Allochimeric therapy induces unique regulatory T cells that mitigate chronic rejection.

Induction of tolerance to an allogeneic graft without the need for nonspecific immunosuppression is a major goal of transplantation therapy. We have shown that treatment with molecularly engineered, allochimeric [alpha(1h)(1/u)]-RT1.Aa class I MHC antigens bearing donor-type Wistar-Furth (WF, RT1.A(u)) or Lewis (LEW, RT1A(1)) amino acid substitutions for host-type ACI (RTI.A(a)) sequences in the alpha(1)-helical region induces donor-specific tolerance to cardiac allografts in rat recipients. The mechanisms involved in the establishment and maintenance of specific allograft tolerance are still not fully understood. It is now clear that acquisition of transplantation tolerance is an active, highly regulated, multistep process. According to the pool size model of allograft tolerance, the allograft outcome, rejection, or tolerance often depend on the balance between cytopathic and regulatory T cells (T-regs). This study examined mechanisms of chronic rejection (CR) development on a model of cardiac transplant tolerance after adoptive transfer of T-regs followed by allochimeric therapy. Generation of T-regs was demonstrated in vitro by MLR coculture and confirmed by adoptive transfer of T cells from primary recipients to secondary hosts. To confirm the true nature of regulatory cells, we performed a second transfer into tertiary recipients. Unlike T-regs from tolerant hosts, T cells from naive rats did not prolong graft survival. Histological evaluation of T-regs-transfected groups showed absence of visible CR. In contrast, T-regs generated in recipients after high-dose cyclosporine treatment failed to inhibit CR in transferred singeneic recipients. Allochimeric therapy triggers generation of unique regulatory lymphocytes that mitigate development of chronic rejection through regulation of anti-inflammatory mechanisms and down-regulation of alloantibody response.

Plasma membrane resident 'fusion complexes' mediate reconstituted exocytosis.

Calcium-triggered exocytosis is thought to be mediated by membrane-associated protein complexes. In sea urchin eggs, high concentrations of calcium activate multiple 'fusion complexes' per cortical vesicle-plasma membrane docking site. Some of these fusion complexes are known to reside in the vesicle membrane. It is not known if fusion complexes also reside in the plasma membrane, or if plasma membrane-resident fusion complexes require cognate partners in the vesicle membrane. Using reconstitution, we show that N-ethylmaleimide treatment of either vesicles or plasma membrane fragments prior to reconstitution does not completely inhibit exocytosis. Treatment of both components did result in complete inhibition. Upon reconstitution, cortical vesicles and the early endosomes formed by compensatory endocytosis both contributed, on average, two fusion complexes per reconstituted docking site. The plasma membrane contributed, on average, two fusion complexes per docking site when assembled with cortical vesicles, but only one complex when reconstituted with endosomes. We conclude that there are at least two types of plasma membrane-resident fusion complexes that participate in reconstituted cortical vesicle-plasma membrane fusion. The activity of one of these fusion complexes is target-specific for cortical vesicles, while the second type also supports fusion with endosomes.

Exocytotic insertion of calcium channels constrains compensatory endocytosis to sites of exocytosis.

Proteins inserted into the cell surface by exocytosis are thought to be retrieved by compensatory endocytosis, suggesting that retrieval requires granule proteins. In sea urchin eggs, calcium influx through P-type calcium channels is required for retrieval, and the large size of sea urchin secretory granules permits the direct observation of retrieval. Here we demonstrate that retrieval is limited to sites of prior exocytosis. We tested whether channel distribution can account for the localization of retrieval at exocytotic sites. We find that P-channels reside on secretory granules before fertilization, and are translocated to the egg surface by exocytosis. Our study provides strong evidence that the transitory insertion of P-type calcium channels in the surface membrane plays an obligatory role in the mechanism coupling exocytosis and compensatory endocytosis.

Calcium influx is required for endocytotic membrane retrieval.

Cells use endocytotic membrane retrieval to compensate for excess surface membrane after exocytosis. Retrieval is thought to be calcium-dependent, but the source of this calcium is not known. We found that, in sea urchin eggs, endocytotic membrane retrieval required extracellular calcium. Inhibitors of P-type calcium channels-cadmium, omega-conotoxin MVIIC, omega-agatoxin IVA, and omega-agatoxin TK-blocked membrane retrieval; selective inhibitors of N-type and L-type channels did not. Treatment with calcium ionophores overcame agatoxin inhibition in a calcium-dependent manner. Cadmium blocked membrane retrieval when applied during the first 5 minutes after fertilization, the period when the membrane potential is depolarized. We conclude that calcium influx through omega-agatoxin-sensitive channels plays a key role in signaling for endocytotic membrane retrieval.

