RT-PCR-based evidence for the in vivo stimulation of renal tubular p-aminohippurate (PAH) transport by triiodothyronine (T3) or dexamethasone (DEXA) in kidney tissue of immature and adult rats

Our previous studies have shown that a pre-treatment of rats with triiodothyronine (T3) or dexamethasone (DEXA) increases renal PAH excretion significantly. This stimulation was accompanied by an enhanced protein synthesis within the renal cortex. To explore the molecular basis for this sub-chronic induction process, we investigated the stimulation of PAH accumulation in renal cortical slices as well as the expression level of organic anion transporter 1 (OAT1), the recently cloned renal basolateral PAH-transporter, using RT-PCR techniques under the applied conditions. 10- and 55-day-old Han:WIST rats were treated in vivo with T3 (20 μg/100 g b.wt.) or DEXA (60 μg/100 g b.wt.), both for 3 days, once daily. Renal cortical slices were incubated for 2 hours in Cross-Taggart medium and PAH uptake into kidney tissue was measured time dependently (slice to medium ratio, Q_S/M). The accumulation capacity is comparable between immature and mature rats (control-Q_S/M: 6.7 ± 0.1 vs. 6.9 ± 0.2, respectively). Both age groups showed a significant increase of PAH accumulation capacity after T3 treatment (10-day-old rats: 15.0 ± 0.2; 55-day-old rats: 11.7 ± 1.3). After DEXA pre-treatment, PAH accumulation was only slightly changed (10-day-old rats: 5.9 ± 0.2; 55-day-old rats: 8.2 ± 1.3). Semi-quantitative measurements of OAT1 mRNA expression level showed a significant increase of OAT1 mRNA after pre-treatment with both T3 and DEXA in the two age groups. Thus, this is the first evidence that T3 and DEXA pre-treatment induces the expression of OAT1.     

SDS-PAGE analysis of the protein layers adsorbing in vivo and in vitro to bone substituting materials

The composition of the protein layer adsorbed to the bone substituting materials, hydroxyapatite, β-whitlockite, titanium and aluminium, in vivo (intramuscularly in guinea pig) and in vitro, was investigated using SOS-gel electrophoresis (SDS-PAGE). After in vivo implantation for 1 d mainly proteins with molecular weights between 10 000 and 20 000 were adsorbed. After 3 months the biolayer of the implanted biomaterials also contained proteins with molecular weights 35 000, 45 000, 60 000 and 200 000. No large qualitative differences in protein composition of the biolayers on the various implanted materials were found.

In vitro incubation with human serum resulted in binding of proteins with estimated molecular weights of 30 000, 60 000 (albumin), 200 000 and > 200 000. It is suggested that the differences between in vivo and in vitro protein adsorption are due to proteolysis occurring in vivo in the vicinity of the implanted material.     

Engineering of extensor tendon complex by an ex vivo approach

Engineering of extensor tendon complex remains an unexplored area in tendon engineering research. In addition, less is known about the mechanism of mechanical loading in human tendon development and maturation. In the current study, an ex vivo approach was developed to investigate these issues. Human fetal extensor tenocytes were isolated, expanded and seeded on polyglycolic acid (PGA) fibers that formed a scaffold with a shape mimicking human extensor tendon complex. After in vitro culture for 6 weeks, 7 cell-scaffold constructs were further in vitro cultured with dynamic mechanical loading for another 6 weeks in a bioreactor. The other 14 constructs were in vivo implanted subcutaneously to nude mice for another 14 weeks. Seven of them were implanted without loading, whereas the other 7 were sutured to mouse fascia and animal movement provided a natural dynamic loading in vivo. The results demonstrated that human fetal cells could form an extensor tendon complex structure in vitro and become further matured in vivo by mechanical stimulation. In contrast to in vitro loaded and in vivo non-loaded tendons, in vivo loaded tendons exhibited bigger tissue volume, better aligned collagen fibers, more mature collagen fibril structure with D-band periodicity, and stronger mechanical properties. These findings indicate that an extensor tendon complex like structure is possible to generate by an ex vivo approach and in vivo mechanical loading might be an optimal niche for engineering functional extensor tendon.     

