Thalamo-hippocampal pathway regulates incidental memory capacity in mice.

Incidental memory can be challenged by increasing either the retention delay or the memory load. The dorsal hippocampus (dHP) appears to help with both consolidation from short-term (STM) to long-term memory (LTM), and higher memory loads, but the mechanism is not fully understood. Here we find that female mice, despite having the same STM capacity of 6 objects and higher resistance to distraction in our different object recognition task (DOT), when tested over 1 h or 24 h delays appear to transfer to LTM only 4 objects, whereas male mice have an STM capacity of 6 objects in this task. In male mice the dHP shows greater activation (as measured by c-Fos expression), whereas female mice show greater activation of the ventral midline thalamus (VMT). Optogenetic inhibition of the VMT-dHP pathway during off-line memory consolidation enables 6-object LTM retention in females, while chemogenetic VMT-activation impairs it in males. Thus, removing or enhancing sub-cortical inhibitory control over the hippocampus leads to differences in incidental memory.

Modulation of perception and brain activity by predictable trajectories of facial expressions.

People track facial expression dynamics with ease to accurately perceive distinct emotions. Although the superior temporal sulcus (STS) appears to possess mechanisms for perceiving changeable facial attributes such as expressions, the nature of the underlying neural computations is not known. Motivated by novel theoretical accounts, we hypothesized that visual and motor areas represent expressions as anticipated motion trajectories. Using magnetoencephalography, we show predictable transitions between fearful and neutral expressions (compared with scrambled and static presentations) heighten activity in visual cortex as quickly as 165 ms poststimulus onset and later (237 ms) engage fusiform gyrus, STS and premotor areas. Consistent with proposed models of biological motion representation, we suggest that visual areas predictively represent coherent facial trajectories. We show that such representations bias emotion perception of subsequent static faces, suggesting that facial movements elicit predictions that bias perception. Our findings reveal critical processes evoked in the perception of dynamic stimuli such as facial expressions, which can endow perception with temporal continuity.

Modifying interleukin-2 concentrations during culture improves function of T cells for adoptive immunotherapy.

BACKGROUND: Adoptive immunotherapy with cytotoxic T cells has shown promising clinical results in patients with metastatic melanoma and post-transplant-associated viral infections. Cell transfer therapies often require the ex vivo expansion of large numbers of reactive lymphocytes. Therefore interleukin-2 (IL-2), a potent T-cell mitogenic cytokine that critically affects the features and effectiveness of T cells, is frequently added to cell culture media. METHODS: We examined the influence of various IL-2 concentrations on cell growth, cytotoxicity, cytokine release and surface marker expression of tumor-infiltrating lymphocytes (TIL) during a standard 14-day rapid expansion phase. The study was conducted under good manufacturing practice (GMP) conditions, using approved reagents in a class 10000 laboratory. RESULTS: T-cell cultures grown in very high IL-2 concentrations (600-6000 IU/mL) expanded massively and maximally secreted interferon (IFN)-gamma in response to antigenic stimulation, but exhibited only low direct cytotoxicity. On the other hand, TIL cultures grown in low concentrations of IL-2 throughout the rapid expansion phase expanded to a lower extent and barely secreted IFN-gamma but displayed high cytotoxic activity. A combined approach of starting with 10-120 IU/mL IL-2 during the first week, followed by increasing the IL-2 concentration to 6000 IU/mL during the second week, results in T cells that expand well, maximally produce IFN-gamma and are highly cytotoxic against tumor cells. DISCUSSION: Fine tuning of the IL-2 concentration during ex vivo expansion of T cells can yield high numbers of T cells with optimal features for clinical use.

Suppressor factor secreted by T-lymphocytes from tumor-bearing mice.

Spleen cells from C57BL mice bearing the syngeneic carcinoma 3LL enhanced tumor growth. Tumor growth was also enhanced by a soluble factor found in the media of cultured spleen cells from tumor-bearing animals. This factor suppressed a protective immune response of the host and was found to be a product of T-lymphocytes. Removal of B-lymphocytes and macrophages did not prevent its appearance in the culture media, whereas removal of T-lymphocytes inhibited its appearance. Similar suppressor factors were obtained from C3H mice bearing the 3LL tumor and from mice with other tumors. The suppressing factor produced after the growth of 3LL tumor also enhanced the growth of other tumors. It could act on strains incompatible with the donor of the factor-producing cells. Hence tumor growth was possibly facilitated by soluble products of T-lymphocytes that were found in spleens of tumor-bearing mice and that nonspecifically suppressed immune defense mechanisms.

