Modulation of ventricular function through gene transfer in vivo

We used a catheter-based technique to achieve generalized cardiac gene transfer in vivo and to alter cardiac function by overexpressing phospholamban (PL) which regulates the activity of the sarcoplasmic reticulum Ca²⁺ ATPase (SERCA2a). By using this approach, rat hearts were transduced in vivo with 5 × 10⁹ pfu of recombinant adenoviral vectors carrying cDNA for either PL, β-galactosidase (β-gal), or modified green fluorescent protein (EGFP). Western blot analysis of ventricles obtained from rats transduced by Ad.PL showed a 2.8-fold increase in PL compared with hearts transduced by Ad.βgal. Two days after infection, rat hearts transduced with Ad.PL had lower peak left ventricular pressure (58.3 ± 12.9 mmHg, n = 8) compared with uninfected hearts (92.5 ± 3.5 mmHg, n = 6) or hearts infected with Ad.βgal (92.6 ± 5.9 mmHg, n = 6). Both peak rate of pressure rise and pressure fall (+3, 210 ± 298 mmHg/s, −2, 117 ± 178 mmHg/s, n = 8) were decreased in hearts overexpressing PL compared with uninfected hearts (+5, 225 ± 136 mmHg/s, −3, 805 ± 97 mmHg/s, n = 6) or hearts infected with Ad.βgal (+5, 108 ± 167 mmHg/s, −3, 765 ± 121 mmHg/s, n = 6). The time constant of left ventricular relaxation increased significantly in hearts overexpressing PL (33.4 ± 3.2 ms, n = 8) compared with uninfected hearts (18.5 ± 1.0 ms, n = 6) or hearts infected with Ad.βgal (20.8 ± 2.1 ms, n = 6). These differences in ventricular function were maintained 7 days after infection. These studies open the prospect of using somatic gene transfer to modulate overall cardiac function in vivo for either experimental or therapeutic applications.     

Heparin-antithrombin III binding. In vitro and in vivo studies

Heparin antithrombin III binding was studied by crossed immunoelectrophoresis. In plasma and purified antithrombin III standard, multicomponent patterns were obtained with low concentrations of mucosal heparin. There is evidence that antithrombin III may bind more than one heparin molecule. At high heparin concentration (greater than 16 U/ml), single symmetrical peaks were obtained. Serum samples showed two antithrombin III peaks due to a decreased heparin binding of the slower peak (2.1-3.9 times), which was probably antithrombin III-activated procoagulant complexes. Heparin analogue (A 73025) also bound antithrombin III in vitro but the mobility of the peak was slower than with mucosal heparin and only a single peak was obtained in serum samples. Radioimmunoassay showed a decreased binding of antithrombin III antibody to heparin-antithrombin III complex. Venous occlusion to the forearm resulted in a slow second peak in the plasma. Heparin therapy gave rise to a double peak in the plasma antithrombin III profile and with continuous infusion, quantitative decreases were noted in all subjects studied, two of whom rethrombosed at the end of 7 days therapy.     

Modulation of bacterial growth by tumor necrosis factor-alpha in vitro and in vivo

Tumor necrosis factor-alpha (TNF-alpha) plays an important role in innate immunity. Recent in vitro studies have shown that TNF-alpha may also serve as a growth factor for some bacteria. We examined the physiologic relevance of this phenomenon both in vitro and in vivo. Recombinant mouse TNF-alpha increased in vitro proliferation of Escherichia coli but not Pseudomonas aeruginosa in a concentration-dependent manner, and this effect was attenuated by anti-TNF-alpha antibodies. However, in vivo, TNF-alpha gene-deficient (TNF-alpha-/-) mice showed higher mortality than wild-type (TNF-alpha+/+) mice after inoculation of intranasal bacteria. An impaired bacterial clearance in TNF-alpha-/- mice was associated with decreased systemic concentrations of chemokine macrophage inflammatory protein-2, reduced pulmonary neutrophil recruitment, and depressed expression of neutrophil CD11b and CD16/CD32, suggesting that the effect of TNF-alpha on E. coli growth was outweighed by the recruited neutrophils. We also demonstrated that neutropenic TNF-alpha+/+ mice had approximately 100-fold higher E. coli counts in their lungs than TNF-alpha-/- mice, although survival rates in both groups were similar. We conclude that TNF-alpha augments E. coli growth in vitro and in vivo. However, in vivo, this effect becomes only apparent in neutropenic animals. The relevance of these findings for immune compromised patients remains to be investigated.     

