Stimulatory and inhibitory activities of lung-conditioned medium on the growth of normal and neoplastic cells in vitro.

Lung-conditioned medium (LCM) was obtained by incubation of BALB/c mouse lung tissue fragments in serum-free Eagle medium for 48 hours and subsequent separation by dialysis or chromatography on a Sephadex G-75 column. LCM fractions were tested for their ability to modulate proliferation of normal human endothelial cells and neoplastic cells (N2a, MCF, HEp-2) in vitro as assessed by plating efficiency and tritiated thymidine incorporation assays. It was found that LCM contained two kinds of factors, either stimulating (molecular weight: 50,000-70,000) or inhibiting (molecular weight: 12,000-20,000 and 3,000-5,000) cell proliferation.

Killing of skin-derived tumor cells by mouse dendritic epidermal T-cells.

Dendritic epidermal T-cells (DETC) are a unique population of T-cells that reside normally in mouse epidermis and express a gamma delta T-cell receptor. We have reported previously that DETC acquire in culture the capacity to lyse the YAC-1 lymphoma, a conventional target for natural killer cells. The aim of the present study was to characterize this cytotoxic potential, using a spectrum of skin-derived mouse tumors. Cytotoxicity was measured by a 51Cr release assay and by the visual assessment of target cell lysis. Long-term DETC lines, established from CBA, AKR, and BALB/c mice by mitogenic stimulation and repeated feeding with interleukin 2 (5 units/ml), were used as effectors. Skin-derived tumor targets included 5 melanoma lines and the transformed keratinocyte line Pam 212. Each DETC line lysed skin-derived tumors as well as YAC-1 targets effectively in the 18-h 51Cr release assay, and target lysis occurred in a non-major histocompatibility complex-restricted manner. By contrast, freshly isolated spleen cells lysed YAC-1 but not skin tumor targets. Moreover, confluent monolayers of melanoma or Pam 212 targets were disrupted completely by added DETC lines but not by spleen cells. The cytolytic activity of DETC appeared to be specific for tumor cells, since normal mouse keratinocyte monolayers remained intact under the same conditions. Finally, DETC freshly isolated from skin failed to exhibit significant cytotoxicity but acquired this capacity 10-14 days after mitogenic stimulation and feeding with interleukin 2 (5 units/ml). We conclude that DETC possess the potential to recognize, bind, and lyse tumor cells that originate in skin.

Lowered angiogeneic capability of peripheral blood lymphocytes in progressive systemic sclerosis (scleroderma).

Peripheral blood lymphocytes isolated from 19 patients with progressive systemic sclerosis (7 with diffuse scleroderma and 12 with CREST syndrome) and from 19 healthy control individuals were tested in a lymphocyte-induced angiogenesis assay. The cells were injected intradermally into x-ray-immunosuppressed mice and their capability to induce new blood vessel formation was assessed by morphologic criteria. The lymphocytes derived from patients with systemic scleroderma showed a significant decrease in angiogeneic capability compared with controls. No significant difference in this capability was found between patients with diffuse scleroderma and those with CREST syndrome. The decrease in the angiogeneic capability of lymphocytes reflects a depression in cell-mediated immunity and might be relevant to the capillary loss observed in systemic scleroderma.

Organ and age distribution of natural anti-chondrocyte cytotoxicity in the mouse.

Natural cell-mediated cytotoxic activity of mouse lymphoid cells against isolated epiphyseal chondrocytes was tested in 18 h chromium-release assay. The highest anti-chondrocyte cytotoxicity was demonstrated with spleen- and peritoneum-derived leukocytes. Bone marrow cells and lymph node cells exerted weak activity while thymocytes and Peyer's patch-derived cells exerted no cytotoxic activity. Slight, but significant, activity appeared in spleen of 1-week-old mice. It increased with age reaching the maximum in about the 8th week which was followed by subsequent decrease. The ability to lyse isolated chondrocytes by peritoneal cells appeared in 3-6th week of life. It reached the maximum on about the 8th week and disappeared completely in about the 12th week. On the contrary, there were no changes in anti-chondrocyte activity of bone marrow cells.

Heterotopic transplantation of syngeneic lymphoid tissues in mice. The ability of graft repopulation cells to enhance cell-mediated immunity as measured in the lymphocyte-induced angiogenesis assay.