Specific cytotoxicity in vitro of lymphocytes sensitized in culture against tumor cells.

The production of tumor-specific cell-mediated cytotoxicity following in vitro sensitization of C57BL spleen cells against a syngeneic 3LL Lewis lung carcinoma was studied. Lymphocytes were sensitized on monolayers of the tumor cells for 4-5 days. The cytotoxicity was assayed by measuring the reduction in 3H-leucine and 3H-thymidine incorporation by target cells after interaction with the sensitized lymphocytes. Spleen lymphocytes sensitized on monolayers of 3LL tumor cells caused a high extent of lysis; such cells tested on C57BL or C3H fibroblast targets evoked only a low level of cytotoxicity. C57BL spleen cells sensitized on C57BL fibroblasts caused a low level of cytotoxicity when tested on a 3LL target. Thus cytotoxicity appeared to be tumor specific. The reduced incorporation into protein and DNA of target tumor cells caused by the sensitized lymphocytes was a measure of cell injury, which was more sensitive than direct cell count or uptake of 51CR. Lymphocytes from syngeneic tumor-bearing mice, tested 13-25 days after tumor inoculation, did not manifest in vitro cytotoxicity. On the contrary, such lymphocytes sometimes appeared to have a promoting effect on the tumor cells.

Immunotherapy of lethal metastases by lymphocytes sensitized against tumor cells in vitro.

To learn whether tumor metastases can be prevented by the immune system, we developed a model for the treatment of mice with syngeneic lymphocytes sensitized against tumor cells in vitro. Mice were given subcutaneously tumor cells that spontaneously metastasized to the lungs. The tumors developing locally were surgically removed and the mice were inoculated with sensitized lymphocytes 1 day later. Prevention of death by lung metastases was the measure of immunotherapy. Only approximately equal to 30-40 percent of mice receiving control treatment survived, whereas approximately equal to 70 percent survived that received lymphocytes sensitized in vitro against the tumor cells. Hence sensitization of syngeneic lymphocytes against tumor cells in vitro and injection of the lymphocytes into the host after removal of a local tumor prevented the development of lethal metastases.

Macrophage-mediated in vitro sensitization of T-lymphocytes. I; Detection of murine leukemia virus-associated antigens.

Cytotoxic effector T-lymphocytes were produced in vitro by sensitization of spleen cells on monolayers of syngeneic macrophages that had been fed with radiation leukemia virus-containing cell extracts or with supernatants of virus-producing cell cultures. The sensitized lymphocytes were cytotoxic to cell lines that expressed viral antigens. Secondary mouse embryo fibroblasts were little affected. Sensitization via macrophages appeared to be a useful system for identification of viral antigens on surfaces of various target cells, as well as for tests of the protective effect of such lymphocytes against tumor growth in vivo.

Spontaneous commitment of murine erythroleukemic cells to terminal differentiation.

Differentiation in murine erythroleukemic cells is arrested at the proerythroblast stage. A small fraction of the population, however, undergoes spontaneous differentiation. This spontaneous differentiation was examined at the individual cell level in relation to cell multiplication, commitment, and maturation. The results indicate that murine erythroleukemic cells destined to undergo spontaneous differentiation first undergo commitment, an irreversible process characterized by cells becoming (a) capable of producing hemoglobin coupled with (b) a loss of their ability to undergo more than six subsequent cell divisions. Commitment is followed by a maturation process which includes the accumulation of erythroid specific markers, e.g., hemoglobin. With respect to commitment, spontaneous differentiation resembles the differentiation produced by most inducers but differs from that evoked by hemin. Serum hemin may therefore be exempt from implication in the spontaneous process. 12-O-Tetradecanoylphorbol-13-acetate, which is known to inhibit murine erythroleukemic cell differentiation, was found to exert its inhibitory effect on both the commitment and maturation steps of spontaneous differentiation. The results further indicate that cells become committed mainly during the logarithmic rather than the stationary phase of the growth cycle. Once committed, however, the cells mature during both the logarithmic and stationary phases. When logarithmic growth was maintained continuously, the rate of the spontaneous differentiation increased (20- to 100-fold) due to the higher probability of cell commitment. A steady state culture was obtained in which the rates of cell multiplication, initiation of commitment, and maturation remained constant.

