Changes in ectonucleotidase activities in rat Sertoli cells during sexual maturation

Sertoli cell maturation is a complex process involving both morphological and biochemical changes. These cells have previously been shown to be targets for extracellular purine structures such as ATP and adenosine. These compounds evoke responses in rat Sertoli cells through the purinoceptor families, P₂X and P₂Y and PA₁. The signals to purinoceptors are usually terminated by the action of ectonucleotidases. In a previous work, we demonstrated that rat Sertoli cells have ecto-ATPdiphosphohydrolase (EC 3.6.1.5), ecto-5-nucleotidase (EC 3.1.3.5) and ecto-adenosine deaminase (ecto-ADA) (EC 3.5.4.4) activities. Here we investigated whether some changes occur during rat Sertoli cell maturation in these activities. Rat Sertoli cells obtained from rats of different ages representing the pre pubertal, mid pubertal and young adult (10-, 18- and 35-day-old, respectively) were cultured and used for different assays. The nucleotide hydrolysis was estimated by measuring the Pi released using a colorimetric method and by HPLC analysis. ATP and ADP hydrolysis was increased 3-fold during sexual maturation. AMP hydrolysis increased 4-fold in 10- to 35-day-old Sertoli cells. Similar results were obtained when we used other substrates to measure the extracellular hydrolysis of nucleotides (GTP, GDP, GMP and IMP). The ecto-ADA activity showed a 2-fold increase in the specific activity (18- to 35-day-old Sertoli cells). The termination of the purine cascade by adenosine degradation was faster in the 35- than in 18-day-old Sertoli cells. Follicle Stimulating Hormone (FSH) influences on the ectonucleotidase activities were investigated in 10- and 18-day-old Sertoli cells and a significant increase in the ATP and ADP hydrolysis was observed. Our results show an increase in the extracellular purine cascade during the Sertoli cell development, indicating a rise in the purine communication inside the seminiferous tubules with rat sexual maturation.     

Oxidation and nitrosylation of oxyhemoglobin by S-nitrosoglutathione via nitroxyl anion

The reaction between low molecular weight S-nitrosothiols and hemoglobin is often used to synthesize S-nitrosohemoglobin, a form of hemoglobin suggested to be involved in the regulation of vascular oxygen delivery. However, this reaction has not been studied in detail, and several groups have reported a variable co-formation of oxidized methemoglobin (metHb) during synthesis. This study examines the mechanism of metHb formation and shows that nitrosylhemoglobin (HbNO) can also be formed. Generation of metHb and HbNO is largely dependent on the presence of protein thiol groups. We present evidence for a mechanism for the formation of metHb and HbNO involving the intermediacy of nitroxyl anion. Specifically, the reaction of nitroxyl with S-nitrosothiols to liberate nitric oxide and reduced thiol is proposed to be central to the reaction mechanism.     

Measles Virus DNA Vaccination: Antibody Isotype Is Determined by the Method of Immunization and by the Nature of both the Antigen and the Coimmunized Antigen

