Caspase-mediated cleavage of raptor participates in the inactivation of mTORC1 during cell death.

The mammalian target of rapamycin complex 1 (mTORC1) is a highly conserved protein complex regulating key pathways in cell growth. Hyperactivation of mTORC1 is implicated in numerous cancers, thus making it a potential broad-spectrum chemotherapeutic target. Here, we characterized how mTORC1 responds to cell death induced by various anticancer drugs such rapamycin, etoposide, cisplatin, curcumin, staurosporine and Fas ligand. All treatments induced cleavage in the mTORC1 component, raptor, resulting in decreased raptor-mTOR interaction and subsequent inhibition of the mTORC1-mediated phosphorylation of downstream substrates (S6K and 4E-BP1). The cleavage was primarily mediated by caspase-6 and occurred at two sites. Mutagenesis at one of these sites, conferred resistance to cell death, indicating that raptor cleavage is important in chemotherapeutic apoptosis.

Preclinical evaluation of two human anti-hepatitis B virus (HBV) monoclonal antibodies in the HBV-trimera mouse model and in HBV chronic carrier chimpanzees.

Two human monoclonal antibodies (mAbs) against hepatitis B surface antigen (HBsAg) generated in the Trimera mouse system are described. Both mAbs 17.1.41 and 19.79.5 are of the IgG1 isotype and have high affinity constants for HBsAg binding in the range of 10(-10) mol/L. Monoclonal antibody 17.1.41 recognizes a conformational epitope on the a determinant of HBsAg whereas mAb 19.79.5 recognizes a linear one. The 2 mAbs bind to a panel of hepatitis B virus (HBV) subtypes with distinct patterns. The neutralizing activity of these antibodies was tested in 2 different animal model systems. Administration of each mAb to HBV-Trimera mice, a system that provides a mouse model for human hepatitis B infection, reduced the viral load and the percentage of HBV-DNA-positive mice in a dose-dependent manner. These 2 mAbs were more effective than a polyclonal antibody preparation (Hepatect; Biotest Pharma, Dreieich, Germany) in both inhibition of HBV liver infection and reduction of viral load. A single administration of a mixture of these mAbs into HBV chronic carrier chimpanzees resulted in immediate reduction in HBsAg levels followed by recurrence to initial levels within few days. Thus, these mAbs may be potential candidates for preventive therapy or in combination with other antiviral agents against HBV. Further studies in humans are needed to assess these mAbs in various clinical indications.

The hepatitis B virus-trimera mouse: a model for human HBV infection and evaluation of anti-HBV therapeutic agents.

Previous studies have demonstrated the feasibility of implantation of human blood cells or tissues in lethally irradiated mice or rats, radioprotected with SCID mouse bone marrow cells: The Trimera system. In the present study, we describe the development of a mouse Trimera model for human hepatitis B virus (HBV) infection. In this model, viremia is induced by transplantation of ex vivo HBV-infected human liver fragments. Engraftment of the human liver fragments, evaluated by hematoxylin-eosin staining and human serum albumin mRNA expression, was observed in 85% of the transplanted animals 1 month postimplantation. Viremia levels were determined in these mice by measuring serum HBV DNA using polymerase chain reaction (PCR), followed by dot-blot hybridization. HBV DNA is first detected 8 days after liver transplantation. Viremia attains a peak between days 18 and 25 when HBV infection is observed in 85% of the transplanted animals. The HBV-Trimera model was used to evaluate the therapeutic effects of human polyclonal anti-HBs antibodies (Hepatect) and of two reverse-transcriptase inhibitors, lamivudine (3TC) and beta-L-5-fluoro-2',3'-dideoxycytidine (beta-L-5FddC). Treatment of HBV-Trimera mice with these drugs effectively reduced both the percentage of infected animals and the viral load in their sera. Treatment cessation resulted in rebound of viral load, indicating HBV replication upon drug withdrawal. These results show that the HBV-Trimera model represents a novel experimental tool for simulating human HBV infection and evaluating potential anti-HBV therapeutic agents.

Human monoclonal antibodies specific to hepatitis B virus generated in a human/mouse radiation chimera: the Trimera system.