Fragments of murine spleen and Peyer's patches were transplanted under the kidney capsule of syngeneic recipients. The ability of graft-repopulating cells to enhance cell-mediated immunity was tested in a lymphocyte-induced angiogenesis assay. The ability to evoke angiogenesis expressed by cells isolated from 10-day-old grafts of both tissues was significantly lower than that of cells isolated from organs in situ and was restored to control levels in both types of grafts 30 days after transplantation. Cells of Peyer's patch grafts showed weaker ability to induce angiogenesis than cells of spleen grafts regardless of the graft's age. Cells isolated from Peyer's patches in situ also showed weaker angiogenesis-inducing ability compared with normal spleen cells. Hence the results suggest that the repopulation of lymphoid tissue graft is specifically influenced by the graft's microenvironment.

Grafting of isolated allogeneic keratocytes in the treatment of experimental defects in the rabbit cornea.

Keratocytes isolated by enzymatic digestion from the rabbit cornea propria were suspended in rabbit plasma clot and then transplanted into deep, experimental defects of the allogeneic recipients cornea. This procedure resulted in improved healing and transparency of the cornea observed up to two years after operation.

Immunocompetence of splenocytes from mice bearing different-aged tumors assessed in lymphocyte-induced angiogenesis assay.

Inbred Balb/c mice bearing 2- or 4-week-old transplantable syngeneic sarcoma L-1, when injected intravenously with the cells of the same tumor, displayed a low or high number of tumor nodules in the lungs, two weeks after the injection. In order to test whether this phenomenon is dependent on changes in host immunity associated with tumor progression, the immunocompetence of spleen cells from tumor-bearing mice was measured in a lymphocyte-induced angiogenesis assay, performed on (C57Bl/Sn X Balb/c)F1 recipients. Splenocytes from mice bearing tumors of different age, whether treated or not with cyclophosphamide, showed, however, the same angiogeneic potency.

Skin disorders associated with rheumatic disease.

Skin manifestations of rheumatic diseases can include a spectrum of changes, ranging from purpura to ulcers. An understanding of the possible causes of these various dermatologic manifestations and a close cooperation between rheumatologist and dermatologist result in prompt diagnosis and treatment.

A new method of the description of the information flow in the brain structures.

The paper describes the method of determining direction and frequency content of the brain activity flow. The method was formulated in the framework of the AR model. The transfer function matrix was found for multichannel EEG process. Elements of this matrix, properly normalized, appeared to be good estimators of the propagation direction and spectral properties of the investigated signals. Simulation experiments have shown that the estimator proposed by us unequivocally reveals the direction of the signal flow and is able to distinguish between direct and indirect transfer of information. The method was applied to the signals recorded in the brain structures of the experimental animals and also to the human normal and epileptic EEG. The sensitivity of the method and its usefulness in the neurological and clinical applications was demonstrated.

Presence of natural killer cells in Peyer's patches of the mouse.

Natural cell-mediated cytotoxicity of normal murine Peyer's patch cells against Ehrlich ascites carcinoma was found in a short-term 51Cr release assay. Peyer's patch and lymph node cells showed natural cytotoxicity at approximately the same level.

In vivo activation of mouse dendritic epidermal T cells in sites of contact dermatitis.

Adult mouse epidermis contains a population of dendritic, Thy-1+, CD3+, CD4-, CD8- and T cell receptor (TcR) V gamma 3/V delta 1+ leukocytes termed dendritic epidermal T cells (DETC). DETC isolated from skin and placed into culture will proliferative vigorously in response to T cell mitogens and T cell growth factors. In the present study, we examined whether DETC can be activated in situ by modulating their epidermal environment. Ear skin of CBA mice was painted with the chemical irritant, croton oil, and the epidermal cells (EC) isolated from such sites were then tested for proliferative responses to exogenous interleukin-2 (IL-2), in the absence of added mitogens. Cells from croton oil-treated skin showed marked IL-2 responsiveness, whereas cells from phosphate-buffered saline-treated skin failed to proliferate. IL-2 responses were seen as early as 2 days after croton oil treatment and peaked between days 5 and 10. gamma delta TcR-bearing cells, most likely resident DETC, were the major population to respond to IL-2, since depletion of gamma delta TcR+ cells, but not alpha beta TcR+ cells, abolished that responsiveness, and since gamma delta TcR+ cell numbers increased markedly in the cultures that contained added IL-2. These results indicate that DETC in normal skin, which are at a state of rest, may be activated when their residential epidermal environment is disrupted externally. This process of DETC activation may be a critical step in the maturation of DETC into effector leukocytes in vivo.