Separation of viable from radiation-induced apoptotic lymphocytes by free-flow electrophoresis.

A human lymphocyte population undergoing apoptosis in vitro due to gamma-irradiation was fractionated by free-flow electrophoresis in triethanolamine--Na-acetate buffers, containing up to 50 mM NaCl, with pH 6.0, 7.2 and 8.5, made isotonic by addition of sucrose. As shown by a flow cytometric analysis of the eluate, the distribution of apoptotic lymphocytes is shifted to the range of higher electrophoretic mobilities relative to that of viable ones at pH 8.5, yielding cell fractions enriched in apoptotic cells by a factor of 3 to 5. The difference in rates of electrophoretic migration observed at a mildly alkaline pH but not at a neutral or mildly acidic one suggests that the surface of apoptotic lymphocytes is more acidic than that of viable ones.

Lymphocyte trafficking and HIV infection of human lymphoid tissue in a rotating wall vessel bioreactor.

The pathogenesis of HIV infection involves a complex interplay between both the infected and noninfected cells of human lymphoid tissue, the release of free viral particles, the de novo infection of cells, and the recirculatory trafficking of peripheral blood lymphocytes. To develop an in vitro model for studying these various aspects of HIV pathogenesis we have utilized blocks of surgically excised human tonsils and a rotating wall vessel (RWV) cell culture system. Here we show that (1) fragments of the surgically excised human lymphoid tissue remain viable and retain their gross cytoarchitecture for at least 3 weeks when cultured in the RWV system; (2) such lymphoid tissue gradually shows a loss of both T and B cells to the surrounding growth medium; however, this cellular migration is reversible as demonstrated by repopulation of the tissue by labeled cells from the growth medium; (3) this cellular migration may be partially or completely inhibited by embedding the blocks of lymphoid tissue in either a collagen or agarose gel matrix; these embedded tissue blocks retain most of the basic elements of a normal lymphoid cytoarchitecture; and (4) both embedded and nonembedded RWV-cultured blocks of human lymphoid tissue are capable of productive infection by HIV-1 of at least three various strains of different tropism and phenotype, as shown by an increase in both p24 antigen levels and free virus in the culture medium, and by the demonstration of HIV-1 RNA-positive cells inside the tissue identified by in situ hybridization. It is therefore reasonable to suggest that gel-embedded and nonembedded blocks of human lymphoid tissue, cocultured with a suspension of tonsillar lymphocytes in an RWV culture system, constitute a useful model for simulating normal lymphocyte recirculatory traffic and provide a new tool for testing the various aspects of HIV pathogenesis.

Experimental HIV infection of human lymphoid tissue: correlation of CD4+ T cell depletion and virus syncytium-inducing/non-syncytium-inducing phenotype in histocultures inoculated with laboratory strains and patient isolates of HIV type 1.

It is now apparent that HIV infection leads to a gradual collapse of a complex system of lymphoid tissue. This collapse tends to be associated with a change in virus tropism from macrophages to T lymphocytes, and a change in phenotype from nonsyncytium inducing (NSI) to syncytium inducing (SI). An experimental system is required to study the role of this change in HIV pathogenesis in lymphoid tissue. Here we describe such a system. Histocultures of human lymphoid tissue preserve their general cytoarchitecture, including a network of follicular dendritic cells (FDCs). Histocultures of tonsils, adenoids, or lymph nodes support productive infection with various laboratory and primary isolates of HIV-1 of different tropism and phenotype and exhibit isolate-dependent CD4+ T lymphocyte depletion. A strong correlation between the extent of CD4+ T cell depletion and the SI/NSI phenotype of the isolates is demonstrated. AZT was used as a model drug to inhibit viral replication and CD4+ T cell depletion in lymphoid histocultures. HIV pathogenesis and the effect of antivirals can now be studied in human lymphoid tissue under controlled conditions in vitro.

Syncytium formation in cultured human lymphoid tissue: fusion of implanted HIV glycoprotein 120/41-expressing cells with native CD4+ cells.