An approach to develop fully human monoclonal antibodies in a human/mouse radiation chimera, the Trimera system, is described. In this system, functional human lymphocytes are engrafted in normal strains of mice which are rendered immuno-incompetent by lethal total body irradiation followed by radioprotection with severe combined immunodeficient (SCID) mouse bone marrow. Following transplantation, human lymphocytes colonize murine lymphatic organs and secrete human immunoglobulins. We have established this system as a tool to develop fully human monoclonal antibodies, and applied it for the generation of monoclonal antibodies specific for hepatitis B virus surface antigen. A strong memory response to hepatitis B surface antigen was elicited in Trimera engrafted with lymphocytes from human donors positive for antibodies to hepatitis B surface antigen. The human specific antibody fraction in the Trimera was 10(2)-10(3)-fold higher as compared with that found in the donors. Spleens were harvested from Trimera mice showing high specific-antibody titres and cells were fused to a human-mouse heteromyeloma fusion partner. Several stable hybridoma clones were isolated and characterized. These hybridomas produce high-affinity, IgG, anti-hepatitis B surface antigen antibodies demonstrating the potential of the Trimera system for generating fully human monoclonal antibodies. The biological function and the neutralizing activity of these antibodies are currently being tested.

Cloning and characterization of the promoter for the human complement factor I (C3b/C4b inactivator) gene.

Complement factor I is a serine proteinase that regulates the classical and alternative pathways of complement by cleaving C3b and C4b and preventing the assembly of C3 and C5 convertase enzymes. In order to understand the regulation of factor I gene expression in liver cells, 4kb of the 5' flanking region of the gene was cloned, and the 1474-bp 3'-end was sequenced and shown to contain a number of transcription factor consensus sequences. A major and two minor transcription start sites were identified, respectively, at 152, 178, and 198bp upstream of the translation start site by primer extension analysis. The transcriptional activity of the 1474-bp fragment was analyzed by fusion of 5' deletion constructs to a cat-encoding gene expression vector and transient transfections into Hep G2 cells. A 273-bp fragment located at -112 to +161 relative to the major transcription start site was sufficient for promoter activity. The 3' fragment spanning +3 to +161 and containing a TATA-like element did not demonstrate promoter activity, suggesting that the core promoter resides in a 115-bp sequence located between -112 and +3. This region contains an Inr-like element overlapping the major cap site and a CTF-NF1 element, two potential CCAAT boxes and an AP-2 element partially overlapping an Sp-1 site. Thus, factor I promoter may belong to the TATA-less Inr-driven class II promoters whose transcription is regulated by Sp-1. The transcriptional activity of the 1474-bp 5' flanking fragment was upregulated by PMA, IL-6 and TNF-alpha, suggesting that factor I may be an acute phase reactant.

Thymocyte-directed enhancement of apoptosis via soluble factor(s) derived from a cortical and a medullary thymic epithelial cell line.

Apoptosis of murine thymocytes was examined either in intact fetal thymus lobes or in thymus cell suspensions, both cultured alone or in the presence of either a cortical (TEC 1.4) or a medullary (TEC 2.3) thymic epithelial cell line. Both TECs induced a pronounced increase of apoptosis in 24-h cultivated single thymus cell suspensions but not in spleen or bone marrow cell cultures. Co-culture of thymocytes with murine fibroblasts did not enhance apoptosis of the thymus cells. A similar enhancement of thymocyte apoptosis was observed with dialysed culture supernatants derived from both TEC lines, the active component(s) having a molecular weight of > 30 kDa. In contrast, the cortical TEC 1.4 had a pronounced apoptosis inducing effect on intact fetal thymus lobes cultivated for six days, whereas the medullary TEC 2.3 had only a marginal influence. TEC 1.4 also induced a significant alteration in the ratio of CD4+CD8+ to CD4-CD8- cells. It is concluded that both the cortical and medullary epithelial cell lines are able to induce thymocyte apoptosis but that a large proportion of the cells within the intact thymus stroma is refractory to the respective signal(s) of the medullary epithelial cell line.

MHC-linked colonization of the thymus and thymocyte development: effects of mature T lymphocytes.

Effects of mature T lymphocytes on thymic colonization by lymphohemopoietic cells were investigated in an in vitro experimental model, using a variety of experimental strategies. Lymphoid-depleted fetal thymus (FT) explants (C57BL/Ka, Thy1.1, H-2b) were incubated with bone marrow (BM) cells from syngeneic (C57BL/Ka; SBM) and allogeneic (BALB/c, Thy1.2, H-2d; ABM) donors. Cocultures of FT with SBM and ABM, depleted of Thy1+ or of CD3+ cells, resulted in equal proportions of lymphocytes from both BM donors. When peripheral blood lymphocytes (PBL) from synegenic or semi-allogeneic donors (F1[C57BL/Ka x C57BL/6J], Thy1.1/Thy1.2); or F1[C57BL/Ka x BALB/c], Thy1.1/Thy1.2, respectively) were added to these cultures, the total lymphocyte count per thymic lobe decreased and a developmental preference of the SBM-derived cells, as compared to the ABM-derived cells, was noted. Cells of the PBL types were also observed in the cultures. Cocultures of FT with ABM and PBL showed reduced proportions of ABM-derived cells and occurrence of cells of the PBL type. Finally, FT explants partially depleted of lymphocytes by irradiation (6 Gy), were cocultured with PBL from either syngeneic or allogeneic donors. In the presence of syngeneic PBL, the total number of cells and the proportion of double-positive (CD4+CD8+) T cells were similar to those in the FT cultured by itself, whereas in the presence of allogeneic PBL these values were reduced. The study suggests that mature T lymphocytes may play a role in the developmental processes in the thymus, and points to MHC-linked selective effects.