Recombinant human interleukin-1 suppresses lipoprotein lipase activity, but not expression of lipoprotein lipase mRNA in mesenchymal rat heart cell cultures.

The effect of human recombinant interleukin-1 (IL-1) on the regulation of lipoprotein lipase (LPL) was studied in rat heart mesenchymal cell cultures. A time-dependent reduction in enzyme activity occurred with a 30% fall after 1 h. The suppression of enzyme activity was accompanied by a commensurate reduction in enzyme mass. The reduction in LPL activity was most prominent in the heparin releasable pool; IL-1 treatment resulted in a 7.2-8.3-fold decrease in the functional compartment and a 2.5-2.8-fold decrease in residual cellular activity. The effect of IL-1 could be prevented by the addition of the IL-1 inhibitor. However, in contradistinction to the effect of tumor necrosis factor (TNF), there was no change in LPL mRNA in cultures treated with IL-1. The present results show that the regulation of LPL in mesenchymal heart cell cultures by IL-1 occurs posttranscriptionally, as has been shown in 3T3 cells. The more pronounced effect on LPL activity in the functional pool suggests that IL-1 treatment might have influenced also the processing and/or transport of the enzyme to the cell surface.

Human monocytes and macrophages: establishment and analysis of cloned populations and functional cell lines.

Human monocytes and macrophages are heterogeneous cell populations, with numerous functions and activities. In the present work we review some new in vitro studies which made possible the development and research of cloned populations of macrophages and functional cell lines. Peripheral blood monocytes were cloned in semisolid media and the cloned monocyte/macrophage populations were analyzed for the expression of various markers. Based on the analysis of cloned populations of monocytes and on additional studies of macrophage functions and markers, we suggest the possibility that the human monocytes represent a unique multifunctional population, expressing reversible changes in functions and markers. The occurrence of stable subsets of cells could not be demonstrated by these methods. Cultures of human monocytes and mature macrophages could be maintained in vitro for long periods of time, but only a few studies reported the continuous proliferation of monocytes in culture. We describe here an approach for immortalization of monocytes by heterologous somatic cell hybridization. This method yielded monocyte hybrid cell lines which preserved some macrophage characteristics. The hybrid cell lines could be used for the isolation and study of monokines and for cytogenetic analysis of macrophage markers and functions.

Marker analysis of cloned populations of human monocytes.

The presence of myelomonocytic progenitor cells in human peripheral blood was used for the analysis of cloned populations of human monocytes. Colonies of granulocytes and macrophages were obtained by plating peripheral blood mononuclear cells (PBM) in methylcellulose containing medium in the presence of medium conditioned by nonstimulated PBM (CM). Following 20-25 days of incubation, most colonies were found to consist of cells with monocyte-macrophage morphology. Cloned populations of monocytes were tested for several monocyte membrane markers and compared to noncloned adherent monocytes. HLA-DR, 63D3, LeuM2 antigens and Fc receptors were expressed on cells from individual colonies in similar proportions to their expression on noncloned monocytes. Some colonies were uniform in their negative expression of the 63D3 antigen, as were the noncloned monocytes. Although the clonality of cells tested was not directly proven, these results indicated that at least for some monocyte markers, heterogeneous expression was obtained in monoclonal populations of monocytes. It is possible, however, that testing of additional markers and functions may reveal homogeneous clones of monocytes and suggest the existence of stable subsets.

A new myelomonoblastic cell line (M20): analysis of properties, differentiation, and comparison with other established lines of similar origin.

