HEMOPOIESIS-STIMULATING ACTION OF ADAMANTYLAMIDE DIPEPTIDE: KINETICS OF INCREASE OF GM-CFC IN FEMUR AND CO-STIMULATING ACTIVITY OF SERUM,ROLE OF BONE MARROW STROMAL CELLS

Chief part of hemopoietic stromal cells in mediating hemopoiesis-stimulating effects of adamantylamide dipeptide (AdDP), a synthetic immunomodulatory compound, has been determined in a series of combined in vivo/in vitro studies. Indirect stimulatory effect of AdDP on proliferation of hemopoietic progenitor cells for granulocytes and macrophages (GM-CFC) was proved to be mediated by the cells of hemopoietic microenvironment growing as adherent stromal cell populations in vitro. These results supplement previously reported findings of a positive role which is played by AdDP at modulating the interplay among stimulatory cytokines and their cellular sources, and are in consent with the idea to introduce AdDP as a constituent of the hemopoiesis- and immunity-stimulating supportive medical care.     

Effects of in vivo administration of adamantylamide dipeptide on bone marrow granulocyte-macrophage hemopoietic progenitor cells (GM-CFC) and on ability of serum of the treated mice to stimulate GM-CFC colony formation in vitro: comparison with muramyl dipeptide and glucan

Adamantylamide dipeptide (AdDP), muramyl dipeptide (MDP), and glucan were shown to increase significantly the numbers of granulocyte-macrophage hemopoietic progenitor cells (GM-CFC) in the bone marrow of mice. However, whereas the sera of mice given MDP or glucan were found to stimulate the growth of colonies from GM-CFC in vitro, i.e. to produce a colony-stimulating activity (CSA), administration of AdDP did not lead to this effect. Nevertheless, when serum of mice given AdDP was added to the cultures concomitantly with a suboptimal concentration of mouse interleukin-3 (mIL-3), a broad spectrum hemopoietic stimulator, counts of colonies from GM-CFC were significantly increased, and accelerated growth of the colonies was found as well. This property of AdDP, i.e. its ability to exhibit co-stimulating activity (CoSA) without being able to exhibit CSA, suggests that AdDP acts in hemopoietic tissues differently as compared with the two other immunomodulators studied. It can be hypothesized that the action of AdDP is more specific when compared with its natural related compound, MDP, as well as with glucan. Our findings prove the possibility to stimulate by AdDP the granulopoietic compartment of hemopoiesis and are in agreement with previous observations concerning the absence of systemic side effects of AdDP. Both these qualities of AdDP may be advantageous when pondering over contingent clinical utilization of AdDP as hemopoietic stimulator.

This work was supported by a grant (PZ-Z2/25/97) from the Ministry of Industry and Trade of the Czech Republic. The research was conducted according to the principles enunciated in the Guide for the Care and Use of Laboratory Animals issued by the Czech Society for Laboratory Animal Science. The authors are indebted to Dr. J. Sandula, Institute of Chemistry, Slovak Academy of Sciences, for the soluble glucan preparation.     

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.     

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.     

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.     

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.     

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.     

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.     

In Vitro Immune Response to Lipopolysaccharide: Thymus-Derived Cells not Required

An in vitro immune response to lipopolysaccharide was obtained using heat-killed Escherichia coli cells as antigen. Spleen cell cultures from phenotypically normal mice responded to sheep erythrocytes and to lipopolysaccharide. Spleen cell cultures from congenitally athymic (nude) mice failed to respond to sheep erythrocytes but did respond to lipopolysaccharide. These results suggest that the in vitro immune response to lipopolysaccharide does not require the participation of thymus-derived cells.     

