Different morphotypes of functional dentition in the lower molar region of tabby (EDA) mice.

OBJECTIVES: To sort and classify the highly variable lower molar dentition in tabby (Ta) mice postnatally. The Ta syndome is homologous to the anhidrotic (hypohidrotic) ectodermal dysplasia (EDA) in human and includes severe developmental defects of teeth, hair and sweat glands. DESIGN: Analysis of tooth shape and cusp pattern and measurement of the mesio-distal crown length. SETTING AND SAMPLE POPULATION: Institute of Experimental Medicine, Academy of Sciences, Prague. Fixed heads of 107 tabby (Ta) homozygous and hemizygous mice and 90 wild type mice aged from post-natal day 11 to adulthood, collected during 1995-2001. OUTCOME MEASURE: Identification of distinct morphotypes of Ta dentition. Reduced tooth length in Ta teeth and specific differences in tooth length between distinct morphotypes. RESULTS: The variable dentitions in the lower molar region of Ta mice were classified in two basic morphotypes I and II. The morphotype I was further subdivided into particular morphotypes Ia, Ib and Ic. Proportion of the basic morphotypes I and II was different in the offspring of heterozygous (84% and 12%) compared with homozygous + hemizygous (45% and 52%) mothers. The proportions of particular morphotypes within a basic morphotype were similar in both offspring groups. CONCLUSION: The identification of the distinct morphotypes made possible to classify the structural variability of the mandibular functional dentition in Ta mice.

Different morphotypes of the tabby (EDA) dentition in the mouse mandible result from a defect in the mesio-distal segmentation of dental epithelium.

OBJECTIVES: Prenatal identification of the different dentition morphotypes, which exist in the lower molar region of tabby (Ta) adult mice, and investigation of their origin. The mouse Ta syndrome and its counterpart anhidrotic (hypohidrotic) ectodermal dysplasia (EDA) in human are characterized by absence or hypoplasia of sweat glands, hair and teeth. DESIGN: Analysis of tooth morphogenesis using serial histological sections and 3D computer aided reconstructions of the dental epithelium in the cheek region of the mandible. SETTING AND SAMPLE POPULATION: Institute of Experimental Medicine, Academy of Sciences, Prague. Heads of 75 Ta homozygous and hemizygous mice and 40 wild type (WT) control mice aged from embryonic day (ED) 14.0-20.5 (newborns), harvested during 1995-2001. OUTCOME MEASURE: Prenatal identification of five distinct morphotypes of Ta dentition on the basis of differences in tooth number, size, shape, position and developmental stage and of the morphology of the enamel knot in the most mesial tooth primordium. RESULTS: The mesio-distal length of the dental epithelium was similar in the lower cheek region in Ta and WT mice. In Ta embryos, there was altered the mesio-distal segmentation of the dental epithelium giving rise to the individual tooth primordia. Prenatally, two basic morphotypes I and II and their particular subtypes (Ia, Ib, Ic, and IIa, IIb, respectively) of the developing dentition were identified from day 15.5. The incidence of the distinct morphotypes in the present sample did not differ from postnatal data. The proportion of the morphotype I and II was dependent on mother genotype. CONCLUSION: The different dentition morphotypes in Ta mice originate from a defect in the mesio-distal segmentation of the dental epithelium in mouse embryos. This defect presumably leads to variable positions of tooth boundaries that do not correspond to those of the WT molars. One tooth primordium of Ta mice might be derived from adjacent parts of two molar primordia in WT mice.

Differential involvement of the P2Y1 and P2YT receptors in the morphological changes of platelet aggregation.

The relative contributions of the P2Y1 and P2YT receptors to the morphological changes induced in platelets by ADP or ADP-releasing agonists were assessed using two P2 antagonists, A2P5P and AR-C67085, selective for P2Y1 and P2YT, respectively. The P2Y1 receptor was found to be involved in i) the centralization of secretory granules elicited by ADP, ii) the formation of filopodia induced by released ADP in weakly activated platelets and iii) actin polymerization and the cytoskeletal translocation of cdc42, rac1 and rhoA, in an integrin alphaIIbbeta3 dependent manner, in ADP-stimulated platelets. In contrast, the P2YT receptor was shown i) to be essential for the formation of stable macroaggregates, ii) to enhance actin polymerization and the cytoskeletal translocation of small GTPases, probably through amplification of platelet aggregation, and iii) not to be involved in the early steps of platelet activation since its blockade did not affect the cytoskeletal translocation of rhoA.