Modulation of doxorubicin-toxicity by tamoxifen in multidrug-resistant tumor cells in vitro and in vivo

Modulation of the resistance of tumors offers new strategies to improve the therapeutical treatment of cancer. In this report, the anti-oestrogen tamoxifen was investigated in multidrug-resistant tumor cells in vitro and in vivo. The doxorubicin-resistance of L 1210/DOX-tumor cells, which express the multidrug-resistance phenotype, could be completely circumvented by addition of 1 g/ml tamoxifen. In contrast, no increased effect could be observed in the parental L 1210 tumor cells or in cytosine arabinoside-resistant L 1210 cells not expressing the multidrug-resistance phenotype. Thus, the enhancing effect of tamoxifen was restricted only to the multidrug-resistant L 1210/DOX tumor cells. Similar to the in vitro experiments, a significant reduction in the growth in solid tumors of mice by the combined treatment of doxorubicin and tamoxifen was again observed only in the multidrug-resistant L 1210/DOX tumors.     

Modulation of in vitro and in vivo T-cell responses by transferrin- gallium and gallium nitrate

Gallium is a group IIIa metal that has efficacy in the therapy of malignant disorders such as lymphoma and urothelial tract tumors. Preclinical studies also indicate a role for gallium in autoimmune disorders, suggesting that gallium is able to modulate T-cell immune reactivity. The purpose of this study was to examine the in vitro and in vivo immunomodulatory action of gallium on T-cell function. Since gallium binds to transferrin in vivo, in vitro studies evaluated the effect of transferrin-gallium (Tf-Ga) on human T cells. Tf-Ga inhibited the mitogen-induced proliferative response of peripheral blood mononuclear cells (PBMC) in a dose-dependent fashion. Alloantigen- induced proliferation was also potently suppressed when evaluated in a mixed lymphocyte culture assay. Tf-Ga affected a significant reduction in the density of IL-2 receptors on activated T cells and a slight reduction in the number of CD3+/CD25+ T cells in PHA-stimulated cultures. Neither secretion of interleukin-2 (IL-2) nor the induction of IL-2-stimulated lymphokine-activated killer activity, however, was inhibited by Tf-Ga. Tf-Ga produced significant upregulation of the transferrin receptor (CD71) in T cells as determined by flow cytometric analysis and northern blot assay, but did not affect the percentage of CD3+/ CD71+ T cells after mitogen stimulation. To assess the in vivo effects of gallium on alloreactive T cells, we evaluated the immunosuppressive effect of gallium in a murine model of graft-versus- host disease (GVHD). Administration of gallium significantly prolonged survival in mice undergoing severe GVHD, suggesting that gallium can ameliorate GVH reactivity. Collectively, these data demonstrate that, at clinically achievable concentrations, Tf-Ga potently inhibits T-cell activation and that this immunosuppressive property of gallium may be of adjunctive therapeutic value in the management of disorders characterized by the presence of autoreactive or alloreactive T-cell populations.     

In vivo and in vitro effects of a novel enterotoxin, STb, produced by Escherichia coli

Escherichia coli may produce a heat-labile enterotoxin (LT) or two heat-stable enterotoxins (STa, STb). STb consistently causes secretion in vivo in 5-hr weaned-pig intestinal loops (P less than .0001). In Ussing chambers in vitro, crude culture filtrates of STb initiate a prompt increase in short circuit current (SCC; P less than or equal to .0001) and potential difference (P less than or equal to .0001) when compared with nontoxigenic culture filtrates. In bidirectional in vitro studies of ion flux (22Na and 36Cl), STb did not alter 22Na or 36Cl unidirectional or net fluxes. The calculated residual ion flux increased significantly (P less than .03), however, in tissues treated with STb and fully accounted for the STb-induced increase in SCC. Measurement of the electrolyte content of ligated intestinal segments in vivo further suggested that STb stimulated bicarbonate secretion. Relative to controls, significant accumulation of Na and Cl also occurred intraluminally in vivo. These data indicate that STb is a unique enterotoxin that causes net secretion in pig jejunum in vivo. In vitro and in vivo studies show that STb stimulates active secretion of nonchloride anion. We postulate that STb causes active bicarbonate secretion in weaned-piglet jejunum.     