Plasmids encoding the measles virus hemagglutinin (HA) and nucleoprotein (NP) proteins inoculated into the skin of BALB/c mice by the gene gun method induced both humoral and cytotoxic lymphocyte class I-restrict- ed immune responses. Although intramuscular immunization induces the immunoglobulin G2a (IgG2a) antibody isotype for both antigens, with gene gun immunization, the NP still generated mainly IgG2a and the major isotype induced by the HA was IgG1. Interestingly, gene gun coimmunization of HA and NP plasmids resulted in a dominant IgG1 HA response and the switching of antibodies generated against the NP to the IgG1 isotype.The initial studies showing that injection of DNA into muscle induces an immune response to the encoded protein opened a new approach to vaccination (for reviews, see references 19 and 22). Recent studies suggest that inoculated muscle cells probably act only as a source of antigen and that immune priming takes place elsewhere in the body (14). For example, excision of an injected muscle a few minutes after DNA inoculation did not affect antibody and cytotoxic T-lymphocyte (CTL) responses (21). Thus, it may be interesting to examine other DNA delivery systems to study how the immune system responds to DNA vaccination. One alternative system involves precipitating DNA onto gold beads which are then propelled into the skin by means of pressurized helium gas (12). When such a system is used, less DNA is required, but unlike the case with intramuscular inoculations, the response is Th2-like, generating immunoglobulin G1 (IgG1) antibodies (17). More recent observations suggest that this is probably due to the mode of inoculation rather than the route (10). We have been studying DNA vaccination against the paramyxovirus measles virus (MV). This disease is one of the primary causes of infant mortality in developing countries, and there is an urgent need for an effective vaccine in infants, as the present live attenuated vaccine is inefficient in the presence of maternal antibodies. Our previous studies established that in a mouse model at least three MV proteins play a role in protection (23). Both glycoproteins, hemagglutinin (HA) and fusion, induce neutralizing antibodies (9, 11), and HA and nucleoprotein (NP) induce CTLs (3, 4), which do not protect against infection but help in recovery (5). In our previous study on DNA vaccination, we showed that intramuscular inoculation of DNAs coding for the MV HA and NP (pV1J-HA and pV1J-NP [6]) induced class I-restricted CTLs and a humoral response corresponding to a Th1 response (6). In the present study, we have extended our observations to compare the same plasmids’ ability to induce an immune response when they are delivered into the skin by a gene gun (Bio-Rad, Ivry sur Seine, France). Gold beads were coated with DNA as follows: approximately 30 mg of gold powder (1.0-μm gold beads; Bio-Rad) was mixed with 100 μl of 0.1 M spermidine (Sigma, L’Isle D’Abeau, France). After sonication, 0.5, 2, or 5 μg of plasmid DNA was added per mg of gold powder, and then 200 μl of 2.5 M CaCl₂ was added to the mixture, with gentle vortexing. Pellets were washed three times and suspended in cold 100% ethanol. Tubes containing dried DNA-coated gold beads were stored at 4°C.

Immune response to MV HA DNA.

Six- to eight-week-old female BALB/c mice (Iffa-Credo, Domaine des Oncins, France) were immunized via the shaved abdominal epidermis one to three times at 21-day intervals with 0.5, 2, or 5 μg of pV1J-HA DNA/mg of gold beads. Two gene gun inoculations (each containing 0.5 mg of gold beads) were given for each dose. The antibody levels measured by enzyme-linked immunosorbent assay, as previously described (6), reached a plateau after two inoculations and did not significantly increase with a third inoculation (result not shown).Our previous studies with intramuscular inoculation established that pV1J-HA induced IgG2a antibodies which are associated with a Th1-type response. When we studied the antibody isotype induced in BALB/c by the gene gun immunization, we observed that it was mainly IgG1 (Fig. ​(Fig.1).1). These data are similar to those described for influenza hemagglutinin by Feltquate et al. (10). The antibody isotype did not vary with time after immunization, number of immunizations, or the amount of plasmid used (data not shown) and was not influenced by genetic background, as pV1J-HA-immunized DBA/2 (H-2^d), C3H (H-2^k), and C57/Black (H-2^b) mice induced mainly the IgG1 isotype (Fig. ​(Fig.11).Open in a separate windowFIG. 1Anti-MV HA isotype of antibodies induced in BALB/c, DBA/2 (H-2^d), C3H (H-2^k), and C57/Black (H-2^b) mice immunized with 0.5, 2, or 5 μg of pV1J-HA by epidermal gene gun. Sera were collected 3 weeks after the immunization. Sera from mice immunized with a control pV1J had means ± standard deviations of 158 ± 198 ng/ml for IgG1 anti-HA antibodies (n = 11) and 10 ± 18 ng/ml for IgG2a anti-HA antibodies (n = 11). Data represent individual animals. To study CTL activity, spleen cells from the immunized mice were stimulated in vitro and analyzed in a cytolytic assay as previously described (6). Despite the apparent Th2-type response, good memory CTL responses were obtained with all protocols used, even when responses were measured just 8 days after a single immunization (Fig. ​(Fig.2),2), and persisted for several months. Open in a separate windowFIG. 2Anti-MV HA and NP CTL response after immunization with pV1J-HA or -NP, respectively. BALB/c mice were immunized with 0.5 (circle), 2 (triangle), or 5 (square) μg of pV1J-HA by epidermal gene gun one (a, d), two (b, e), or three (c, f) times at 3-week intervals. The spleen cells were removed 3 weeks (continuous line) or 8 days (dotted line) after the last immunization. After in vitro stimulation with P815-HA or -NP cells, respectively, lysis was measured on P815-HA or -NP cells, and P815 cells were used as a negative control. The results show the specific lysis of targets at graded effector/target ratios. Each curve represents an individual animal.