Some cellular and molecular characteristics of high and low tumorigenicity variants of polyoma-virus transformed cells

We analyzed several cellular and molecular properties of BALB/c 3T3 cellular clones transformed in vitro with polyoma virus and exhibiting a high or low tumorigenicity phenotype. We also analyzed the same clones after a single in vivo passage in syngeneic mice. This passage invariably induced and/or selected variants exhibiting a very high tumorigenicity phenotype.

BALB/c mice bearing tumors induced by the inoculation of the above cells, regardless of their tumorigenicity phenotype, have a lower number of L3T4 positive splenocytes than appropriate controls. The response to Con-A of spleen cells from such mice was also suppressed. Concomitantly, an increase in Mac-1 positive splenocytes could be measured. In spite of the non-specific suppression of T cells, spleen cells from tumor-bearers showed a specific proliferative response to polyoma antigens.

Molecular analysis of polyoma transformed cells showed no differences between the various cells with respect to integration of the polyoma viral genes or with respect to src, myc and fos proto-oncogenes. In vitro maintained cells and in vivo passaged cells seemed to differ, however, in the content of polyoma middle T.

Whereas polyoma virus transformed cells maintained only in culture never expressed low affinity receptors for IgG (FcγRII), certain in vivo passaged cells did. This expression could be measured both at the protein and the mRNA level. Those in vivo passaged cells which expressed F RII gave tumors following a long latency period.

Ongoing experiments will indicate whether or not FcγRII expression is linked to long latency of tumor development.     

Differential immunotoxic effects of the environmental chemical benzo[a]pyrene in young and aged mice

Young (3–6 months), middle-aged (16–18 months) and aged (23–26 months) mice were exposed in vitro and in vivo to the immunotoxic environmental chemical benzo[a]-pyrene. The generation of antibody producing cells to the T-dependent antigens of sheep erythrocytes was observed to be suppressed in all age groups. Significantly, aged mice were shown to exhibit a greater percent suppression of antibody responses than young or middle-aged mice both in vitro and in vivo. The results presented provide the first evidence that the degree of immunological toxicity of environmental chemicals may be partially dependent upon the chronological and immunological age of the animal.     

Stimulation of mouse macrophage antigen presentation by cocaine

Cocaine has been demonstrated to have multiple effects on the immune system. Here, we determined the effects of cocaine on macrophage antigen presentation, using an in vitro antigen presentation assay after macrophages were treated with cocaine both in vitro and in vivo. Our results showed that in vitro treatment of macrophages with cocaine significantly enhanced macrophage's ability to present ovalbumin (OVA) and the enhancement was also demonstrated in the macrophages of cocaine-injected mice. The presentation of an OVA-derived antigenic peptide (OVA_323-339), however, was not affected. In vitro cocaine treatment neither affected antigen uptake nor major histocompatibility complex (MHC) II expression and the expression of co-stimulatory molecules B7. These results suggest that cocaine may act on an early event in the antigen handling by accessory cells.     

Antioxidant and anti-inflammatory activities of the plant Andrographis paniculata Nees

In this study, we explored the antioxidant and anti-inflammatory properties of the medicinal herb Andrographis paniculata using in vitro as well as in vivo systems. Methanolic extract of Andrographis paniculata was found to inhibit formation of oxygen derived free radicals such as superoxide (32%) hydroxyl radicals (80%) lipid peroxidation (80%) and nitric oxide (42.8%) in in vitro system. In vivo studies using BALB/c mice models also showed significant inhibition in PMA induced superoxide (32.4%) and nitric oxide (65.3%) formation. Interestingly we also found that, administration of Andrographis paniculata extract produced complete inhibition of carageenan induced inflammation compared with control models.     

Increase of Natural Killer Activity of Mouse Lymphocytes Following in vitro and in Vivo Treatment with Lithium

The in vivo and in vitro influence of lithium lactate on mouse natural killer activity was investigated. In vitro exposure of effector-target mixture to graded concentrations of lithium did not substantially modify the natural killer activity of mouse splenocytes, untreated or pretreated with cyclophosphamide. However in vitro treatment of effector splenocytes increased the frequency of NK-percursor cells.

The in vivo treatment with lithium lactate greatly increased the natural killer activity in intact mice, whereas it did not improve this cytotoxic function in host immunodepressed by cyclophosphamide.

These data suggest that lithium salts produce a modulation of natural killer activity of mouse spleen cells, probably through a mechanism involving the increase of the number of NK-precursors in hosts not subjected to cytotoxic chemotherapy.     