Modulation of in vivo cardiac function by myocyte-specific nitric oxide synthase-3

Nitric oxide (NO) functions principally as a diffusible paracrine effector. The exception is in cardiomyocytes where both NO synthases (NOS) and target proteins coexist, allowing NO to work in an autocrine/intracrine fashion. However, the most abundant myocyte isoform (NOS3) is far more expressed in vascular endothelium; thus, the in vivo contribution of myocyte-NOS3 remains less clear. The present study tested this role by transfecting whole hearts of NOS3-null (NOS3(-/-)) mice with adenovirus-expressing NOS3 coupled to a alpha-MHC promoter (AdV(NOS3)), comparing results to hearts transfected with marker-gene beta-galactosidase (AdVbeta(gal)). Total myocardial NOS3 protein and activity were restored to near wild-type (WT) levels in NOS3(-/-)+AdV(NOS3) hearts, and NOS3 relocalized normally with caveolin-3. Ejection function by pressure-volume analysis was enhanced in NOS3(-/-)+AdVbeta(gal) over WT or NOS3(-/-)+AdV(NOS3). More prominently, isoproterenol (ISO)-stimulated systolic and diastolic function in WT was amplified in NOS3(-/-)+AdVbeta(gal), whereas NOS3(-/-)+AdV(NOS3) returned the response to control. ISO-activated systolic function was inhibited 85% by concomitant muscarinic stimulation (carbachol) in NOS3(-/-)+AdV(NOS3) but not NOS3(-/-)+AdVbeta(gal) hearts. Lastly, NOS3(-/-)+AdVbeta(gal) mice displayed enhanced inotropy and lusitropy over WT at slower heart rates but a blunted rate augmentation versus controls. A more positive rate response was restored in NOS3(-/-)+AdV(NOS3) (P<0.001). Thus, myocyte autocrine/intracrine NOS3 regulation in vivo can underlie key roles in beta-adrenergic, muscarinic, and frequency-dependent cardiac regulation.     

Effect of shigella enterotoxin 1 (ShET1) on rabbit intestine in vitro and in vivo.

BACKGROUND: Shigella enterotoxin 1 is a novel enterotoxin elaborated by Shigella flexneri 2a that causes fluid accumulation in rabbit ileal loops and a rise in short circuit current in Ussing chambers. AIMS: To gain insights into the mechanism of action of shigella enterotoxin 1. METHODS: Supernatants from genetically engineered clones either overexpressing shigella enterotoxin 1 or producing deletion mutants of the toxin were tested in rabbit ileum both in vitro and in vivo. RESULTS: In rabbit ileum shigella enterotoxin 1 induced an irreversible rise in short circuit current that was not mediated by any of the recognised intracellular mediators of secretion. Deletion of 90% of the A subunit of the holotoxin ablated its enterotoxicity. In the in vivo perfusion model, the toxin induced a time dependent decrease in water absorption, whereas no changes were detected in the segment perfused with supernatants obtained from the deletion mutant. Finally, partially purified toxin induced a dose dependent increment in short circuit current that reached its plateau at a toxin concentration of 4 x 10(-6) M. CONCLUSIONS: Shigella enterotoxin 1 induces a time and dose dependent intestinal secretion in the rabbit animal model, suggesting that it may be responsible for the watery phase of Shigella flexneri 2a infection.     

Modulation of intestinal epithelial wound healing in vitro and in vivo by lysophosphatidic acid.

BACKGROUND & AIMS: Lysophosphatidic acid (LPA) is assumed to play an important role in the modulation of injury and tissue repair in nonepithelial tissues. The effects of LPA on intestinal epithelial wound repair in vitro and in vivo were characterized. METHODS: Effects of LPA on intestinal epithelial restitution and proliferation were assessed by using an in vitro wounding model with confluent intestinal epithelial cell line 6 (IEC-6) monolayers and colorimetric thiazolyl blue (MTT) assays. In addition, LPA signaling pathways were characterized. Effects of LPA on intestinal wound healing in vivo were studied by using the trinitrobenzene model of colitis in rats. RESULTS: LPA significantly enhanced migration and inhibited cell proliferation of IEC-6 cells in vitro. The effects on intestinal epithelial cell migration and proliferation were mediated through transforming growth factor beta (TGF-beta)-independent pathways and binding to a G-protein receptor. In addition, LPA significantly ameliorated intestinal epithelial injury in the trinitrobenzene model of colitis in rats. CONCLUSIONS: These findings suggest that LPA enhances intestinal epithelial wound healing by modulation of intestinal epithelial cell migration and proliferation through TGF-beta-independent pathways. Thus, exogenous administration of LPA may provide a new approach for modulating intestinal injury in vivo.     