Carbon monoxide induces murine thymocyte apoptosis by a free radical-mediated mechanism.

Carbon monoxide (CO) induces acute or chronic toxicity, according to the level and duration of the exposure. Since chronic CO exposure was shown to have immunosuppressive effects (as it decreases the frequency of rat splenic immunocompetent cells and immunoglobulin production), we investigated the effect of CO on thymocytes, since these are the most sensitive cells to oxidative damage from the lymphoid lineage. We exposed thymocytes to CO, then determined their apoptotic index after 6 h of incubation at 37 degrees C using the fluorochrome Hoechst 33342 and electron microscopy and found an increase of apoptosis in CO-exposed thymocytes. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), an antioxidant vitamin E analog, decreased CO-induced thymocyte apoptosis unlike methylene blue, L-nitroarginine methyl ester or pyrrolidine dithiocarbamate. We also observed that lipid peroxidation was increased in the CO-exposed thymocytes and that it was inhibited by Trolox. Our results suggest that CO induces thymocyte apoptosis by a free radical-mediated mechanism which can be inhibited by Trolox but which does not involve the activation of the guanylyl cyclase-cGMP pathway.

Productive infection of primary cultures of endothelial cells from the cat liver sinusoid with the feline immunodeficiency virus.

Given the similarities between the two viruses, the feline immunodeficiency virus (FIV) is becoming an interesting animal model for human immunodeficiency virus (HIV) studies. To explore the still controversial role of the liver in the development of HIV infection, sinusoidal endothelial cells (SEC) were isolated, and primary cultures were infected with the FIV Villefranche IFFA strain. The isolated cells were characterized by their typical fenestrations, the presence of von Willebrand factor (vWf), and their ability to take up acetylated low-density lipoproteins and denatured collagen. Two weeks after infection, significant amounts of FIV p24 antigen were detected by immunofluorescence in both multinucleated giant and single cells and by enzyme-linked immunosorbent assay in the culture medium. High amounts of viral particles were observed together with different steps of budding at the plasma membrane or at the membrane of intracytoplasmic vacuoles. The released viral particles were shown to be infectious for a permissive cell line. During the first 3 weeks of infection, the only cytopathic effect of FIV was syncytia formation. No noticeable impairment of the pattern of fenestrations and the modulation of their number by a cytoskeleton-mediated process occurred. The productive infection of SEC may contribute to the progression of the infection.

Kinetics of nitric oxide synthase induction by Propionibacterium avidum and lipopolysaccharide.

Conditions for the induction of rat liver Ca2(+)-independent nitric oxide synthase were determined with killed Propionibacterium avidum, and compared with lipopolysaccharide endotoxin. Similar maximal induction was obtained intraperitoneally with the two types of inducers but killed Propionibacterium avidum gave a long-lasting induction while lipopolysaccharide displayed a rapid and short response. Moreover, the induction resulting from an intravenous administration of killed Propionibacterium avidum reached 60 times that of the control whereas lipopolysaccharide treatment induced a 24-fold stimulation only. It is noteworthy that with the first inducer the nitric oxide activity was stable with time whereas with the second one it dropped after 8 h. Whatever the route of administration of killed Propionibacterium avidum, some huge vacuolated Kupffer cells were found in the liver whose parenchyma was almost normal. Numerous monocytes, and unaltered Kupffer cells, were observed. Kupffer cells were identified to be responsible for the uptake of killed Propionibacterium avidum.

Mouse hepatitis virus type 3 infection provokes a decrease in the number of sinusoidal endothelial cell fenestrae both in vivo and in vitro.

Fenestrations of hepatic endothelial cells play an active role as a sieving barrier allowing extensive exchange between the blood and liver parenchyma. Alteration of these structures may be induced in the course of various pathological events and provoke important perturbations of liver function. We demonstrate here that sinusoidal endothelial cells are permissive for mouse hepatitis virus 3 (MHV3) in vivo and in vitro and that this infection leads to a striking decrease in the number of fenestrae. The disappearance of these structures observed under scanning electron microscopy or in cryofracture preparations in vivo and in vitro cannot be reversed by the action of cytochalasin B on the microfilament network. The decrease in the porosity seems to be related directly to the productive infection of the endothelial cells, because it was not observed in A/J mice resistant to the virus and in susceptible BALB/c mice immunized with a thermosensitive mutant in which no viral replication occurs. In conclusion, a viral infection of liver endothelial cells may cause extensive loss of the fenestrations and thus lead to important functional pertubations.