MHC recognition in colonization of the thymus by bone marrow cells.

The role of major histocompatibility complex (MHC) class I and II molecules in the process of colonization of the thymic microenvironment by lymphohemopoietic cells was analyzed in an in vitro experimental model. When lymphoid-depleted fetal thymus (FT) explants were cocultured with a mixture of bone marrow (BM) cells, from donors syngeneic and allogeneic to the FT, the cells syngeneic to the FT showed a developmental preference. Treatment of these cocultures with antibodies to MHC class I (H-2D, H-2K) or class II (I-E, I-A) molecules of the syngeneic cells led to preferential development of the allogeneic donor type cells. Incubation of either the FT or the BM cell inoculum with the antibodies prior to coculture indicated that the effect was exerted on the BM cells rather than on the thymic stroma.

Aging in the T lymphocyte compartment. A developmental view.

A decline in the capacity of bone marrow cells to differentiate to T lymphocytes was found when cells from young and old donors were seeded onto an alymphoid fetal thymus. A step-by-step analysis of cell-cell interactions of the lymphohemopoietic cells and the thymic stroma indicated an effect of age on a variety of cell differentiation parameters. These included a decrease in the affinity of bone marrow cells to the stroma, and in their capacity to compete with the thymic lymphoid resident cells on colonization of the thymus. There was a significant decrease in the ability of cells of old donors to replicate sequentially within the thymic microenvironment. There was a reduced capacity of bone marrow cells from aging mice to express a developmental preference after seeding onto a syngeneic fetal thymus in a mixture with cells from allogeneic donors. We addressed the question whether the aging thymus contains increased levels of immature cells that fail to differentiate in the involuted thymic microenvironment by seeding thymocytes from young and old donors onto the fetal thymic stroma. The values of T cells that developed from the old donor inoculum were lower under these conditions. Our studies suggest that at least some of the manifestations of aging in the T cell compartment are related to developmentally programmed events in the lymphohemopoietic cell compartment.

Expression of the amiloride-blockable Na+ channel by RNA from control versus aldosterone-stimulated tissue.

The amiloride-blockable Na+ channel was expressed in Xenopus oocytes injected with total RNA isolated from the toad urinary bladder. This system was used to investigate mechanisms that mediate the natriferic action of aldosterone. Incubation of the epithelium with aldosterone for 3 h doubled its channel activity but did not increase the ability of isolated RNA to express functional channels in oocytes. A 20-h incubation with the hormone produced an additional increase of Na+ transport across the intact epithelium and also augmented the channel activity expressed in oocytes by nearly 10-fold. The data are in agreement with our model that aldosterone enhances the apical Na+ permeability of tight epithelia by a short term activation of pre-existing channels, followed by chronic induction of new channel protein. Blocking methyl transfer reactions, previously shown to inhibit the natriferic action of aldosterone in tight epithelia, did not alter the basal or aldosterone-induced response in oocytes.

NaCl-dependent expression of amiloride-blockable Na+ channel in Xenopus oocytes.

RNA was isolated from chicken lower intestine (both colon and coprodeum) and injected into Xenopus oocytes. 22Na+ fluxes measured after 1-4 days demonstrated the induction of an amiloride-blockable pathway. The Na+ transporter expressed by the exogenous RNA had a high affinity to amiloride (inhibitory constant less than 0.1 microM), but was insensitive to ethylisopropyl amiloride, i.e., it is likely to be the apical Na+ channel. Functional channels were readily expressed in oocytes injected with RNA derived from chickens fed a low-NaCl diet. On the other hand, no channel activity was detected in oocytes injected with RNA isolated from chickens fed a high-NaCl diet. Thus the previously reported regulation of transport by the dietary NaCl intake involves modulations in the level of mRNA that codes either for the Na+ channel or a posttranscriptional regulator of the channel.

Quantitative analysis of bone marrow thymic progenitors in young and aged mice.