Modulation of transcription factor NF-kappa B binding activity by oxidation-reduction in vitro.

NF-kappa B is a widely used regulator of inducible and tissue-specific gene control. In the cytosol, when complexed to an inhibitory molecule, I kappa B, NF-kappa B is in an inactive form and cannot bind DNA. Activation of cells with appropriate stimuli results in the dissociation of NF-kappa B from I kappa B and its translocation to the nucleus as an active binding protein. We now demonstrate that NF-kappa B binding in vitro can be inhibited by agents that modify free sulfhydryls. Binding is eliminated after treatment with N-ethylmaleimide, an alkylating agent, and diamide, an oxidizing agent. The diamide effect can be reversed by 2-mercaptoethanol. Further, 2-mercaptoethanol acts synergistically with deoxycholate plus Nonidet P-40 in converting inactive cytosolic NF-kappa B to an active DNA-binding form. It is therefore possible that modulation of the redox state of NF-kappa B could represent a post-translational control mechanism for this factor.     

Modulation of actin structure and function by phosphorylation of Tyr-53 and profilin binding

On starvation, Dictyostelium cells aggregate to form multicellular fruiting bodies containing spores that germinate when transferred to nutrient-rich medium. This developmental cycle correlates with the extent of actin phosphorylation at Tyr-53 (pY53-actin), which is low in vegetative cells but high in viable mature spores. Here we describe high-resolution crystal structures of pY53-actin and unphosphorylated actin in complexes with gelsolin segment 1 and profilin. In the structure of pY53-actin, the phosphate group on Tyr-53 makes hydrogen-bonding interactions with residues of the DNase I-binding loop (D-loop) of actin, resulting in a more stable conformation of the D-loop than in the unphosphorylated structures. A more rigidly folded D-loop may explain some of the previously described properties of pY53-actin, including its increased critical concentration for polymerization, reduced rates of nucleation and pointed end elongation, and weak affinity for DNase I. We show here that phosphorylation of Tyr-53 inhibits subtilisin cleavage of the D-loop and reduces the rate of nucleotide exchange on actin. The structure of profilin–Dictyostelium-actin is strikingly similar to previously determined structures of profilin–β-actin and profilin–α-actin. By comparing this representative set of profilin–actin structures with other structures of actin, we highlight the effects of profilin on the actin conformation. In the profilin–actin complexes, subdomains 1 and 3 of actin close around profilin, producing a 4.7° rotation of the two major domains of actin relative to each other. As a result, the nucleotide cleft becomes moderately more open in the profilin–actin complex, probably explaining the stimulation of nucleotide exchange on actin by profilin.     

Endocrine testicular function in vivo and in vitro in infertile men.

In order to detect any possible Leydig cell dysfunction associated with male infertility, the endocrine capacity of the tests was investigated in vivo and in vitro in 21 infertile men. Plasma testosterone was determined before and after 3 days of hCG stimulation. Testicular tissue obtained by bilateral biopsies was subjected to (1) histological examination, (2) determination of basal testosterone concentration and (3) incubation with hCG. Patients were grouped according to histology. In vitro basal and stimulated testicular testosterone was similar in patients with normal histology, Sertoli-cell-only syndrome and spermatogenic arrest. Tissue from patients with Leydig cell hyperplasia showed 3-fold higher basal testosterone levels and a greater response to hCG. All patients had plasma testosterone levels and responses to hCG in the normal range. There was no significant correlation between the data obtained in vivo and in vitro, indicating that testosterone determinations in peripheral blood do not necessarily reflect the intratesticular situation. There was no evidence for gross abnormality in Leydig cell function accompanying disturbed spermatogenesis.     

Insulin action and binding in adipocytes exposed to endotoxin in vitro and in vivo

We investigated in vitro and in vivo effects of endotoxin on glucose oxidation and insulin binding in rat adipocytes. Exposure of adipocytes to endotoxin (500 micrograms/ml) in vitro resulted in 48% increase in basal glucose oxidation after 30 minutes, 63% and 65% increase after 90 and 150 minutes, respectively. Adipocytes from rats injected 6 hours previously with endotoxin IV (2 mg/100 gm) also showed elevated (42%) basal glucose oxidation. Both the absolute levels of stimulation and sensitivity to insulin stimulation were less than in control cells. A tendency for higher insulin binding affinity at low ambient insulin concentrations was noted. At higher insulin concentrations this difference disappeared. Our results demonstrate an insulin-like effect of endotoxin, as well as perturbation by endotoxin of insulin action at the cellular level. Changes in receptor affinity are also suggested, implying that the insulin receptor may be a site for modulation of target cell sensitivity in endotoxemia.     

