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.

The role of homophilic binding in anti-tumor antibody R24 recognition of molecular surfaces. Demonstration of an intermolecular beta-sheet interaction between vh domains.

The murine antibody R24 and mouse-human Fv-IgG1(kappa) chimeric antibody chR24 are specific for the cell-surface tumor antigen disialoganglioside GD3. X-ray diffraction and surface plasmon resonance experiments have been employed to study the mechanism of "homophilic binding," in which molecules of R24 recognize and bind to other molecules of R24 though their heavy chain variable domains. R24 exhibits strong binding to liposomes containing disialoganglioside GD3; however, the kinetics are unusual in that saturation of binding is not observed. The binding of chR24 to GD3-bearing liposomes is significantly weaker, suggesting that cooperative interactions involving antibody constant regions contribute to R24 binding of membrane-bound GD3. The crystal structures of the Fabs from R24 and chR24 reveal the mechanism for homophilic binding and confirm that the homophilic and antigen-binding idiotopes are distinct. The homophilic binding idiotope is formed largely by an anti-parallel beta-sheet dimerization between the H2 complementarity determining region (CDR) loops of two Fabs, while the antigen-binding idiotope is a pocket formed by the three CDR loops on the heavy chain. The formation of homophilic dimers requires the presence of a canonical conformation for the H2 CDR in conjunction with participation of side chains. The relative positions of the homophilic and antigen-binding sites allows for a lattice of GD3-specific antibodies to be constructed, which is stabilized by the presence of the cell membrane. This model provides for the selective recognition by R24 of cells that overexpress GD3 on the cell surface.

Psoriasis: pathophysiology and oral manifestations.

Psoriasis is a chronic, remitting and relapsing inflammatory skin disorder with a strong genetic predisposition. Psoriasis affects 1-3% of the world's population in their early lives representing a disabling condition with significant social and economic impact. Despite a great deal of research on the etiology and tissue destruction mechanisms, the disease is not well understood. The purpose of this paper is to provide current information from the literature with a special focus on oral manifestations. The major signs and symptoms presented in the oral environment of a psoriasis patient may include geographic tongue, fissure tongue, gingival and/or mucosal lesions. Inflammatory temporomandibular joint lesions have been reported in less than 5% of psoriasis patients. Multiple treatment strategies, be they topical or systemic, have been applied to these patients for symptom relief but not for cure.

Does laparoscopy adversely affect gas exchange and pulmonary mechanics in the newborn? An experimental study.

Notwithstanding the clinical acceptability of pediatric laparoscopy, the physiological consequences of carbon dioxide (CO(2)) peritoneal insufflation on lung mechanics and gas exchange are not established. This study seeks to establish the affects of increased intraabdominal pressure (P ABD) from CO(2) insufflation on lung mechanics, the compliance of the respiratory system (CRS stat), and gas exchange. Ten anesthetized tracheostomized, and paralyzed rabbits (2.5-3.1 kg) were examined. Mechanical ventilation was established to produce normal ventilation (PaCO(2) 35-45 Torr). Continuous pulse, blood pressure (BP), and serial arterial blood gas measurements were obtained by femoral arteriotomy. The animals were examined at baseline conditions (P ABD = 0 mm Hg), at 2 levels of external compression (P ABD = 6 and 12 mm Hg, respectively), and at two levels of intraperitoneal CO(2) insufflation (P ABD=6 and 12 mm Hg, respectively). Increased P ABD, whether applied externally or by intraperitoneal insufflation, did not alter the CRS stat (ANOVA p = 0.557), nor systematically change the pH, PaCO(2) or PaO(2) (ANOVA p = 0.541, p = 0.545, p = 0.446, respectively). Neither BP nor pulse rate showed any change. Under conditions encountered during laparoscopy, P ABD of 0-12 mm Hg did not have a deleterious effect on ventilation and gas exchange in the ventilated and paralyzed small animal.

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.

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.

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.

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.

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.

Studies of bronchoalveolar lavage cells and fluids in pulmonary sarcoidosis. I. Enhanced capacity of bronchoalveolar lavage cells from patients with pulmonary sarcoidosis to induce angiogenesis in vivo.

Both allogeneic immunocompetent CD4+ lymphocytes and activated macrophages of mice can induce neovascularization when inoculated intradermally into host animals. Because sarcoidosis is associated with an increase in both activated macrophages and CD4+ effector lymphocytes in the lung, we carried out experiments in which cells obtained by bronchoalveolar lavage (BAL) of patients with pulmonary sarcoidosis were tested in a murine intradermal angiogenesis assay. BAL cells from patients with pulmonary sarcoidosis induced a significantly greater degree of angiogenesis than those from normal volunteers or from patients with other lung diseases. Moreover, the degree of angiogenesis induced by BAL cells from patients with sarcoidosis correlated positively with the severity of the disease. When BAL cells were separated into macrophage and lymphocyte subpopulations by flow cytometric techniques, the observed angiogenic activity was restricted primarily or exclusively to macrophages; lymphocytes were unable to induce angiogenesis in this xenogeneic assay system. These experiments suggest that pulmonary macrophages may play a role in the pathogenesis of sarcoidosis by inducing changes in the pulmonary microvasculature. Moreover, we hypothesize that these vascular changes may be induced not only in the lung but also in other organ systems such as skin, muscle, and eye in which microangiopathies are associated with sarcoid disease.

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.

The prognosis of carotid siphon stenosis.

We retrospectively reviewed the clinical course and angiograms of 15 patients with carotid siphon stenosis of 50% or greater. Fourteen had less than 50% stenosis at the origin of the ipsilateral internal carotid artery, and one had a greater degree of stenosis but underwent endarterectomy after an initial angiogram. Angiograms were examined for evidence of hemodynamic abnormalities in addition to residual lumen diameter. Seven patients initially had TIAs, 5 had strokes, and 3 were asymptomatic. In an average followup of 51 months (range 4-123 months) subsequent cerebral ischemic events occurred in 6 (40%), but only 1 had a stroke with a persisting neurological deficit that could be directly attributed to the siphon stenosis. Stenoses were hemodynamically significant by angiography in 5 of 7 TIA patients, and only 1 of 5 stroke patients. The incidence of subsequent ischemic events in this study was similar to 2 previous studies of siphon stenosis, however in this study most of the events ipsilateral to the siphon stenosis were TIAs or minor strokes. The association of hemodynamic angiographic abnormalities and initial TIAs but not strokes suggests that the mechanism producing ischemic symptoms may differ in patients with TIA and stroke who have carotid siphon stenosis.

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.

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.

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.

Inhibition of tumour-induced angiogenesis by systemically administered protamine sulphate.

Systemic administration of protamine sulphate significantly decreased the intensity of angiogenesis induced in X-ray immunosuppressed (BALB/c X DBA/2W) F1 mice by either HEp-2 (human larynx carcinoma) cells or semi-syngeneic splenocytes injected intradermally. In vitro experiments have shown that protamine sulphate markedly decreases the proliferation of human endothelial cells as assessed by 3H-TdR incorporation assay. In contrast, the proliferation of HEp-2 cells was not affected, and only slight inhibition of normal human fibroblasts could be demonstrated. Heparin abolished the inhibitory effect of protamine sulphate, both in vivo and in vitro. These results suggest that the observed inhibitory effect of protamine sulphate on angiogenesis in vivo may be at least partially due to the interaction of this compound with endothelial cells.

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.

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.

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.