Our studies on the capacity of bone marrow (BM) to generate T lymphocytes in aging have revealed that under the competitive conditions of thymic reconstitution, cells of aged mice are significantly inferior to those of the young. The present study was designed to further investigate the basis of this age-related change. Two mechanisms were considered: (a) The potential of BM-derived T cell precursors from aged mice to proliferate and differentiate in the thymic microenvironment is impaired. (b) The frequency of T cell precursors is reduced in BM of aged mice, thus affecting their ability to compete efficiently in reconstituting the thymus. These possibilities were studied in vitro by colonizing thymocyte-depleted fetal thymic lobes with BM cells from aged (24-month) and young (3-month) C57BL/6 mice. By determining the cell cycle duration of BM-derived cells which have seeded the thymic lobes, we found that cells originating from aged mice proliferate in the thymus at the same rate as those from young mice. Reconstitution with limiting numbers of BM cells indicated that the frequency of thymic progenitors in the BM is significantly reduced in aged as compared to young mice. We thus conclude that aging is associated with a quantitative reduction in the frequency of thymic progenitors in the BM.

Selective accumulation of lymphocyte precursor cells mediated by stromal cells of hemopoietic origin.

Thymocytes were propagated in long-term cultures supported by stromal cells of both bone marrow and thymus origin. Interleukin 2 (IL-2) supplementation augmented the cell yield and allowed detailed phenotype analysis. Within 2-3 months of culture a cell population was selected in which the expression of Thy-1 antigen persisted, CD4 and CD8 antigens gradually declined, and Pgp-1 antigen, found on less than 5% of fresh thymocytes, was strongly increased. This cultured cell population (Thy-1.2 origin) contained no detectable spleen colony-forming units (CFU-S) but efficiently repopulated the thymus of Thy-1.1-irradiated congenic mice, indicating the precursor T-cell nature of the population. Upon removal from the stroma, the T cells exhibited poor cytotoxicity towards syngeneic tumor cells. Further propagation with IL-2 in the absence of stroma resulted in the acquisition of cytotoxic ability. Replacement of the horse serum used in the above experiments with fetal calf serum resulted in accumulation of cells expressing B220 antigen. This experimental model provides the means to maintain lymphocyte precursor cells in long-term culture and to further study their differentiation in the absence of stroma, both in vitro and in vivo.

Syngeneic preference manifested by thymic stroma during development of thymocytes from bone marrow cells.

The question whether major histocompatibility complex (MHC) recognition is expressed in interactions between thymocyte progenitors and thymic stroma cells was investigated in an organ culture system, in which inductive interactions between thymic stroma cells and thymocyte progenitors of different MHC haplotypes could be measured. Thymocyte-depleted fetal thymuses were reconstituted with mixtures of syngeneic and allogeneic bone marrow cells, which also differed in their Thy-1 allele. The relative repopulating ability of the cells was estimated by determining the percentage of emerging Thy-1.1+ vs. Thy-1.2+ thymocytes. Similar values of Thy-1+ cells of the bone marrow donor type developed when the thymus were reconstituted by bone marrow from donors which were either syngeneic or allogeneic to the thymic explants. However, when a 1:1 mixture of syngeneic and allogeneic cells was applied to the thymus, a syngeneic preference was manifested in development of Thy-1+ cells. When mixtures of bone marrow cells from C57BL/Ka (Thy-1.1) and B10.A MHC-congenic (Thy-1.2) mice were used, this developmental preference was found to map to the I-E region. Thymocytes derived from bone marrow cells allogeneic to the stroma, seeded on their own, manifested an advantage over allogeneic bone marrow cells from a different MHC haplotype, in a secondary reconstitution. This suggested that allogeneic bone marrow progenitor cells can be "educated" by the host thymic stroma to behave, in the competitive reconstitution, like syngeneic cells.

Age-related changes in the capacity of bone marrow cells to differentiate in thymic organ cultures.

The capacity of the bone marrow to give rise to T cells in advanced age was studied in vitro by reconstituting fetal thymic lobes from 14-day C57BL/Ka (Thy-1.1) mice with bone marrow cells from old (24-month) or young (3-month) C57BL/6 (Thy-1.2) mice. The use of these congenic strains enabled distinguishing between donor and host contribution to the developing T cells. We found that bone marrow cells from aged mice maintained their capacity to reconstitute fetal thymic explants and to differentiate into various T-cell subsets as assessed by distinct T-cell-specific surface markers (Thy-1, Lyt-1, Lyt-2, and L3T4) and functions (concanavalin A-induced proliferative and cytotoxic responses). However, when mixtures of old and young bone marrow cells reconstituted fetal thymic explants, the cells of old mice were less efficient than those of young in their capacity to give rise to T cells. These results indicate that bone marrow cells from aged mice can reconstitute the thymus and differentiate into T cells; however, their reconstituting capacity is inferior to that of bone marrow cells from young mice.