The effect of in vivo endotoxin on myocardial function in vitro

Isolated perfused working hearts from male adult Sprague Dawley rats were used to examine myocardial performance following acute in vivo endotoxin administration (LD50-6-hour). Three hours after endotoxin administration, cardiac output and peak systolic pressure in the isolated perfused working heart were depressed 25-50% over a range of left atrial filling pressures (preload) from 10 to 30 cm of water. Linear regression analysis of the relationship between myocardial work indices, oxygen uptake, and glucose oxidation indicated that the hearts from the endotoxin-treated animals required more oxygen and glucose to perform the same amount of work. Levels of cyclic 3'5' adenosine monophosphate were 72% higher in ventricular tissue from the endotoxin-treated group compared to the hearts of controls. Isoproterenol (10(-9) mol/min) raised levels of the nucleotide to the same final concentration in both groups of hearts, whereas myocardial pressure work in hearts from endotoxin-treated rats was only 70% that of control. Provision of isoproterenol, while increasing mechanical work by the hearts in the endotoxin-treated group, did not induce an increase to the same performance levels as that of control hearts not treated with isoproterenol. These data are consistent with the concept that endotoxemia produces an intrinsic defect in myocardial mechanical performance.     

In vitro and in vivo effects of desmopressin on platelet function.

BACKGROUND AND OBJECTIVE: Desmopressin (DDAVP) may shorten bleeding time in patients with disorders of platelet function, but its mechanism of action in these conditions is still a matter of debate. In particular, contrasting results have been obtained concerning the ability of DDAVP to interact with platelets and to activate them directly. To gain further information on the DDAVP-platelet interaction, we studied the in vitro and ex vivo effects of DDAVP on platelet function. DESIGN AND METHODS: Platelet responses to DDAVP both as a single agent and in conjunction with agonists of platelet activation were investigated. For in vitro experiments platelets were obtained from healthy adult volunteers, while the ex vivo effects of DDAVP were studied in 12 patients with a bleeding disorder receiving a test dose of this drug. RESULTS: DDAVP in vitro did not induce either platelet aggregation or surface expression of the activation-dependent antigens; it did, however, greatly inhibit platelet aggregation response to vasopressin (AVP) and increased the maximal extent of platelet aggregation induced by collagen and ADP. DDAVP infusion did not promote the expression of activation antigens, but significantly enhanced ex vivo platelet aggregation stimulated by ADP and collagen. This priming effect was observed in patients with von Willebrand's disease, hemophilia A, May-Hegglin anomaly, gray platelet syndrome and Ehlers-Danlos syndrome. In all these patients bleeding time was shortened by DDAVP infusion. In contrast, neither platelet aggregation nor bleeding time was modified in two subjects with Glanzmann's thrombasthenia. INTERPRETATION AND CONCLUSIONS: Our in vitro experiments indicate that DDAVP interacts directly with platelets and facilitates their activation via other agonists. In vivo results suggest that this effect occurs and is clinically relevant in patients with platelet dysfunction responding to DDAVP with a shortening of bleeding time.     

In vivo and in vitro effects of different anaesthetics on platelet function

Different effects of thiopental, propofol and sevoflurane on platelets have been reported. Patients undergoing thyroid surgery were anaesthetized with thiopental-fentanyl-sevoflurane (n = 11) or propofol-fentanyl-sevoflurane (n = 9). Platelet aggregation and thromboxane A2 generation were studied at baseline, and at the end of anaesthesia induction and surgery. Dose-response experiments were also performed in vitro with single agents. Thiopental-fentanyl-sevoflurane significantly reduced collagen-induced aggregation by the end of induction, while ADP-induced aggregation and thromboxane generation were unaffected. Propofol-fentanyl-sevoflurane had no effect on platelets. Thiopental dose-dependently inhibited platelets in vitro, while fentanyl or propofol did not. In conclusion, thiopental reduces platelet function both ex vivo and in vitro and propofol might be considered haemostatically safer.