Mixed Leukxyte Culture Reactions in Mouse Thymus Cells

Parenchymal thymus cells from several strains of normal, non-immunized mice responded to histoincompatible thymus cells with increased incorporation of ³H-thymidine in vitro. The response was most pronounced with mixtures of AKR + CBALB/c thymocytes which were mutually stimulatory, as determined by experiments in which one of the cell populations had been rendered unresponsive by irradiation. Reactivity was not restricted to cell combinations bearing major histo-incompatibilities; increases in ³H-thymidine uptake also occurred in cell mixtures with θ isoantigen differences. The culture technique described allows the assessment of the interaction of antigen with native thymocytes in a completely in vitro system.     

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.     

Effect of Δ9-Tetrahydrocannabinol on in Vitro Sensitization of Mouse Splenic Lymphocytes

The effects of Δ⁹-tetrahydrocannabinol (Δ⁹-THC) on the immune response of murine cells sensitized in vitro was determined using a plaque-forming cell (PFC) assay. Splenic lymphocytes from mice injected with Δ⁹-THC showed a depressed immunologic response when compared with cells from control animals which were identically semitized in vitro with sheep erythrocytes (SRBC). The direct addition of Δ⁹-THC to the culture media altered the im-munological response as demonstrated by a reduction in the number of PFC.     

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.     

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.     

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.     

Effects of Pseudostellaria Heterophylla on Proliferation and Differentiation of Murine Bone Marrow Cells

The incorporation of tritiated thymidine into the DNA of murine bone marrow cells could be stimulated by fraction PH-I Ba separated from the roots of Pseudostellaria heterophylla in a dose-dependent manner in vitro. Moreover, PH-I Ba could induce the differentiation of murine bone marrow cells from pluripotent haemopoietic stem cells into macrophages-like cells in vitro. Autocrine or paracrine stimulation of granulocyte-macrophage colony stimulating factor was likely to underly the induction of differentiation. Therefore, PH-I Ba was proved to be an immunostimulating agent for mouse marrow hematopoiesis and was found to be a polysaccharide. The sugar components were analyzed by Gas Liquid Chromatography of their alditol acetates.     

Cytolytic Activity in Vitro of Lymph Node Cells After Exposure in Vivo to Tumor Cells Suspended in Blocking Sera

The in vivo effect of blocking sera (bs) on both tumor growth and subsequent in vitro cytolytic activity of regional lympth node cells was determined following injection of hepatoma cells suspended in normal sera (ns) or bs into the hind footpads of guinea pigs. Tumor growth was unaffected by bs but the primary response to tumor by LNC draining tumor/bs sites was significantly lower in i/6 experiments as compared to cells from lymph nodes draining tumor/ns sites.     

B-Cell Activation In Vitro by Helper T Cells Specific to a Protein Region that is Recognized Both by T Cells and by Antibodies

This laboratory had previously mapped the regions of T and B cell recognition on sperm whale myoglobin (Mb). Mb has five regions (E1-E5) that are recognized by both T cells and B cells (i.e. antibodies, Abs) and an additional region (E6) that is recognized exclusively by T cells (i.e., T_E6) and to which no Abs are detectable. The responses to the site are each under separate genetic control. Recently, we showed in an H-2^d haplotype that T_E6 cells preferentially activated Mb-primed B cells (B_Mb) that made Abs against sites within E3 and E4 on the same protein. In the present work, we established, from Mb-primed SJL mice, an E4-specific T cell line (T_E4) by passage in vitro with synthetic peptide E4. At relatively low numbers, these T cells activated syngeneic B_Mb cells in vitro to produce anti-Mb Abs that recognized each of the antigenic sites within regions E1, E2, E3, E4 and E5. We confirmed the ability of T_E4 to activate B cells that produce Abs against each of these regions by allowing T_E4 to activate in vitro syngeneic B cells that had been primed with E1, E2, E3, E4 or E5. The helper activity of T_E4 cells was dependent on the in vitro concentration of the challenge Ag (intact Mb or peptide E4). Thus, T cells against an epitope may provide help restricted to B cells that make Abs against selected antigenic sites or they may activate B cells that make Abs against all the antigenic sites of a protein. This might depend on the site-specificity of the T cell and/or on the host.     

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.     

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.