A new myelomonoblastic cell line (M20) was established from the peripheral blood of a ten-year-old child with acute myeloblastic leukemia, using an improved method for supporting the initial stages of cell proliferation. The addition of irradiated macrophage monolayers to the proliferating cells appeared to overcome the deterioration of the primary cultures and enable them to continue proliferating until they became independent of this environment. The cell line that developed consisted of myeloblasts and promyelocytes characterized by light and scanning electron microscopy, cytochemistry, and enzymatic activities. The cells expressed Fc receptors and WT1 antigens but did not exhibit HLA-DR, HMA1, Epstein-Barr virus nuclear antigen, and surface Ig. The M20 cells produced colonies when cultured in semisolid medium and secreted lysozyme, prostaglandin E2, and interleukin 1. An attempt was also made to analyse the position of the M20 cells in the scheme of differentiation of the myelomonocytic lineage using different approaches. Treatment of the cells with 12-O-tetradecanoyl phorbol 13-acetate induced their adherence to plastic surfaces and partial maturation to macrophages as judged by morphological criteria, cytochemistry, and enzyme activities. However, comparison of the M20 cells to other well-established myelomonoblastic cell lines did not reveal any pattern suggesting a possible relationship between surface markers, cell function, and differentiation pathway of the various cell lines tested. Establishment of additional cell lines and identification of new markers may assist in defining the mechanisms involved in normal differentiation and malignant transformation of this cell lineage. In addition, such cell lines may also provide a tool for the quantitative recovery of a variety of monokines.

Primary cultures of human spleen macrophages in vitro.

A method for establishing and maintaining primary cultures of human spleen macrophages is described. The cultured cells have been characterized as macrophages by virtue of their adherence, morphology, phagocyte activity, surface Fc receptor, and the production of lysozyme.

The M20 IL-1 inhibitor. I. Purification by preparative isoelectric focusing in free solution.

An interleukin-1 (IL-1) inhibitor produced by the M20 myelomonocytic cell line has been shown to be active in various in vitro and in vivo IL-1 induced parameters. This inhibitor has been purified from the conditioned medium by gel filtration through a Sephacryl S-300 column or dye ligand chromatography on Affi-Gel blue column, followed by isoelectric focusing in free solution in the pH range 3-5 using the Rotofor cell. When gel filtration by FPLC with the Superose 12 column was used as the final step, the combined sequence of purification procedures resulted in a 1600-fold purification of the IL-1 inhibitor. The purified IL-1 inhibitor has a molecular weight of approximately 52 +/- 4 kDa and a pI of 4.15 +/- 0.1. By SDS-PAGE analysis the inhibitor preparation thus obtained showed the presence of two protein bands, while a few closely spaced protein bands were seen by analytical isoelectric focusing in polyacrylamide gels (pH 3-6). Some of these bands in PAGIF might correspond to different degrees of glycosylation of the inhibitory protein. Although the M20 IL-1 inhibitor has not yet been purified to homogeneity, it should be stressed that the procedures used, allowed us to remove the great majority of the proteins present in the medium in which the M20 cells were cultured, and to recover in satisfactory yield the inhibitor which we consider likely to be present in the conditioned medium in subnanomolar concentrations.

The M20 IL-1 inhibitor. II. Biological characterization.

Interleukin-1 (IL-1) is an important mediator in inflammation and immunological processes. The findings of native IL-1 inhibitors suggest a negative feedback mechanism to down-regulate IL-1 mediated acute inflammation. IL-1 inhibitors were also found elevated in disease states associated with high IL-1 levels. We have previously described one such IL-1 inhibitor derived from the human M20 myelomonocytic cell line. In this paper we present several biological and biochemical characteristics of the M20 IL-1 inhibitor. Various in vitro activities of the inhibitor are described and its IL-1 specificity in these assays is demonstrated. Purification of the inhibitor was performed by DEAE-high performance liquid chromatography, isoelectric focusing, gel filtration and dye ligand chromatography column. This protein factor has a MW of 52 +/- 4 kDa and a pI of 4.15 +/- 0.1. The inhibitor has no cross-reactivity against a panel of known cytokines (IL-1 alpha, IL-1 beta, IL-2, sIL-2R, IL-6, tumor necrosis factor (TNF), interferon-gamma (IFN-gamma)) and is distinct from the IL-1 receptor antagonist (IL-1ra). The purified IL-1 inhibitor was destroyed by trypsin, 2-mercaptoethanol, sodium dodecyl sulfate and extremes in pH and in temperature. Only IL-1 induced (but not the IL-2, IL-6 or TNF induced) thymocyte proliferation and PGE2 production by fibroblasts were inhibited by the inhibitor, thus showing specificity to IL-1 in these assays.