Permissiveness of Kupffer cells for simian immunodeficiency virus (SIV) and morphological changes in the liver of rhesus monkeys at different periods of SIV infection.

The pathogenesis of liver injury, which remains unclear in the course of human immunodeficiency virus infection, can be investigated in simian immunodeficiency virus-infected macaques, which develop an immunodeficiency disease resembling human acquired immune deficiency syndrome (AIDS). We studied the livers of 21 monkeys infected with simian immunodeficiency virus (SIVmac251) for 4 days to 39 months and detected viral antigens in Kupffer cells, macrophages, and lymphocytes in 65% of the livers tested. Virus-containing cells were present in 5 out of 9 livers tested as early as 4 days postinoculation. The number of positive cells as well as their content in viral proteins substantially increased in sinusoidal cells with the progression of the disease. Morphological features and double immunolabeling indicated that Kupffer cells constituted the predominant cell type containing viral antigens. The presence of multinucleated giant cells displaying the ultrastructural features of resident liver macrophages was another sign of the productive infection of Kupffer cells in vivo, which was attested by the observation of budding, immature, and mature SIV particles. Kupffer cell hyperplasia and hypertrophy were evident and appeared to be related to the development of SIV infection, because a close correlation was found between antigenemia and the surface area occupied by these cells. The Kupffer cells contained apoptotic lymphocytes, indicating that resident liver macrophages could play a role in the uptake of such cells from the blood. The production of tumor necrosis factor alpha (TNF alpha) and, possibly, interferon-alpha by Kupffer cells, the expression of vascular adhesion molecule-1, (VCAM-1), intralobular and periportal inflammation, and the proliferation and expansion of bile duct cells were other signs of liver involvement in SIV infection.

Morphological changes in lymph nodes and expression of VCAM1 and cytokines at the late stages of SIV-induced disease in rhesus monkeys.

Four patterns of structural alterations were found in lymph nodes (LNs) from rhesus monkeys 17 to 34 months after infection with simian immunodeficiency virus (SIV-mac251). SIV p27gag antigen and viral particles were localized either between the processes of follicular dendritic cells (FDCs) or in the cytoplasm of macrophages. In hyperplastic follicles, enlarged germinal centres contained numerous Ki67+ proliferating centroblasts which were rather rare in light zones occupied by the CD23+ FDC network. Involuted follicles contained a small number of Ki67+ centroblasts and the CD23 labelling was limited to a very small apical zone. A correlation was found between the morphological characteristics of the follicles (hyperplasia-involution) and the level of expression of the vascular cell adhesion molecule 1 (VCAM1) on FDCs. A gradient in VCAM1 intensity with no expression in the subcapsular-intermediary sinuses, low membrane labelling in the mantle and strong expression in the FDC network was observed. IL1 alpha+ and IL6+ (interleukin) cells (lymphocytes and macrophages) were detected in the mantle, the interfollicular area and the medulla of LNs. Expression of the tumour necrosis factor alpha and ultrastructural markers of interferon alpha production were found in a few FDC and macrophages. Our findings indicate a close relationship between the morphofunctional properties of FDC and the LN structure in SIV infection.

Presence of virion protein x (Vpx) of simian immunodeficiency virus SIVmac 251 in target cells in vivo.

Localization of virion-associated protein x (Vpx) of SIVmac 251 was studied in lymph nodes and liver of six SIVmac-infected monkeys. Vpx was found associated with the network of follicular dendritic cells and macrophages in lymph nodes and/or livers from five out of six animals by immunohistochemistry. Although the humoral response to Vpx occurs in only 50% of the animals, the presence of Vpx in target cell or antibodies to Vpx in all the monkeys studied, suggests that Vpx may be necessary for viral replication in vivo.

Feline immunodeficiency virus can productively infect cultured endothelial cells from cat brain microvessels.

Feline immunodeficiency virus (FIV) provokes a disease in cats characterized by histopathological lesions similar to those observed in AIDS patients. In order to determine whether endothelial cells from brain microvessels are involved in the central nervous system disease to the same extent as macrophages and microglia, cells were isolated from healthy cat brains, cultured and infected in vitro with the FIV Villefranche IFFA 1/88 strain. The isolated cells displayed typical endothelial cell ultrastructural features and were characterized further by von Willebrand factor-labelling and the binding of specific lectins such as Ulex europaeus lectin on their membrane. They were also able to take up acetylated low density lipoproteins. Two weeks after infection, significant amounts of FIV p24 antigen were detected by indirect immunofluorescence in syncytia and single cells. Concomitantly, the same antigen could be detected in the culture medium of the infected cells by an ELISA technique. Numerous viral particles as well as different steps in the process of viral budding were observed under transmission electron microscopy. The synthesis of FIV p24 antigens still occurred in cells in which replication was blocked in the G2 phase with taxol. Our results suggest the possibility of a productive infection of brain microvascular endothelial cells by FIV in vivo, which could lead to important perturbations in the functions of the blood-brain barrier.