Immune response to MV NP DNA.

BALB/c mice were immunized with pV1J-NP with the gene gun and a similar schedule of immunizations. The antibody response with the different number of doses and different plasmid concentrations was similar to that observed for HA, i.e., increased levels after one boost. Similar antibody levels were induced in the range of 0.5 to 5 μg of DNA (data not shown). As was previously shown by intramuscular inoculation (6), the antibody isotype induced was mainly IgG2a (Fig. ​(Fig.3),3), in contrast to the HA results. One explanation for this could be that as the NP is a cytosolic protein and the HA is membrane bound, the potential processing and presentation of the two proteins may be different. However, the same argument would be valid for intramuscular inoculation. Furthermore, it has been reported that gene gun immunization with influenza NP induces a Th2 response (17), so clearly the directed differentiation of T cells is more complicated than a simple distinction between cytosol and membrane-bound proteins. The two methods of immunization (intramuscular versus gene gun) target different cell types, possibly influencing the T-cell response. Furthermore, 9 weeks after immunization, one-third of the 18 mice analyzed showed increased levels of anti-NP IgG1 over IgG2a, regardless of the quantity of DNA injected or the number of inoculations (data not shown). CTL responses were also high, even after a single inoculation (Fig. ​(Fig.2).2). Open in a separate windowFIG. 3Anti-MV NP antibody response as measured by enzyme-linked immunosorbent assay in BALB/c mice immunized with 5 μg of pV1J-NP by epidermal gene gun. Sera were collected 3 weeks after immunization. Each pair of bars represents an individual animal. Sera from mice immunized with a control pV1J had means ± standard deviations of 13 ± 45 ng/ml of IgG1 anti-NP antibodies (n = 11) and 83 ± 276 ng/ml of IgG2a anti-NP antibodies (n = 11).

Coimmunization of HA and NP DNA.

