Adipocytokines may delay pubertal maturation of human Sertoli cells.

Reproduction is an important target of obesity complications, including adverse effects on spermatogenesis and steroidogenesis. Adipocytokines are key mediators in various complications of obesity. Our aim was to study the potential of adipocytokines to affect Sertoli cell function, which is crucial for spermatogenesis, and possibly link these findings to the observed attenuation of spermatogenesis in obese males. Testicular biopsies were obtained from healthy donors. Highly purified adult human Sertoli cells (HSCs) were isolated by fluorescence-activated cell sorting. Cells were cultured and exposed to different concentrations of adipocytokines (10-1000ngmL-1 ) for 2-7 days. Expression of selected Sertoli cell genes was quantified by quantitative polymerase chain reaction. Long-term treatment (7 days) of HSCs with higher concentrations of chemerin, irisin, nicotinamide phosphoribosyltransferase (Nampt), resistin and progranulin significantly suppressed FSH receptor expression (by 79%, 83%, 64%, 71% and 26% respectively; P P invitro , may negatively affect Sertoli cell maturation and retain these cells in a more prepubertal stage. This could negatively affect testis function and add to fertility problems in obese adults.

Antibody landscapes after influenza virus infection or vaccination.

We introduce the antibody landscape, a method for the quantitative analysis of antibody-mediated immunity to antigenically variable pathogens, achieved by accounting for antigenic variation among pathogen strains. We generated antibody landscapes to study immune profiles covering 43 years of influenza A/H3N2 virus evolution for 69 individuals monitored for infection over 6 years and for 225 individuals pre- and postvaccination. Upon infection and vaccination, titers increased broadly, including previously encountered viruses far beyond the extent of cross-reactivity observed after a primary infection. We explored implications for vaccination and found that the use of an antigenically advanced virus had the dual benefit of inducing antibodies against both advanced and previous antigenic clusters. These results indicate that preemptive vaccine updates may improve influenza vaccine efficacy in previously exposed individuals.

Induction of stable prenatal tolerance to beta-galactosidase by in utero gene transfer into preimmune sheep fetuses.

The successful transduction of hematopoietic stem cells and long-term (28 months) transgene expression within the hematopoietic system following the direct injection of high-titer retroviral vectors into preimmune fetal sheep was previously demonstrated. The present studies extended these analyses for 40 months postinjection and evaluated whether the longevity of transgene expression in this model system was the result of induction of prenatal tolerance to the transgene product. The intraperitoneal injection of retroviral vectors into preimmune sheep fetuses transduces thymic epithelial cells thought to present antigen and thus define self during immune system development. To directly demonstrate induction of tolerance, postnatal sheep were boosted with purified beta-galactosidase and showed that the peripheral blood lymphocytes from in utero-transduced sheep exhibited significantly lower stimulation indices to transduced autologous cells than did control animals and that the in utero-transduced sheep had a reduced ability to mount an antibody response to the vector-encoded beta-galactosidase protein compared with control sheep. Collectively, our results provide evidence that the direct injection of retroviral vectors into preimmune sheep fetuses induces cellular and humoral tolerance to the vector/transgene products and provide an explanation for the duration and stability of transgene expression seen in this model. These results also suggest that even relatively low levels of gene transfer in utero may render the recipient tolerant to the exogenous gene and thus potentially permit the successful postnatal treatment of the recipient. (Blood. 2001;97:3417-3423)

Chemical hypoxia-ischemia induces apoptosis in cerebromicrovascular endothelial cells.