The isolation and purification of human peripheral blood monocytes in cell suspension.

A simple method for the isolation of human peripheral blood monocytes in cell suspension is described. The method is based on the adherence and detachment characteristics of monocytes during their in vitro cultivation and does not require any gradient centrifugation or modulating substances. The mean percentage of monocytes recovered from different donors was 81.9% (60-100%) of the original phagocytic cells found in the peripheral blood of the tested donors and the cell suspensions obtained consisted of more than 95% of monocytes. The monocytes isolated in suspensions show high activity in various monocyte functions tested.

Development of macrophage and granulocyte colonies from human peripheral blood.

The presence of macrophage and granulocyte progenitor cells in the human peripheral blood enabled the establishment of colonies from this accessible tissue and obviated the need for bone marrow to achieve this task. We have developed a method of obtaining reproducible growth of macrophage/granulocyte colonies from human peripheral blood. Colonies of macrophages and granulocytes were obtained by plating peripheral blood mononuclear cells (PBM) in methylcellulose containing medium in the presence of medium conditioned by nonstimulated PBM (CM). At early stages of colony growth both macrophage and granulocyte colonies were detected while following 20-25 days in culture all colonies tested revealed monocyte-macrophage morphology. To obtain higher numbers of colonies, we tested different cell sources, different CM preparations and the effect of steroid hormones on colony development. We found that the mononuclear cells obtained from cord blood (CB) or from some patients with inflammatory bowel disease yielded much higher numbers of colonies than PBM from normal individuals. Colony development from these two sources did not depend on an external source of colony stimulating factor (CSF) but was augmented as a result of CSF supplementation. CM obtained from CB mononuclear cells as well as supernatants from some human monoblastic cell lines were similar in their CSF activity to CM from normal PBM and made possible the development of macrophage/granulocyte colonies. Higher numbers of colonies were induced by including physiological concentrations of estradiol in the culture medium, in the absence of external sources of CSF. The system described above enabled the analysis of cloned macrophages and their circulating progenitor cells as well as the assay of different preparations of CSF.

Transition from T cell protection to T cell enhancement during tumor growth in an allogeneic host.

The cell-mediated immune response of mice toward a lethal allogeneic tumor was investigated during tumor development. The activity of spleen cells from the tumor-bearing mice was studied by transferring them together with 3LL tumor cells into normal C3H/eb recipient mice. The activity depended upon the time interval between inoculation of the tumor and transfer. Spleen cells taken relatively early, 1 week after tumor inoculation, mediated protection against tumor growth. In contrast, spleen cells taken 4 weeks after tumor inoculation markedly enhanced tumor growth. The tumor-enhancing cells, like the tumor-protecting cells, appeared to be T lymphocytes. The enhancing activity could be transferred by extra cellular medium prepared by incubating the enhancing T cells. Protecting activity could not be transferred by cell-free medium prepared from the protecting T cells. Both activities were found to exist to a relatively slight degree in populations of spleen cells from normal mice. The transition from T cell protection to T cell enhancement might be a determining factor in the outcome of the host-tumor relationship.

Correlations and the encoding of information in the nervous system.

Is the information transmitted by an ensemble of neurons determined solely by the number of spikes fired by each cell, or do correlations in the emission of action potentials also play a significant role? We derive a simple formula which enables this question to be answered rigorously for short time-scales. The formula quantifies the corrections to the instantaneous information rate which result from correlations in spike emission between pairs of neurons. The mutual information that the ensemble of neurons conveys about external stimuli can thus be broken down into firing rate and correlation components. This analysis provides fundamental constraints upon the nature of information coding, showing that over short time-scales correlations cannot dominate information representation, that stimulus-independent correlations may lead to synergy (where the neurons together convey more information than they would if they were considered independently), but that only certain combinations of the different sources of correlation result in significant synergy rather than in redundancy or in negligible effects. This analysis leads to a new quantification procedure which is directly applicable to simultaneous multiple neuron recordings.