Our results show that when injected by the gene gun, the different MV proteins induce different antibody isotypes. This phenomenon has been suggested to parallel the induction of Th1 and Th2 pathways (1). The pathway taken has been shown to be influenced by the induction of certain cytokines. To determine if coimmunization of these two plasmids would influence the isotype of the antibody response, BALB/c mice were immunized with a mixture of pV1J-HA and pV1J-NP in ratios of 1:1, 4:1, or 1:4 while the total amount of DNA was kept constant (5 μg).Measurement of the anti-HA isotype antibody in mice vaccinated with the different mixtures showed it to be mainly IgG1, similar to that for HA alone (data not shown). In contrast, the anti-NP antibodies switched from the IgG2a to the IgG1 isotype after coimmunization (Fig. ​(Fig.4).4). The proximity of expression of the two antigens was not important in this switching effect, as when pV1J-HA and -NP were inoculated separately in different areas of the skin, the antibody response induced 3 weeks later was the same as that induced when the mixture was inoculated (Fig. ​(Fig.4).4). When analyzed 6 weeks later, only one of six mice showed IgG2a predominance. Open in a separate windowFIG. 4Relationship between the isotype of anti-NP antibodies in sera from mice immunized with 5 μg of pV1J-NP or mixtures of pV1J-HA and pV1J-NP at ratios of 1:1, 4:1, and 1:4 so that the total quantity of DNA/mg gold beads was 5 μg, or pV1J-HA and pV1J-NP injected in different skin area. BALB/c mice were immunized by epidermal gene gun. Sera were collected 3 weeks after immunization. Data are results for individual animals.Cytokines have been used to direct the immune response in several studies. Expression of interleukin-12 either alone or with immunizing antigens can increase protection against microbial pathogens (2) or tumors in animal models (13, 18), in parallel with a Th1 response. Expression or addition of interleukin-4 with the immunogen induces a Th2 response (16, 20). The local concentrations of the cytokines in the initial priming of the immune response are probably critical, as once the T cells have been committed, they cannot be modified. Although some studies have suggested the possibility of Th1 and Th2 switching, a more recent study has shown that once differentiated, T cells cannot switch (15). In agreement with this, Feltquate et al. (10) have shown that initial immunization establishes the Th-cell type of the immune response and that this is not modified by subsequent alternative methods of immunization.Acute viral infections induce a Th1 response, whereas soluble proteins favor a Th2 response (7). When tetanus toxoid was administered 1 day after viral infection, the response to this soluble protein changed from Th2 to Th1 (8). Presumably, this change is due to the domination by the cytokines induced by the viral infection of those produced by the tetanus toxoid. In our studies, we observed that after the coexpression of MV HA and NP, the HA-induced Th2 response was dominant. These observations will obviously have an impact on DNA vaccination, as DNAs coding for several pathogens should ideally be administered concomitantly.     

4-Hydroxybenzoate 3-geranyltransferase from Lithospermum erythrorhizon: purification of a plant membrane-bound prenyltransferase

Geranyldiphosphate:4-hydroxybenzoate 3-geranyltransferase is a regulatory enzyme in the biosynthesis of shikonin, a phytoalexin and pharmaceutical produced by cell cultures of Lithospermum erythrorhizon Sieb. et Zucc.. In Linsmaier-Skoog medium, the activity of this enzyme could be enhanced more than 200-fold by addition of methyl jasmonate, and this culture material was used for the solubilization and purification of the enzyme. Of various detergents examined, digitonin was the most suitable for the solubilization of the enzyme. The solubilized enzyme was purified 800-fold by chromatography over diethylaminoethyl (DEAE)-Sephacel, Heparin-Sepharose, Reactive Green 19-Agarose, and Cholic Acid-Agarose. The purified enzyme required magnesium ions as cofactor and was highly specific for geranyldiphosphate (GPP) and 4-hydroxybenzoate (4HB) as substrates. The K _m values for 4HB and GPP were calculated by the method of Lineweaver and Burk as 18.4 μM and 13.8 μM, respectively. Received: 2 July 1997 / Accepted: 14 October 1997     

Dissecting the mechanism of torso receptor activation

Regulated activation of receptor tyrosine kinases depends both on the presence of the receptors at the cell surface and on the availability of their ligands. In Drosophila the torso (tor) tyrosine kinase receptor is distributed along the surface of the embryo but it is only activated at the poles by a diffusible extracellular ligand generated at each pole which is trapped by the receptor, thereby impeding further diffusion. However, it is not well understood how this signal is generated, although it is known to depend on the activity of many genes such as torso-like (tsl) and trunk (trk). To further investigate the mechanism involved in the local activation of the tor receptor we have altered the normal expression of the tsl protein by generating females in which the tsl gene is expressed in the oocyte under the control of the tor promoter rather than in the ovarian follicle cells. Analysis of the phenotypes generated by this hybrid gene and its interactions with mutations in other genes in the pathway has enabled us to further dissect the mechanism of tor receptor activation and to define more precisely the role of the different genes acting in this process.     

Four-Dimensional Characterization of Thrombosis in a Live-Cell,Shear-Flow Assay: Development and Application to Xenotransplantation

Background

Porcine xenografts are a promising source of scarce transplantable organs, but stimulate intense thrombosis of human blood despite targeted genetic and pharmacologic interventions. Current experimental models do not enable study of the blood/endothelial interface to investigate adhesive interactions and thrombosis at the cellular level under physiologic conditions. The purpose of this study was to develop and validate a live-cell, shear-flow based thrombosis assay relevant to general thrombosis research, and demonstrate its potential in xenotransplantation applications.

