LOXL in elastic fiber formation, together with LOX, and within the epidermis where both enzymes should play a role in post-translational modification of yet unknown substrates.

In the interstitium of the alveolar septa in the peripheral parts of the lung, four molecular types of collagen (I, III, IV and V) each with different morphological appearances, can be identified. The structural integrity of collagens accounts for the physiological efficiency of the lung. Fibrous thickening of alveolar septa is an invariable result of various diseases affecting the interstitium of the lung. The light and electron microscopic findings, and the immunological typing of collagens in six cases of fibrotic alveolar disease, are described. In the alveolar septa, two different compartments (the alveolo-capillary junction and the supportive axis) were affected by fibrosis: the alveolo-capillary junction was widened by the addition of interstitial collagens to basement membranes. In the axis, the increase of interstitial (types I and III) collagen gave rise to different patterns of connective matrix organization, graded as Loose or Dense depending on quantitative alterations of the type I/III ratio. The mode of organization of the fibrotic lung connective matrix, which depends on the quality of deposits in the matrix, may be correlated with the evolution of interstitial pulmonary fibrosis, in terms of its stability, remodelling ability and reversibility.

Poster P10; International audience