Cerebral endothelial cell (CEC) death from ischemia may exacerbate brain injury by altering microvascular integrity, but little is known concerning the pattern of CEC death and disruption of tight junction between two CECs to ischemia. To address these questions, CECs were isolated from bovine, cultured, and placed in glucose-free medium containing sodium cyanide. Trypan blue staining shown that sodium cyanide resulted in a dose-dependent insult of CECs (10-80 mM). CEC injury increased progressively with the duration of 20 mM cyanide exposure, becoming significant (71%) after 12 h. The mode of cell death induced by cyanide is clearly apoptosis in CECs, as shown by Hoechst 33,342 staining and transmission electron microscope, i.e. cyanide induced condensation and margination of chromatin, nuclear fragmentation and shrinkage of cell body and condensed apoptotic bodies in CECs. Most importantly, we found that the tight junction between two CECs was disrupted 12 h after chemical-ischemia, i.e. when CECs underwent apoptosis, the tight junctional complexes became thinner and rough; the cleft of tight junction between two CECs became blurred and more wider, and membranes of tight junction were course and irregular; and the adherens junctions were damaged. These results indicate that chemical hypoxia-ischemia induces apoptotic cell death in CECs and alters the microvascular integrity by disrupting tight junction complexes, and suggest that CEC apoptotic death and disruption of tight junction may exacerbate ischemic insults to brain. Thus, prevention of CEC apoptotic death may contribute to improvements of ischemic insults.

Neonatal gene therapy. transfer and expression of exogenous genes in neonatal sheep following direct injection of retroviral vectors into the bone marrow space.

We investigated whether gene transfer into hematopoietic cells could be achieved by direct injection of retroviral vector supernatant into the bone marrow space of newborn sheep. Six sheep (5 weeks old) were injected bilaterally with either 1 mL of G1nBgSvNa8.1 vector supernatant (titer: 1 x 10(7)) in each hip (n = 5) or with 3 mL of the same vector preparation/hip (n = 1). In addition, one 3-month-old sheep was injected unilaterally with 1 mL of the same vector preparation. Blood and marrow of these animals were analyzed for the transgene before injection and at intervals thereafter. At 1 week postinjection, an average of 11.6% of the lymphocytes and 25.5% of the granulocytes/monocytes in the marrow, and an average of 0.9% of the lymphocytes and 1.8% of the granulocytes/monocytes in the blood contained and expressed the LacZ gene. The presence/expression of the transgene has persisted for at least 13 months within the blood and bone marrow of these animals. These findings demonstrate that the direct injection of small volumes of high-titer retroviral supernatant into the bone marrow of newborn sheep results in transduction of hematopoietic cells that persists for at least 13 months postinjection.

In utero transfer and expression of exogenous genes in sheep.

OBJECTIVE: We have previously reported that directly injecting low-titer retroviral vector supernatant into pre-immune sheep fetuses resulted in the transfer and long-term expression of the bacterial NeoR gene within the hematopoietic system of these animals for over 5 years. In the present studies, we investigated whether using a higher titer vector would enable more efficient transduction and expression of the transgenes within the hematopoetic cells in sheep injected in utero. MATERIALS AND METHODS: Sixteen pre-immune sheep fetuses were injected intraperitoneally with the G1nBgSvNa8.1 helper-free retroviral vector supernatant encoding the bacterial NeoR and LacZ genes (titer: 1x10(7) cfu/mL). RESULTS: Over the 2-year time course of these studies, the presence and expression of the NeoR and LacZ genes were demonstrated in 12 of the 14 animals evaluated by several immunological and biochemical methods. Seven of the 12 sheep examined by flow cytometric analysis contained > or =6% transduced peripheral blood lymphocytes. Vector distribution was widespread without any detectable pathology. Importantly, PCR analyses and breeding experiments demonstrated that the germ line was not altered. CONCLUSIONS: These studies confirmed that direct injection of an engineered retrovirus is a feasible means of safely delivering foreign genes into a developing fetus and thus achieving long-term expression of the transgenes within the recipient's hematopoietic cells. Furthermore, expression of the NeoR gene from these studies was higher than that reported in our previous study in which a lower titer vector was used.

Transforming growth factor-beta mediates astrocyte-specific regulation of brain endothelial anticoagulant factors.