Methodology/Principal Findings

Confluent wild-type (WT, n = 48) and Gal transferase knock-out (GalTKO, which resist hyperacute rejection; n = 11) porcine endothelia were cultured in microfluidic channels. To mimic microcirculatory flow, channels were perfused at 5 dynes/cm² and 37°C with human blood stained to fluorescently label platelets. Serial fluorescent imaging visualized percent surface area coverage (SA, for adhesion of labeled cells) and total fluorescence (a metric of clot volume). Aggregation was calculated by the fluorescence/SA ratio (FR). WT endothelia stimulated diffuse platelet adhesion (SA 65 ± 2%) and aggregation (FR 120 ± 1 a.u.), indicating high-grade thrombosis consistent with the rapid platelet activation and consumption seen in whole-organ lung xenotransplantation models. Experiments with antibody blockade of platelet aggregation, and perfusion of syngeneic and allo-incompatible endothelium was used to verify the biologic specificity and validity of the assay. Finally, with GalTKO endothelia thrombus volume decreased by 60%, due primarily to a 58% reduction in adhesion (P < 0.0001 each); importantly, aggregation was only marginally affected (11% reduction, P < 0.0001).

Conclusions/Significance

This novel, high-throughput assay enabled dynamic modeling of whole-blood thrombosis on intact endothelium under physiologic conditions, and allowed mechanistic characterization of endothelial and platelet interactions. Applied to xenogeneic thrombosis, it enables future studies regarding the effect of modifying the porcine genotype on sheer-stress-dependent events that characterize xenograft injury. This in-vitro platform is likely to prove broadly useful to study thrombosis and endothelial interactions under dynamic physiologic conditions.     

Prognostic Value of Malic Enzyme and ATP-Citrate Lyase in Non-Small Cell Lung Cancer of the Young and the Elderly

Background

Lung cancer is the leading cause of death among malignancies worldwide. Understanding its biology is therefore of pivotal importance to improve patient’s prognosis. In contrast to non-neoplastic tissues, cancer cells utilize glucose mainly for production of basic cellular modules ‘(i.e. nucleotides, aminoacids, fatty acids). In cancer, Malic enzyme (ME) and ATP-citrate lyase (ACLY) are key enzymes linking aerobic glycolysis and fatty acid synthesis and may therefore be of biological and prognostic significance in non-small cell lung cancer (NSCLC).

Material and Methods

ME and ACLY expression was analyzed in 258 NSCLC in correlation with clinico-pathological parameters including patient’s survival.

Results

Though, overall expression of both enzymes correlated positively, ACLY was associated with local tumor stage, whereas ME correlated with occurrence of mediastinal lymph node metastases. Young patients overexpressing ACLY and/or ME had a significantly longer overall survival. This proved to be an independent prognostic factor. This contrasts older NSCLC patients, in whom overexpression of ACLY and/or ME appears to predict the opposite.

Conclusion

In NSCLC, ME and ACLY show different enzyme expressions relating to local and mediastinal spread. Most important, we detected an inverse prognostic impact of ACLY and/or ME overexpression in young and elderly patients. It can therefore be expected, that treatment of NSCLC especially, if targeting metabolic pathways, requires different strategies in different age groups.     

“It’s My Secret”: Fear of Disclosure among Sub-Saharan African Migrant Women Living with HIV/AIDS in Belgium

Patients with HIV not only have to deal with the challenges of living with an incurable disease but also with the dilemma of whether or not to disclose their status to their partners, families and friends. This study explores the extent to which sub-Saharan African (SSA) migrant women in Belgium disclose their HIV positive status, reasons for disclosure/non-disclosure and how they deal with HIV disclosure. A qualitative study consisting of interviews with twenty-eight SSA women with HIV/AIDS was conducted. Thematic content analysis was employed to identify themes as they emerged. Our study reveals that these women usually only disclose their status to healthcare professionals because of the treatment and care they need. This selective disclosure is mainly due to the taboo of HIV disease in SSA culture. Stigma, notably self-stigma, greatly impedes HIV disclosure. Techniques to systematically incorporate HIV disclosure into post-test counseling and primary care services are highly recommended.     