Simultaneous Analysis of In Vivo CD8+ T Cell Cytotoxicity Against Multiple Epitopes using Multicolor Flow Cytometry

CD8+ T cells play a critical role in host defense against infections and tumors. Analysis of cytotoxic function of antigen-specific CD8+ T cells in animal models would be important in optimizing vaccine design against infections and tumors. In vivo cytotoxicity assays using fluorescent cellular dyes have been used as a popular alternative to traditionally used in vitro ⁵¹Cr-release assays. With the identification of multiple epitopes in various pathogen models, methods to simultaneously analyze cytotoxicity of CD8+ T cells to multiple epitopes in vivo would assist studies which aim to generate protective CD8+ T cell immunity to multiple epitopes. In this study, we evaluate the use of multiple fluorescent cellular dyes for the in vivo cytotoxicity assay. The use of 3 dyes allowed us to analyze the cytotoxicity of antigen-specific CD8+ T cell populations to multiple epitopes generated by virus infections, as well as their functional avidity, in vivo. Our studies extend the use of in vivo cytotoxicity assays to allow direct comparisons of cytotoxicity to various epitopes in the same animal and may also be applicable to assessment of in vitro cytotoxicity of human CD8+ T cells specific for multiple viral or tumor antigens in clinical settings.     

IN VITRO RESPONSE OF v-Ha-ras TRANSGENIC MOUSE LYMPHOCYTES AFTER IN VIVO TREATMENT WITH ALCOHOL

Oncomouse is a transgenic mouse carrying an activated v-Ha-ras oncogene under the control of the mouse mammary tumor virus promoter. The objective of this paper was to learn if the in vitro secretion of IL-2 and IFN-γ and the release of sIL-2R by Oncomice splenocytes and thymocytes depended on the presence of the oncogene product, on the in vivo pretreatment with alcohol, or on the in vitro treatment with cocaine or morphine. Oncomice thymocytes released less sIL-2R than FVB thymocytes. Alcohol did not increase sIL-2R release in Oncomice as it did in FVB mice thymocytes. Oncomice thymocytes secreted more IFN-γ than FVB thymocytes, their secretion was downregulated by in vivo treatment with alcohol, while it was upregulated in FVB thymocytes. IFN-γ secretion was lower in Oncomice splenocytes from animals receiving alcohol. Oncomice thymocytes and splenocytes responded in a nearly opposite fashion to their FVB counterparts. Therefore, the in vivo treatment with alcohol modified the in vitro response to cocaine or morphine in an oncogene-dependent and -independent manner. Hence, our results further emphasize the role of v-Ha-ras oncogene in defining the host immune response, and of alcohol in modulating such response.     

Human bone marrow stromal cells hamper specific interactions of CD4 and CD8 T lymphocytes with antigen-presenting cells

Bone marrow stromal cells (BMSCs) may inhibit T-cell functions in vitro and thus have been proposed as immunoregulators to control in vivo graft-versus-host disease (GVHD) in haploidentical hemopoietic stem cell transplants. To better investigate this phenomenon, we used a defined experimental system in which responding T cells are antigen-specific and devoid of alloreactivity against BMSC from a different subject. Thus, we established antigen-specific human CD4 and CD8 T-cell lines as the readout system. Antigen-dependent proliferation was reduced with both T-cell subsets cultured on confluent BMSCs, and also on confluent human skin fibroblasts (HSF) inhibited T-cell proliferation with similar efficiency. Morphological observations of the cocultures showed impairment of physical interactions between T-cell and antigen-presenting cells in the presence of BMSC, with lack of formation of antigen-dependent clusters of T cells and antigen-presenting cells (APCs). In contrast, no effects were seen with BMSC-conditioned medium. Since suppression was seen only with confluent mesenchymal cells, this phenomenon may not be relevant in vivo, where BMSCs are at low frequency. In addition, if the reported suppressive effect of BMSCs on GVHD in vivo is confirmed, a different in vitro system should be envisaged to better understand and exploit the underlying mechanism.     