While glycoprotein gp120/41 clearly causes HIV-infected cells to form syncytia in monolayers and in suspension, there is unfortunately scant knowledge on syncytium formation in tissues. We implanted gp120/41-expressing cells labeled with fluorescent particles inside blocks of human lymphoid tissue kept in long-term histoculture. Observed by confocal microscopy, together with immunohistochemical and morphological analysis of implanted cells, more than one-third of these gp120/41-expressing cells fused with native CD4+ cells of the host tissue, yielding small (three to five nuclei) syncytia. Such widespread fusion of gp120/41-expressing cells in tissue in vitro, together with the finding of increased virulence of syncytium-inducing isolates of HIV, support the hypothesis that syncytium formation within lymph tissue of HIV-infected individuals contributes to AIDS pathogenesis. This system and the methods developed may provide a way to study HIV-infected cells inside the very tissue whose destruction may prevent immune system repopulation.

Dye-coupling in three-dimensional histoculture of rat lingual frenulum.

A three-dimensional histoculture of wet stratified squamous epithelium of rat lingual frenulum was cultured on a liquid-air interface. The tissue retained its morphology for many days in culture. During this period the vast majority of the epithelial cells remained viable and exhibited dye (lucifer yellow) coupling in all living epithelial strata. Dye coupling was determined using two methods: the conventional intracellular injection method, and a new method--"cut-loading." In the cut-loading method, an incision is made in the epithelium in the presence of dye, and intracellular diffusion of dye throughout the epithelium was measured using confocal microscopy. The basolateral surface of the lingual frenulum also acted as a substrate for neuroblastoma cells to grow without exogenously added trophic factors. These neuroblastoma cells grow neurites that establish contacts with epithelial cells. This preparation can serve as a model for investigating interactions among epithelial cells and between nerves and epithelial cells.

Confocal microscopy of cells implanted into tissue blocks: cell migration in long-term histocultures.

In three-dimensional tissues in vivo, cells find themselves in a unique, heterogeneous microenvironment among various cellular and noncellular elements. Cells are greatly affected by and contribute to their physical and chemical microenvironments. However, live cells are currently studied predominantly in homogeneous monolayer cultures where newly established contacts might be fundamentally different from contacts in vivo. Several systems have been suggested to simulate the three-dimensional environment of real tissue. In this report, we describe a new system for studying cell behavior inside real tissues in vitro. By fluorescently labeling mouse tumor cells, them implanting them into cultured tissue blocks (histocultures), we have observed cellular location and followed their locomotion, within tissues in vitro for days. We discuss the potential of the described system for studying different aspects of cell behavior in a nativelike microenvironment.

[Cell growth and motility in culture (in vitro) under microgravity conditions. The Fibroblast Experiment]. / Rost i podvizhnost' kletok v kul'ture (in vitro) v usloviiakh mikrogravitatsii. Eksperiment "Fibroblast".

The experiment "Fibroblast" was performed in 1992 on biosatellite "Cosmos-2229" in onboard device "Biobox" designed by the order of European Space Agency. The main objective was elucidation of the mechanisms responsible for the effect of space flight factors, mostly microgravity, on cell culture. We studied time-related changes in growth, motility and some morphological characteristics of the cells in monolayer cultures on a solid substrate and in three-dimensional cultures supported by sponge gels. Studies were carried out on connective tissue cells isolated from the mouse embryos. Comparative after-flight analysis of the cell cultures exposed to space flight and of those under the normal gravity conditions (1 g) on the Earth has revealed some differences. The space flight conditions, mainly microgravity, induced marked changes in morphological characteristics and functional activity of the cultured fibroblasts: changes in the nucleus size and shape, retardation of cell growth and division rate. We believe that these changes may be due to weakening of intercellular contacts and cell adhesion to the substrate. These findings are important both for general biology and space medicine, specifically for the problems of tissue regeneration and wound healing under the conditions of long-term space flight.

Organotypic growth and differentiation of human mammary gland in sponge-gel matrix supported histoculture.

Blocks of breast tissue obtained during radical mastectomies from 23 patients with mammary gland carcinomas were used for cultivation in native-state, gel-supported histocultures. We show that the human mammary gland can be successfully maintained in this system so that normal epithelial breast structures proliferate and undergo differentiation for several weeks and a well-developed system of ducts and lobules is formed. Using antibodies to individual keratins 17 and 8 we have shown for the first time that ducts and alveoles developing in vitro undergo differentiation into the lining epithelium and myoepithelium in the same way as mammary gland epithelium in vivo. Growth of epithelial structures in vitro is also accompanied by the development of continuous basal membrane.