Analysis of mesial temporal seizure onset and propagation using the directed transfer function method.

The directed transfer function (DTF) method, a multichannel parametric method of analysis based on an autoregressive model, is a newly developed tool that permits determination of patterns of flow of activity. The DTF method of analysis was applied to seizures originating from mesial temporal lobe structures in 3 patients recorded by combined subdural grid and depth electrode arrays. These first applications to human intracranial recordings demonstrated that the DTF method can accurately determine patterns of seizure onset and propagation. In addition the DTF method can provide evidence regarding patterns of flow of seizure activity that are not readily apparent from visual inspection of the EEG recordings. Important considerations for appropriate application of the DTF method for the analysis of intracranial ictal recordings are discussed.

Natural cell-mediated cytotoxic activity against isolated chondrocytes in the mouse.

The ability of lymphoid cells from normal mice to exert a natural cytotoxic activity against isolated syngeneic and allogeneic epiphyseal chondrocytes was studied by means of 51Cr release assay. We found, that both spleen and peritoneal cells, but not thymocytes, exerted an anti-chondrocyte cytotoxic effect. Addition of unlabelled chondrocytes markedly reduced experimental 51Cr release and the inhibitory effect was proportional to the number of 'cold' cells added. This indicate, that chondrocyte lysis was due to specific effector-target interaction. As no cytotoxicity was observed against isolated fibroblasts, our results could not be explained by lysis of fibroblasts contaminating chondrocyte cultures.

Angiogenesis-inducing ability of human bladder epithelium cell lines and "spontaneously" transformed murine fibroblasts.

Ten human bladder epithelium cell lines were tested for their ability to induce blood vessel formation after intradermal injection into irradiated ST/a mice. Cell lines that were shown to be tumorigenic in nude mice, were able to evoke angiogenesis of a higher intensity than nontumorigenic cell lines. No difference was observed between the angiogeneic ability of tumorigenic cells originating from tumors and from in vitro transformed urothelium of nontumor origin. Similarly the origin of nontumorigenic urothelial cell lines did not show any influence on their angiogeneic abilities, but nontumorigenic cell lines which had undergone "infinite growth transformation" exhibited a higher angiogeneic activity than nontumorigenic cell lines with a finite life. The angiogeneic reaction evoked by human bladder epithelium cell lines showed cell dose- and time-dependence; but it was unrelated to the growth potential of the cultured cells. Two "spontaneously" altered sarcoma-producing murine cell lines showed a higher angiogeneic activity than tumorigenic human bladder epithelial cells. The angiogeneic response to these two murine cell lines was unrelated to morphological signs of transformation and to differences in growth rate, serum requirement, saturation density, anchorage dependence, and isoimmunizing properties.

Cultured brain endothelium inhibits the cytocidal action of natural killer cells on glioma.

The killing of GL26 and YAC-1 cells by natural killer cells (NKC) is reduced in the presence of a monolayer of endothelial cells. This reduction in cytotoxicity correlates with the degree of adhesion between the tumor cells and the endothelial monolayers. The cytotoxicity of NKC toward glioma was 10% when carried out on plastic, but a monolayer of endothelium derived from brain inhibited the cytotoxicity by about 90%. Endothelium from thoracic duct and lung also inhibited cytotoxicity by about 90%, endothelium from aorta inhibited by 55% and that from ovary by only 45%. Cytotoxicity of NKC toward YAC-1 (a control NK target) was 40% on plastic, but a monolayer of endothelium from thoracic duct inhibited the cytotoxicity by 75%. Endothelium from brain and lung inhibited cytotoxicity by about 60%, aorta by 50%, and ovary by 40%. Interactions between tumor cells and the host-organ microvascular endothelium appear to protect neoplastic cells from natural surveillance mechanisms and may play a role in the formation of metastatic tumor deposits.