Neural dynamics in cortex-striatum co-cultures-I. Anatomy and electrophysiology of neuronal cell types

An in vitro system was established to analyse corticostriatal processing. Cortical and striatal slices taken at postnatal days 0–2 were co-cultured for three to six weeks. The anatomy of the organotypic co-cultures was determined using immunohistochemistry. In the cortex parvalbumin-positive and calbindin-positive cells, which resembled those seen in vivo, had laminar distributions. In the striatum, strongly stained parvalbumin-positive cells resembling striatal GABAergic interneurons and cholinergic interneurons were scattered throughout the tissue. The soma area of these iterneuron classes was larger than the average striatal soma area, thus enabling visual selections of cells by class before recording. Cortical neurons with projections to the striatum showed similar morphological features to corticostriatal projection neurons in vivo. No projections from the striatum to the cortex were found. Intracellular recordings were obtained from 94 neurons. These were first classified on the basis of electrophysiological characteristics and the morphologies of cells in each class were reconstructed. Two types of striatal secondary neurons with unique electrophysiological dynamics were identified: GABAergic interneurons (n = 17) and large aspiny, probably cholinergic, interneurons (n = 15). The electrophysiological and morphological characteristics of cortical pyramidal cells (n = 27), cortical interneurons (n = 1), as well as striatal principal neurons (n = 34), were identical to those reported for similar ages in vivo.

Organotypic cortex-striatum co-cultures are therefore suitable as an in vitro system in which to analyse corticostriatal processing. The network dynamics, which developed spontaneously in that system, are examined in the companion paper.     

Enhancement by Muramyl Dipeptide of in vitro Nude Mice Responses to a T-Dependent Antigen

N- acetyl-muramyl-L-alanyl-D-isoglutamine (referred to as MDP for muramyl dipeptide) has been shown to enhance in vivo and in vitro immune responses to various antigens. It has previously been reported that in the case of T-de pendent antigens, the adjuvant: activity of MDP was mediated by a helper T-cell. Our present findings demonstrate that in vitro responses of nude mice spleen cells to T - independent, TNP-PAA or T-dependent SRBC can also be markedly increased by this synthetic adjuvant. Moreover, under the same conditions, MDP produced polyclonal activation.     

Inhibitory effect of mast cell-mediated immediate-type allergic reactions by sulfapyridine

We examined the effect of sulfapyridine on mast cell-mediated immediate-type allergic reactions. Sulfapyridine (1 and 10 μg/kg) significantly inhibited systemic allergic reaction induced by compound 48/80 in rats. Sulfapyridine (1 and 10 μg/kg) also inhibited significantly local mast cell-mediated immediate-type allergic reactions activated by anti-dinitrophenyl (DNP) IgE. Moreover, sulfapyridine inhibited histamine release dose-dependently in the rat peritoneal mast cells (RPMC) activated by compound 48/80 or anti-DNP IgE. When sulfapyridine was added, the level of cAMP in RPMC, transiently and significantly increased about 4-fold compared with that of basal cells. These results indicate that sulfapyridine inhibits mast cell-mediated immediate-type allergic reactions in vivo and in vitro.     

IN VITRO RESPONSE OF v-Ha-ras TRANSGENIC MOUSE LYMPHOCYTES AFTER IN VIVO TREATMENT WITH COCAINE

Oncomouse is a transgenic mouse carrying an activated v-Ha-ras oncogene under the control of the mouse mammary tumor virus promoter. The objective of this paper was to learn if the in vitro secretion of IL-2 and IFN-γ and the release of sIL-2R by Oncomice spleen and thymus cells depended on the presence of the oncogene product, on the in vivo pretreatment with cocaine, or on the in vitro treatment with cocaine or morphine. Oncomice thymocytes from different experimental groups released less sIL-2R than FVB thymocytes. Oncomice thymocytes secreted more IFN-γ than FVB thymocytes. Oncomice thymocytes cultured in the presence of Con A and cocaine showed a diminished release of sIL-2R and a lower secretion of IFN-γ, a phenomenon not observed in FVB thymocytes. IFN-γ secretion was lower in Oncomice splenocytes. In general, Oncomice thymocytes and splenocytes responded in a nearly opposite fashion to their FVB counterparts. In this study, the in vitro response to mitogens, cocaine or morphine depended on genetic background and not on the in vivo pretreatment with cocaine. Our results emphasize the role of the v-Ha-ras oncogene in defining the host immune response.     