A polycation causes migration of negatively charged phospholipids from the inner to outer leaflet of the liposomal membrane.

Aggregation of the negatively charged liposomes caused by the addition of the linear polycation, poly-N-ethyl-4-vinylpyridine bromide, was studied. At the point of maximal size and zero electrophoretic mobility of aggregates, the concentration of positive charges brought in by the adsorbed polycation was found to be equal to the total concentration of negative charges both on the outer and inner surface of the lipid bilayer. Since polycation saturation of the liposomal negative charges was found to occur without disruption of the membrane, it was concluded that the polycation induced migration of negatively charged phospholipid molecules from the inner to outer leaflet of the bilayer.

Organotypic growth and differentiation of human mammary gland in sponge-gel matrix supported histoculture.

Blocks of breast tissue obtained during radical mastectomies from 23 patients with mammary gland carcinomas were used for cultivation in native-state, gel-supported histocultures. We show that the human mammary gland can be successfully maintained in this system so that normal epithelial breast structures proliferate and undergo differentiation for several weeks and a well-developed system of ducts and lobules is formed. Using antibodies to individual keratins 17 and 8 we have shown for the first time that ducts and alveoles developing in vitro undergo differentiation into the lining epithelium and myoepithelium in the same way as mammary gland epithelium in vivo. Growth of epithelial structures in vitro is also accompanied by the development of continuous basal membrane.

Antitumor effect of k+/h+-antiporter nigericin on human lung-carcinoma grown in in-vivo-like histocultures.

Small shifts of intracellular pH (pH(i)) play a crucial role in many cellular functions, in particular progression through the cell cycle. We present results demonstrating that in the majority of cases, nigericin shows cytostatic effect on tumor cells in the in vivo-like 3-dimensional histocultures of human lung tissues while the normal tissue remains morphologically intact.

Restoration of adhesive potentials of Ehrlich ascites carcinoma cells by modification of plasma membrane.

A novel technique for modulating the spreading of ascites cells has been developed. Plasma membranes of Ehrlich ascites carcinoma cells were modified in two different ways: 1) biotin residues were covalently coupled to membrane components; 2) biotinylated lipid was introduced into plasma membrane. Adhesion and spreading of modified cells on avidin-coated substrates were studied and compared to those of non-modified cells. Both types of membrane alteration were shown to induce specific (biotin-dependent) interaction with immobilized avidin with resultant cell spreading. Spread cells attained epithelioid-like morphology with the formation of wide thin lamellae, focal contacts with substrate, and circular actin bundles. The process of spreading was shown to be energy-dependent: it could be blocked by metabolic inhibitors and by low temperature. Formation of extended lamellae was prevented by preincubation of cells in the presence of cytochalasin B. The effects of metabolic poisons, low temperature, and microfilament--disruptive drugs were reversible and after the restoration of physiological conditions the cells resumed the spreading process. Immunoprecipitation of biotinylated cell lysates with antiserum to cytoplasmic domain of beta 1-integrin subunit revealed a major 110 kD avidin-binding component. We conclude that lack of spreading of ascites carcinoma cells may be explained by the lack of functionally active adhesion- and spreading-competent cell-surface receptors, but may not be attributed to the defects in intracellular function or organization. Intracellular machinery of cell spreading is preserved in these ascites cells and could be turned on by cell attachment to the substrate via artificial adhesive site incorporated into plasma membrane.

Influence of cytochalasin D on the lipid transfer between cells and liposomes.

Cytochalasin D stimulates the binding of dipalmitoylphosphatidylcholine liposomes to bovine lens epithelial cells in vitro. Lipid transfer between plasma membranes and egg phosphatidylcholine liposomes is also increased.

Adhesion defect of ascites cells corrected with membrane-bound attachment molecules.

Adhesion is obligatory for cell proliferation in most types of cells. This function becomes defective after malignant transformation. An extreme example is ascites cells which proliferate in suspension. The nature of their defects remains obscure. Here we show that the linking of biotin molecules to the Ehrlich ascites carcinoma cells enables these cells to spread normally on an avidin-coated substrate. The spreading was the result of specific avidin-biotin interaction. The morphology of the spread cells and sensitivity to different inhibitors are similar to those of normal epithelia. Thus it is enough to supply appropriate substrate-adhesive molecules to the ascites cell surface to normalize their adhesion.