Dendritic epidermal gamma/delta T cells (DETC) activated in vivo proliferate in vitro in response to Mycobacterium leprae antigens.

BACKGROUND: gamma/delta T-cell receptor (TCR)+ dendritic epidermal T cells (DETC) are part of a primitive defense system in the skin; they are capable of responding only to a limited number of antigens. The aim of the present study was to test whether DETC can proliferate in vitro in response to antigens of Mycobacterium leprae. METHODS: DETC were obtained from CBA mouse ear skin by trypsinization and Histopaque gradient centrifugation. The resulting epidermal cell suspension contained up to 20% DETC, as analyzed by the fluorescence activated cell sorter (FACS) after staining with anti-Thy-1 or anti-gamma/delta TCR monoclonal antibodies (mAbs). The freshly isolated cells, or DETC cultured up to 4 weeks with interleukin-2 (IL-2), were exposed in vitro for up to 6 days to varying doses of the following M. leprae antigens: (1) integral (live) M. leprae bacilli; (2) Dharmendra antigen; and (3) PGL-1 (phenolic glycolipid of M. leprae). The DETC response was assessed by tritiated thymidine (3H-TdR) incorporation. RESULTS: The freshly isolated DETC, or DETC cultured up to 4 weeks with IL-2, did not respond significantly to any of the M. leprae antigens, although at the same time they were able to respond vigorously to concanavalin A (Con A), as positive control. If, however, DETC were isolated from skin, painted 7 days before with croton oil (10 microL/cm2 to cause irritant dermatitis, they were able to respond to all M. leprae antigens by a 3-4-fold incrase in the 3H-TdR uptake. The most effective stimulator was a 1 : 1 mixture of Dharmendra and PGL-1 (0. 01 microg/mL), which was as effective as 10-fold higher doses of either antigen alone. Cell counts confirmed that increased DNA synthesis was associated with cell proliferation. Experiments employing alpha/beta-TCR CBA murine spleen cells and epidermal cell suspension treated with anti-gamma/delta or antialpha/beta mAbs + C' proved that only the gamma/delta DETC were the responder cells to M. leprae antigens. CONCLUSIONS: The results suggest that activation of DETC in vivo may make them responsive to M. leprae antigens. A significant increase in the number of class II major histocompatibility complex (MHC) positive, nondendritic cells was observed in the croton oil-treated epidermis. We hypothesize that croson oil-induced upregulation of class II MHC expression, which endows epidermal cells with antigen-presenting capabilities, might be an important factor in vivo in delivering an immunogenic signal to resident DETC in the skin.

Human peripheral blood T lymphocyte subpopulations isolated on the basis of their affinity for sheep red blood cells differ in angiogenesis-inducing capability.

Human peripheral blood lymphocytes injected intradermally into X-ray immunosuppressed mice were tested for angiogenesis-inducing capacity. Both T and B lymphocytes evoked angiogenesis of the same intensity. The total T cell population was fractionated into three subpopulations on the basis of their different affinities for sheep red blood cells (SRBC). Cells belonging to the subpopulation of T lymphocytes displaying moderate affinity for SRBC induced angiogenesis of the higher intensity, higher than that induced by cells of the total T lymphocyte population. However, lymphocytes both with the highest and with moderate affinity for SRBC, mixed together, evoked angiogenesis no different from that evoked by cells of the total T lymphocyte population, suggesting that inhibitory interactions occur among T cells.

Protease-mediated enhancement of lymphocyte-induced angiogenesis in X-ray irradiated mice.

Angiogenesis was induced in mice by intradermal injection of semi-syngeneic splenocytes, and after three days the number of newly formed blood vessels at the injection site was counted. When recipients were total-body irradiated with 700 R 2 hours before the lymphocyte injection, the angiogenesis was significantly higher than in non-irradiated mice. The angiogenesis enhancement was of a systemic (not local) character as revealed in experiments with shielding of irradiated animals. This enhancement was not due to X-ray dependent immunosuppression, as shown in experiments with non-irradiated, pharmacologically immunosuppressed mice. Decreased angiogenesis was observed in irradiated mice after treatment with cortisone acetate, aprotinin, and EACA. The results suggest that proteases might be involved in mediating the angiogenesis enhancement after X-irradiation.