BACKGROUND AND PURPOSE: Astrocytes are potent regulators of brain capillary endothelial cell function. Recently, astrocytes were shown to regulate brain capillary endothelial expression of the fibrinolytic enzyme tissue plasminogen activator (tPA) and the anticoagulant thrombomodulin (TM). To study the mechanism of this process, we examined the hypothesis that astrocyte regulation of endothelial tPA and TM is mediated by transforming growth factor-beta (TGF-beta). METHODS: Brain capillary endothelial cells were grown in blood-brain barrier models. We examined astrocyte-endothelial cocultures, endothelial monocultures, and astrocyte-conditioned media (ACM) for the expression of TGF-beta. We also incubated endothelial cells with ACM to determine the role of TGF-beta. Following 24 hours of incubation, we assayed for tPA and TM mRNA, as well as tPA and TM activity. RESULTS: Astrocyte-endothelial cocultures and ACM exhibited significantly higher levels of active TGF-beta than brain endothelial monocultures and endothelial cells grown in nonconditioned media, respectively. Brain endothelial cells incubated with ACM exhibited reduced tPA and TM mRNA and activity. Treatment with exogenous TGF-beta produced dose-dependent reductions in tPA and TM. The effects of ACM on both tPA and TM were blocked by TGF-beta neutralizing antibody. CONCLUSIONS: These data indicate that TGF-beta mediates astrocyte regulation of brain capillary endothelial expression of tPA and TM.

Rat brain capillary thrombomodulin: structure and function.

The anticoagulant transmembrane glycoprotein thrombomodulin (TM) is expressed at the luminal surface of vascular endothelial cells. Recently, we showed that TM antigen and TM mRNA are expressed in brain microvessels in several species and that brain capillaries have the capability to activate protein C. The activation of protein C in brain microcirculation was greatly impaired by major stroke risk factors in rats due to downregulation of TM. In this study, a partial sequence of TM was determined from TM mRNA from brain capillaries examined in brain capillaries of the rat, a species that provides a useful model to investigate stroke mechanisms in relation to brain hemostasis. The predicted deduced amino acid sequences for rat TM were compared with other TM sequences. Particularly high homology (77-100%) among functional domains of the protein, i.e., the epidermal growth factor repeats (EGFRs) 1-6 and the transmembrane region, was observed between mice and rats. Somewhat less degree of homology was observed for bovine and human EGFRs 1-6, while the homology of the transmembrane region was 92-96%. All cysteine residues were conserved among the TM sequences, and specific amino acids previously suggested to be essential for activation of protein C by thrombin TM were highly conserved. We conclude that the highly conserved mRNA and protein sequences may reflect a similar anticoagulant role of TM in brain endothelial and systemic vascular endothelial cells across different species.

Astrocyte regulation of endothelial tissue plasminogen activator in a blood-brain barrier model.

Expression of tissue plasminogen activator (tPA) substantially determines endothelial-dependent fibrinolysis. We used a blood-brain barrier (BBB) model to analyze regulation of brain capillary endothelial tPA and its inhibitor, plasminogen activator inhibitor-1 (PAI-1). This model consists of coculture of murine astrocytes with bovine brain capillary endothelial cells grown as capillary-like structures (CS); after 1 week, astrocytes become extensively associated with CS, and the BBB-associated enzyme gamma-glutamyl transpeptidase is present. We measured tPA and PAI-1 mRNA and tPA activity in this model. Reverse transcription-polymerase chain reaction (RT-PCR) studies showed similar tPA and PAI-1 mRNA levels after 1 day mono-culture (endothelial cells only) versus astrocyte-endothelial coculture preparations. After 7 days (i.e., when elements of the BBB are present), astrocyte-endothelial cocultures (compared with endothelial mono-cultures) showed a 50.7%+/-27.1% (mean +/- SD) reduction in tPA mRNA (P < 0.03) and a 183.3%+/-86.9% increase in PAI-1 mRNA expression (P < 0.02). Moreover, 7-day cocultures demonstrated reduced tPA activity compared with mono-cultures (14.6+/-2.9 IU/mL versus 30.2+/-7.7 IU/mL, P < 0.01); 1-day cocultures and mono-cultures had similar tPA activity. These findings demonstrate that astrocytes regulate brain capillary endothelial expression of tPA when elements of the BBB phenotype are present in this model. These data suggest an important role for astrocytes in the regulation of brain capillary endothelial fibrinolysis.