Carbamazepine Affects Neutrophil Function through an Action on Peripheral Benzodiazepine Receptors

The aims of this study were to assess the possible role of peripheral benzodiazepine receptors (pBZrs)¹ in mediating the in vitro effects of carbamazepine (CBZ) on some neutrophil functions in healthy volunteers and to investigate neutrophil function and pBZr expression in patients with epilepsia on CBZ monotherapy for at least 1 year. In vitro CBZ (42-168 μM) concentration-dependently inhibited chemotaxis induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP) or lipopolysaccharide (LPS)-activated human serum. CBZ did not affect random migration, phagocytosis index, phagocytosis frequency, NBT reduction frequency, C. albicans lethality index and resting superoxide production. The pBZr antagonist PK 11195 (1 μM, per se ineffective) reversed the inhibitory effect of CBZ on chemotaxis induced by endotoxin-activated serum or FMLP. The pBZr agonist Ro 5-4864 (10-100 μM) mimicked the effect of CBZ on chemotaxis induced by endotoxin-activated serum or FMLP and had no effect on the other parameters. Neutrophils from epileptic patients on chronic CBZ monotherapy had impaired FMLP- and serum-induced chemotaxis and enhanced expression of pBZrs on neutrophils. These data strongly suggest an involvement of pBZrs in mediating the in vitro effects of CBZ on chemotaxis; furthermore, impairment of the same neutrophil function parameters and overexpression of pBZrs in patients are consistent with the hypothesis of an in vivo interaction of CBZ with pBZrs.     

Chronic aluminum toxicity: effects of aluminum on neurotransmitters

Available data suggest that chronic neurotoxicity may be associated with repeated dietary exposure to extremely low concentrations of aluminum in susceptible populations. Adverse effects of aluminum on central neurotransmitter function have been demonstrated both in vivo and in vitro with several different aluminum salts.     

Analysis of lymphocyte heterogeneity in carp, Cyprinus carpio L., using monoclonal antibodies

Several in vivo and in vitro studies on functional aspects of fish lymphocytes have led to the assumption that lymphoid cell populations analogous to T- and B-cells in higher vertebrates are also present in teleosts. However, formal proof of such heterogeneity based on structure-function correlations is lacking. In this report, data on the reactivity of a number of monoclonal antibodies made against thymocytes or serum immunoglobulin of carp (Cyprinus carpio L.) are reviewed. The data suggest that the use of monoclonal antibodies will provide powerful tools with which to identify and separate structurally heterogeneous populations of fish lymphocytes so that their relative roles in the defense system of these vertebrates can be explored and exploited.     

In vivo validation of signaling pathways regulating human monocyte chemotaxis

Identification of novel signal transduction pathways regulating monocyte chemotaxis can indicate unique targets for preventive therapies for treatment of chronic inflammatory diseases. To aid in this endeavor we report conditions for optimal transfection of primary human monocytes coupled with a new model system for assessing their chemotactic activity in vivo. This method can be used as a tool to identify the relevant signal transduction pathways regulating human monocyte chemotaxis to MCP-1 in the complex in vivo environment that were previously identified to regulate chemotaxis in vitro. MCP-1-dependent chemotaxis of monocytes is studied in an adoptive transfer model where human monocytes transfected with mutant cDNAs are transferred to mice followed by initiation of peritonitis. Harvesting peritoneal cells at 24 h diminishes the contribution of immunologic responses to the cross-species transfer. Validation of relevant regulatory molecules in vivo is critical for understanding the most relevant therapeutic targets for drug development.     

Immunotoxic Effects of Copper and Cadmiun the Sea Bass Dicentrarchus Labrax

Two phagocytes-mediated activities of the sea bass Dicentrarchus labrax were examined after exposure to sublethal concentrations of copper and cadmium: (a) phagocytosis (measured by phagocytotic index), and (b) the production of reactive oxygen intermediates (luminoldependent chemiluminescence) in response to bacteria Aeromonas salmonicida. In vivo exposure for 48 h to each metal separately by intraperitoneal injection did not affect the quantity of phagocytes of pronephros and their viability but inhibited, in dose-dependent manner, phagocytosis and chemiluminescence of these cells. The half-inhibition value was 250 µ;gkg^-1 for copper and 1 mgkg^-1 for cadmium. In vitro exposure to copper for 30 min had the same immunomodulatory effect on macrophage chemiluminescence as that observed in vivo, whereas treatment with cadmium under the same conditions had a dose-dependent effect opposite to that observed in vivo.

Assessement of these two macrophage-mediated functions could therefore be used as early bioindicators of the marine pollution.