HIV-1 infection induces functional alterations in human liver endothelial cells in primary culture.

OBJECTIVES: Since human liver endothelial cells allow HIV-1 multiplication in vitro, we investigated whether HIV induced functional alterations in these cells in primary culture. DESIGN: Direct evidence of the replication of HIV in endothelial cells is sparse, but clotting abnormalities and thrombi, which suggest the existence of an endothelial dysfunction, have been observed in HIV-infected patients. We therefore studied the storage and release of endothelial-specific factors in primary cultures of liver endothelial cells infected with HIV, as well as their cytoskeleton, pinocytic and phagocytic properties. METHODS: Intracellular storage of von Willebrand's factor (vWF) was determined by immunofluorescence and computer image analysis. Excretion of vWF, protein S and endothelin-1 was measured using an enzyme-linked immunosorbent assay and radioimmunoassay. Cytoskeletal constituents were studied by light microscopy. The pinocytosis of acetylated low-density lipoproteins and the phagocytosis of latex beads were analysed under light and electron microscopy. RESULTS: The synthesis of vWF is markedly decreased in HIV-infected liver endothelial cells, as is the excretion of endothelin-1. In contrast, the excretion of protein S remains unaffected and the cytoskeletal network appears to be unaltered. Pinocytosis and phagocytosis are preserved. CONCLUSIONS: HIV infection triggers non-lethal functional alterations in cultured human liver sinusoidal endothelial cells, with a selective impairment in the storage and/or the excretion of endothelial-specific factors such as vWF. This functional modulation could play a role in the pathophysiology of HIV-induced disease.

Signs of Kupffer cell involvement in productive simian immunodeficiency virus infection in monkey liver.

The livers of 21 rhesus monkeys inoculated with SIVmac251 were examined at 4 days to 39 months after infection. SIV antigens were detected in the cytoplasm of Kupffer cells (KC), macrophages and lymphocytes in two-thirds of the livers tested. The number of cells containing viral proteins substantially increased during the development of the disease, and KC were the main cell type displaying SIV proteins at an advanced stage of infection. Mature and immature lentiviral particles were found in cytoplasmic vacuoles or associated with worm-like structures in KC, indicating that SIV replication could occur within resident liver macrophages. Another sign of the permissiveness of KC was the formation of multinucleated giant cells within the hepatic sinusoids. Some of these cells containing 3-6 nuclei still retained ultrastructural features of KC. Most of them contained a high quantity of viral particles. Numerous lymphocytes displaying signs of apoptosis were taken up by KC, especially at the beginning of infection. Hyperplasia and hypertrophy of KC were noted in the course of SIV disease in the liver. The present data indicate that KC can be infected in vivo and may serve as a reservoir for SIV during the progression of the disease.

Human endothelial cells isolated from the hepatic sinusoids and the umbilical vein display a different permissiveness for HIV1.

Endothelial cells are now considered as potential targets for HIV infection, together with other non-lymphoid cells. We previously demonstrated that endothelial cells isolated from the human liver sinusoids (SEC) are permissive for HIV1 in vitro (Steffan et al., 1992), whereas human umbilical vein endothelial cells (HUVEC) do not allow replication of seven HIV1 and HIV2 strains under the same infection conditions (Lafon et al., 1992). The aim of our work was to compare the permissiveness of SEC and HUVEC and to determine whether the lack of HIV replication in HUVEC, which do not possess CD4 receptors (Lafon et al., 1992), could be related to the absence of virus penetration. The bypass of the early events in the virus life cycle, including the stage of fusion, by transfection of HUVEC with the HTLV-IIIB provirus led to one cycle of viral replication. Moreover, a low level of viral replication was observed in HUVEC infected with the NDK HIV strain, known to penetrate its target cell in a CD4-independent fashion. These results indicate that the block of infection in HUVEC takes place during the early stages of the viral replicative cycle. However, given the low level of NDK replication in HUVEC (0.02% infected cells) in comparison with NDK-infected SEC (30 to 50% infected cells), the absence of the CD4 receptor on HUVEC may not be the only limiting factor for viral growth. Although these two types of endothelial cells display a very different permissiveness for HIV, the present data suggest that they could both serve as a viral reservoir.