Measurement of thrombomodulin mRNA expression in brain capillaries by polymerase chain reaction.

Thrombomodulin (TM), an endothelial integral membrane protein, is a potent activator of the protein C anticoagulant pathway. TM protein expression is limited and regionally distributed in the brain. Recent investigations have demonstrated low TM mRNA expression by brain endothelium, corresponding to its distribution at the protein level. To facilitate the study of TM expression at the transcriptional level, we measured TM mRNA by quantitative-competitive polymerase chain reaction (QC-PCR) and by standard densitometric analysis of reverse transcriptase-PCR products (RT-PCR) in different regions of bovine brain. QC-PCR demonstrated differential TM mRNA expression in the pons (100+/-9%), cerebellum (359+/-103%), and cortex (441+/-24%). We compared these results with those of RT-PCR and found similar differences in relative TM mRNA expression in the pons (100+/-44%), cerebellum (343+/-8%), and cortex (404+/-62%). Data derived by QC-PCR and RT-PCR were highly correlated (r=0.99, p<0.03). These findings indicate that either QC-PCR or RT-PCR can be used to accurately quantify TM mRNA.

Thrombomodulin expression in bovine brain capillaries. Anticoagulant function of the blood-brain barrier, regional differences, and regulatory mechanisms.

Thrombomodulin (TM), a key cofactor of the TM-protein C pathway, is of major biologic significance for the antithrombotic properties of endothelial cells. Yet, there is uncertainty whether TM is expressed in brain and what mechanisms govern brain endothelial anticoagulant activity. In this study, bovine brain capillaries were used as an in vitro model of the blood-brain barrier to determine factors involved in the regulation of TM expression in cerebral vasculature. Quantitative competitive-polymerase chain reaction assay revealed significant regional differences in the amount of brain capillary TM mRNA, i.e., cortical > cerebellar > pontine, consistent with the reverse transcription-polymerase chain reaction findings in which the abundance of TM mRNA was analyzed relative to beta-actin mRNA. Regional differences in TM mRNA brain capillary level correlated well with differences in protein C activation. The TM mRNA and activity were not detectable in brain parenchyma. Pathogenic mediators of ischemic stroke, interleukin 1 beta (10 U/mL), and tumor necrosis factor alpha (10 U/mL), produced a time-dependent decrease in brain capillary TM mRNA (t1/2 of 2.1 and 3.9 hours, respectively) and reduced endothelial TM activity. Incubation of brain capillaries with retinoic acid (10 mumol/L) and dibutyryl cAMP (3 mmol/L) resulted in a 4-fold increase in TM mRNA at 4 and 8 hours, respectively, followed by an increase in protein C activation. We conclude that TM at the blood-brain barrier is likely to be an important physiologic anticoagulant in brain microcirculation. Its downregulation by cytokines may contribute to ischemic brain damage and potentially could be counteracted by retinoic acid and cAMP.

Regulation of brain capillary endothelial thrombomodulin mRNA expression.

BACKGROUND AND PURPOSE: Endothelial cells regulate hemostasis in part via expression of thrombomodulin, a potent anticoagulant protein. The purpose of this study was to analyze brain capillary endothelial cell expression of thrombomodulin mRNA. METHODS: Bovine brain capillary endothelial cells were grown in a blood-brain barrier model in which endothelial cells form capillary-like structures. In situ hybridization and polymerase chain reaction (PCR) were used to examine thrombomodulin expression. Endothelial cells were then cocultured with astrocytes. We examined both coculture and monoculture preparations for gamma-glutamyl transpeptidase (GGTP), a marker of the blood-brain barrier. We then used quantitative-competitive PCR to compare thrombomodulin expression in endothelial monocultures and astrocyte-endothelial cocultures after 1 and 7 days of culture. RESULTS: Both in situ hybridization and PCR studies demonstrated thrombomodulin mRNA expression by endothelial cells. During 1 week of astrocyte-endothelial coculture, there was (1) progressive association of astrocytes with capillary-like structures and (2) expression of GGTP; endothelial monocultures did not express GGTP. There was no significant difference in thrombomodulin mRNA expression for cocultures versus monocultures after 1 day. After 1 week, however, astrocyte-endothelial cocultures had markedly decreased thrombomodulin mRNA compared with monocultures (9 +/- 2 versus 189 +/- 62 pg/mL; P < .025). This thrombomodulin mRNA decrease thus occurred when elements of the blood-brain barrier phenotype were demonstrable, ie, when astrocyte association with capillary-like structures was maximal and when GGTP was expressed in cocultures. CONCLUSIONS: These findings indicate astrocyte regulation of thrombomodulin mRNA expression in vitro and suggest an important role for the blood-brain barrier in the regulation of thrombomodulin.