Activities of Wogonin Analogs and Other Flavones against Flavobacterium columnare

In our on‐going pursuit to discover natural products and natural product‐based compounds to control the bacterial species Flavobacterium columnare, which causes columnaris disease in channel catfish (Ictalurus punctatus), we synthesized flavone and chalcone analogs, and evaluated these compounds, along with flavonoids from natural sources, for their antibacterial activities against two isolates of F. columnare (ALM‐00‐173 and BioMed) using a rapid bioassay. The flavonoids chrysin ( 1a ), 5,7‐dihydroxy‐4′‐methoxyflavone ( 11 ), isorhamnetin ( 26 ), luteolin ( 27 ), and biochanin A ( 29 ), and chalcone derivative 8b showed strong antibacterial activities against F. columnare ALM‐00‐173 based on minimum inhibition concentration (MIC) results. Flavonoids 1a, 8, 11, 13 (5,4′‐dihydroxy‐7‐methoxyflavone), 26 , and 29 exhibited strong antibacterial activities against F. columnare BioMed based upon MIC results. The 24‐h 50% inhibition concentration (IC₅₀) results revealed that 27 and 29 were the most active compounds against F. columnare ALM‐00‐173 (IC₅₀ of 7.5 and 8.5 mg/l, resp.), while 26 and 29 were the most toxic compound against F. columnare BioMed (IC₅₀ of 9.2 and 3.5 mg/l, resp.). These IC₅₀ results were lower than those obtained for wogonin against F. columnare ALM‐00‐173 and F. columnare BioMed (28.4 and 5.4 mg/l, resp.). However, based on MIC results, none of the compounds evaluated in this study were as active as wogonin (MIC 0.3 mg/l for each F. columnare isolate). Further modification of the wogonin structure to enhance antibacterial is of interest.     

Survey of Sand Flies (Diptera: Psychodidae) in an Environmentally Protected Area in Brazil

Brazil is one of the most important endemic areas for leishmaniasis worldwide. Protected areas that are tourist attractions likely present an important risk of transmission of cutaneous leishmaniasis (CL). Furthermore, with the geographical expansion of visceral leishmaniasis (VL), several studies have recorded the occurrence of its vector, Lutzomyia longipalpis, and cases of human and canine VL in such tourist areas. The Parque Estadual do Sumidouro is an environmentally protected area located in the Brazilian Cerrado biome and in an important area endemic for leishmaniasis in the state of Minas Gerais. The purpose of this study was to monitor the sand fly fauna in areas of tourist activity in the park. Sampling was performed every month, from September 2011 to August 2013, using CDC light traps at six sites of differing environmental characteristics. Sampled specimens were identified following Galati (2003), and females were submitted to molecular techniques for the detection and identification of Leishmania DNA. A total of 4,675 sand fly specimens of 25 species belonging to nine genera were collected. The most abundant species were Micropygomyia quinquefer, Lutzomyia renei and Pintomyia pessoai, although only Pi. pessoai is implicated in the transmission of Leishmania braziliensis. The species accumulation curve reached saturation on the 16th sampling event. Species richness, diversity and evenness differed among the sampled areas. The seasonal curve was not determined by a single unique species, and no single species was the most abundant in all environments sampled. The main vector of Leishmania (Leishmania) infantum, Lutzomyia longipalpis, accounted for only 5.35% of the specimens collected. Proven or suspected vectors of Leishmania (Viannia) braziliensis were recorded, and one female of the cortellezzii complex tested positive for Le. braziliensis DNA. Even with a low infection rate (0.62%), these data indicate the circulation of the parasite and reinforce the need for entomological and epidemiological surveillance in the park and its surroundings.