Ontogeny of T cells: development of pre-T cells from fetal liver and yolk sac in the thymus microenvironment.

The patterns of development of T cells from the very early stem cells that settle in the embryonic thymus have been studied. For this purpose, mouse embryonic thymuses (14 days) depleted of thymocytes were reconstituted with hemopoietic stem cells from fetal liver (FL) and yolk sac (YS) and T-cell development was followed in vitro in organ culture. It was found that cells derived from FL and YS of 10- to 14-day-old embryos were capable of reconstituting depleted thymic explants and exhibiting membrane markers in a pattern similar to that of thymocytes developing in intact thymic explants. Furthermore, these cells responded to concanavalin A in proliferative and cytotoxic assays as measured by limiting-dilution analysis. Thus, lymphohemopoietic stem cells emerging in the embryo prior to thymus lymphoid development are capable of differentiation in the thymus microenvironment into T cells, identified by phenotypic markers and functions that are characteristic of cells developing in the intact embryonic thymus.

Inhibition of the formation of lipid-linked intermediates in normal and transformed cells by a purified tunicamycin homologue.

The effects of a purified homologue of tunicamycin (B2-tunicamycin) on the biosynthesis of lipid-linked intermediates participating in protein glycosylation in normal embryonic fibroblasts, 3T3 and virally transformed (simian virus 40 and polyoma virus) mouse fibroblasts grown in culture were investigated. Long incubations (20 h) with the antibiotic caused a higher degree of inhibition of sugar incorporation into glycoproteins in transformed cells. However, the formation of lipid-linked intermediates was inhibited to a similar level in both cell types. When time dependent inhibition experiments were carried out using transformed cells, an earlier and stronger inhibition of the formation of lipid-oligosaccharides occurred (70% inhibition at 30 min). In 3T3 cells, prolonged incubation (6-8 h) was necessary in order to reach a similar degree of inhibition. Formation of lipid-sugar was also inhibited to a greater extent by B2-tunicamycin in transformed cells. This inhibition was not clearly time dependent. Analysis of the newly synthesized glycolipids in 3T3 and in transformed cells after B2-tunicamycin treatment have shown reduction in dolichyl-P-P-sugars as well as in other glycolipids. Dimethylsulfoxide (10%) and linoleic acid (0.5 mg/ml) markedly increased the level of tunicamycin activity in 3T3 cells while phosphatidylcholine (2 mg/ml) partially reversed it. The stronger and faster inhibition of the formation of lipid intermediates of the dolichyl-phosphate cycle caused by B2-tunicamycin in transformed cells, described here for the first time, may therefore be due to differences in penetration of the antibiotic into these cells.

Differentiation and activity of mast cells following immunization in cultures of lymph-node cells.

An extensive clonal differentiation into mast cells from primitive blast cell precursors occurred when lymph node cells obtained from mice immunized with horse serum were cultured on mouse embryonic skin monolayers. Horse serum was always present in the culture as a constituent of the nutritional medium. Mast cells developed to lesser extent also in cultures prepared from non-immunized mice. However, a clear difference in mast cell-granule ultrastructure and in histamine content was noted between the two. In cultures of lymph nodes cells from non-immunized mice the granules were tiny and uniform in size and in staining density; whereas granules in the immune cultures were larger and non-uniform in size and in staining density, and the intragranular organization manifested alterations of various forms. The content of intracellular histamine per 10(6) mast cells was about equal in both cultures. However, much more free histamine (per 10(6) mast cells) gradually accumulated in cultures of the immune lymph node cells, indicating higher rates of synthesis and release of histamine. The mast cells were readily degranulated by heat-inactivated (IgG1) sera of the mice used as donors of the lymph node cells. 92% of the mast cells were degranulated and as much as 80% of the histamine was released. The degranulation was accompanied by an immediate (albeit reversible) response of the fibroblast cells in the monolayer. A shift of the well-stretched cytoplasm of the fibroblasts opened numerous 'window' over the whole monolayer. The degranulated mast cells survived the process and could be maintained further in the cultures. Moreover, they were capable of repeated degranulation, releasing 50% of their histamine, even after four degranulation cycles performed over a 7 days' period of culture. No cytotoxic effect on the mast cells was noted and the histamine content in culture, 3 days after degranulation, seemed to be higher than in the undergranulated control cultures--suggesting an intensified rate of histamine synthesis.