p53-deficient mice are protected against adrenalectomy-induced apoptosis.

The p53 tumor suppressor gene, an important regulator of the cell cycle, has been implicated in apoptotic cell death in vitro, and more recently in neuronal degeneration in vivo. The present study investigated the importance of p53 expression in the apoptotic death of hippocampal granule cells following adrenalectomy. Mice, either homozygous or heterozygous for the p53 null allele and wild-type controls were sacrificed 16 days after adrenalectomy. Hippocampal morphology was assessed in paraffin sections stained with hematoxylin and eosin. Cells exhibiting features characteristic of apoptosis were evident in hippocampi from wild-type mice. A significant decrease in the number of apoptotic cells was observed in both homozygous and heterozygous mice. These findings demonstrate that absence or attenuation of p53 expression protects granule cells from adrenalectomy-induced apoptosis and, combined with the results of other studies, suggest that p53 is required for certain types of neuronal degeneration.

Synthesis of a secondary N-desmethyl and a tertiary N-cyclopropylmethyl bridged hexahydroaporphine as precursors to bicyclic opioid ligands.

In an attempt to generate a bicyclic 5,8-ethano derivative of N-methylmorphinan, an isomeric bicyclic hexahydroaporphine 2 was synthesized. The phenolic analogue of 2 has demonstrated affinity for mu opioid receptors in vitro and, along with 2, provided weak, primarily nonopioid analgesic action when injected intracerebroventricularly in mice. It was of interest to assess the potential opioid antagonist action of bicyclic hexahydroaporphine analogues containing cyclopropylmethyl and allyl nitrogen substituents. As the first steps in the generation of these potential opioid antagonists, the secondary bicyclic hexahydroaporphine 3 and its N-cyclopropylmethyl congener 4 were synthesized. N-Demethylation of 2 was initially attempted via the von Braun reaction, but acid-catalyzed hydrolysis of the crude N-cyano intermediate resulted in product decomposition. A successful approach to 3 involved the hydrolysis of the N-formyl precursor 1 in ethanolic potassium hydroxide. Direct alkylation of the secondary amine 3 utilizing cyclopropylmethyl bromide and sodium bicarbonate successfully generated the alkylated derivative 4. Both products were purified in hydrochloride salt form and characterized by standard analytical and spectroscopic methods. The free base form of 3 was highly sensitive to photooxidation. Opioids are known to oxidize to 10-keto structures, and secondary amines can oxidize to hydroxylamines. Infrared analysis of the decomposition product indicated the presence of both hydroxy and carbonyl groups which were absent in the spectrum of the salt. Structures of potential oxidation products are proposed.

An enzyme electrode for specific determination of L-lysine: A real-time control sensor.

Lysine decarboxylase (L-lysine carboxylyase, E.C.4.1.1.18) is immobolized on a carbon dioxide gas-sensing electrode, by copolymerization with gelatin using the bifuncitional agent glutaraldehyde. The enzyme electrodes thus prepared are used in a continuous flow system to measure the concentration of L-lysine in a mixture of amino acids. The measuring time for each sample is about 3 min, including response and rinsing times. The electrode response is linear between 0.01-1 g/L and has a high specificity for L-lysine. The enzyme electrode response to lysine at concentrations below 0.5 g/L is stable on repeated use for at least 500 assays.