Primary cultures of endothelial cells from the human liver sinusoid are permissive for human immunodeficiency virus type 1.

Human endothelial cells isolated from hepatic sinusoids were infected in vitro with human immunodeficiency virus type 1 (HIV-1). An early sign of infection occurring in the culture was the formation of multinucleated cells. By double-labeling immunofluorescence, 5-15% of the cells recognized as endothelial cells owing to the presence of von Willebrand factor were found to contain HIV p24 and gp120 antigens after 2 weeks. Reverse transcriptase activity was released into the medium, and different steps in the process of viral budding were observed by electron microscopy. The virus produced by the endothelial cells was found to be infectious for CEM cells, a human T-cell line. CD4 molecules are present at the surface of the endothelial cells, as demonstrated by immunogold-silver staining and backscattered electron imaging. Treatment with an anti-CD4 antibody abolished productive infection of the sinusoidal endothelial cells. The possibility that endothelial cells of the liver sinusoid are infected in vivo with HIV remains to be clearly shown.

Characterization of trans-immortalized hepatic cell lines established from transgenic mice.

Hepato-specific regulatory (promoter/enhancer) DNA sequences were used for targeting the expression of onc genes, such as murine c-myc and Simian Virus 40 T Antigen, to hepatocytes of transgenic mice which subsequently developed hepatocellular carcinomas after a variable period of time (depending on the type of onc gene employed). Several trans-immortalized cell lines were established and compared with respect to the expression of adult hepatic markers and response to growth factors. Despite the morphological differences observed between trans-hepatomas, owing to the expression of the two different onc genes, all tumor-derived cell lines behaved in a comparable fashion during long-term culture displaying an adult hepatic phenotype for at least 40 passages. They differed, however, in response to epidermal growth factor. When the gene coding for human alpha 1-antitrypsin was placed under the control of the same hepato-specific promoter/enhancer, high levels of the human recombinant protein could be harvested from the supernatants of trans-hepatoma-derived cell lines.

Comparative ultrastructural study of rat livers preserved in Euro-Collins or University of Wisconsin solution.

University of Wisconsin solution greatly lengthens the time liver storage is possible compared with all previous solutions used. To test whether this improvement is related to better preservation of the endothelial cell, which is thought to be the most vulnerable cell type in cold storage, we compared time-related ultrastructural changes in rat livers stored in this solution or in Euro-Collins solution. Rat livers were harvested after combined arterial and portal perfusion with the cold-storage solution. They were then preserved for different lengths of time in the same solution at 4 degrees C before being perfusion-fixed and processed for light and electron microscopy. The first preservation damage was noted in endothelial cells; the time course of the lesions was similar in both solutions. After 2 hr of storage, enlarged and ruptured fenestrae with many gaps were observed. Swollen at 4 hr, the endothelial cells became stringlike at 10 hr, leading to stripped sinusoidal walls. Hepatocytes appeared better preserved in University of Wisconsin solution. The amount of glycogen, maintained near the control level at 24 hr in the latter, decreased dramatically between 0 and 4 hr in Euro-Collins solution, as ultrastructurally observed and biochemically confirmed. Furthermore, sinusoidal obstruction by blebs originating from the hepatocytes and quantified by image analysis on electron micrographs was markedly delayed. It was significantly less pronounced in University of Wisconsin solution at 24 hr than in Euro-Collins solution at 2 hr (p less than or equal to 0.05). Our findings confirm that endothelial cells are highly susceptible to preservation damage and show that University of Wisconsin solution does not improve preservation during storage.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of cultured human Kupffer cells with HIV-infected CEM cells: an electron microscopic study.

Evidence has accumulated indicating that macrophages could play a role in the physiopathology of AIDS. We recently demonstrated that cultured human liver macrophages, the so-called Kupffer cells (KC), are permissive for HIV. Their infection in vivo would lead these cells to constitute a target for the virus and a reservoir as well. Since they occupy a strategic position within the liver sinusoid, their opportunity to interact with blood-borne virus or already infected T lymphocytes may be very high. In the present study, we investigated the possibility for KC to be infected via HIV-infected CEM cells, a lymphoid cell line. Therefore, we cocultured both cell types for various times before fixing them for electron microscopy. Syncytia appeared within 20 h of infection as well as a large amount of virus particles. HIV in the way of budding was also easily observed. This has to be compared to the direct infection of KC with free virus which needs, at least, about 10 days to give the same results.