Search

We investigated the effects of nonlethal gamma radiation on the metastatic potential of the murine tumor cell line, B16 melanoma. The ability of B16 cells to adhere to fibronectin, which is in part mediated by the alpha IIb beta 3 integrin receptor, is predictive of metastatic potential. We determined that exposure to 0.25-2.5 Gy gamma radiation significantly enhanced B16 cell adhesion to fibronectin. The radiation-enhanced adhesion was dependent on enhanced expression of the alpha IIb beta 3 integrin. We observed that 15 min after 0.5 Gy radiation, 99% of irradiated B16 tumor cells were positively labeled with monoclonal antibodies directed against alpha IIb beta 3 compared to 22% of sham-irradiated cells. Radiation-enhanced expression of the alpha IIb beta 3 receptor is reversible and down-regulation begins within 2-4 h postirradiation. Finally, we found that irradiation significantly enhanced the ability of B16 cells to form metastases in a lung colony assay. It is concluded that a relationship exists between radiation effects on the B16 tumor cells, alpha IIb beta 3 receptor expression, adhesion in vitro, and metastasis in vivo. We suggest that low-dose radiation, at levels comparable to those used in fractionated or hyperfractionated radiotherapy, may alter the metastatic phenotype and potential of surviving tumor cells via a rapid alteration in their surface expression of alpha IIb beta 3 integrin receptors.

A critical component of immune responsiveness is the localization of effector cells at sites of inflammatory lesions. Adhesive molecules that may play a role in this process have been described on the surfaces of both lymphocytes and connective tissue cells. Adhesive interactions of T lymphocytes with fibroblasts or endothelial cells can be inhibited by preincubation of the fibroblasts or endothelial cells with antibody to intercellular adhesion molecule 1 (CD54) or by preincubation of the T cells with antibody to lymphocyte function-associated Ag 1 (CD11a/CD18), molecules shown to be important in several other cell-cell adhesive interactions. Here we show that gamma-irradiation of human T lymphocytes impaired their ability to adhere to both fibroblasts and endothelial cells. This impairment was not associated with a loss of cell viability or of cell surface lymphocyte function-associated Ag 1 expression. gamma-Irradiation of T cells is known to result in the activation of ADP-ribosyltransferase, an enzyme involved in DNA strand-break repair, causing subsequent depletion of cellular nicotinamide adenine dinucleotide (NAD) pools by increasing NAD consumption for poly(ADP-ribose) formation. Preincubation of T cells with either nicotinamide or benzamide [corrected], both known inhibitors of ADP-ribosyltransferase, completely reversed the suppressive effects of gamma-irradiation on T cell adhesion. The maintenance of adhesion was accompanied by inhibition of irradiation-induced depletion of cellular NAD. These experiments suggest that the impairment of cellular immune function after irradiation in vivo may be caused, in part, by defective T cell emigration and localization at inflammatory sites.

The effects of gamma-irradiation on the properties of microvessel endothelial cells were studied in vitro. After incubating confluent endothelial cell monolayers in low serum-containing medium for 24 h, the monolayers were irradiated with 137Cs. Survival of rat lung microvessel endothelial (RLE) and mouse brain microvessel endothelial (MBE) cells were similar after irradiation (Do = 2.17 and 1.75 Gy, Dq = 4.44 and 5.67 Gy, and n = 7.8 and 25 for RLE and MBE cells, respectively). We examined the effects of gamma-irradiation on endothelial cell morphology, adhesion of syngeneic rat lung or mouse brain metastasizing tumor cells, release of the subendothelial matrix-degrading enzyme heparanase, and secretion of soluble mitogenic factors that stimulated the growth of syngeneic metastatic tumor cells. The effects of gamma-irradiation were not apparent until several hours after irradiation, and by 24 h doses of greater than or equal to 10 Gy caused limited endothelial cell retraction and reorganization of the endothelial monolayer. By 24 h after irradiation there was also increased adhesion of metastatic tumor cells to RLE but not MBE cells. We also examined the effects of gamma-irradiation on the release from endothelial cells of enzymes that solubilize the subendothelial matrix. Radiation resulted in a significant increase in the release of matrix-degrading enzyme (heparanase) that solubilized [35S]-labeled heparan sulfate from subendothelial matrix. This was most pronounced in the 24 h sample from gamma-irradiated endothelial cells. Finally, we examined the gamma-irradiation-induced release of mitogenic factors from endothelial cells that could stimulate the growth of metastatic cells in serum-limiting medium. The medium from RLE but not MBE cells stimulated the growth of a rat mammary carcinoma cell line. The results suggest that gamma-irradiation of microvessel endothelial cells can affect the interactions of tumor cells with endothelial cells and their subendothelial matrix; these processes could facilitate metastasis formation in irradiated tissues such as the lung.

Baker et al. [Cancer Res. 46: 1263-1274, 1986] developed an adhesive tumour cell culture system (ATCCS) using a culture surface coated with a cell attachment matrix (CAM). The ability of CAM-coated plates to support the growth of cells from human tumour biopsies has been evaluated. Successful growth was obtained in 9/22 samples (41%), but fibroblasts, rather than tumour cells, grew in the majority. Comparison of CAM with other surfaces showed that CAM was no better for establishing tumour cell growth than the presence of feeder cells or an alternative attachment factor vitronectin.

Deep ultraviolet (UV) irradiation is shown to modify organosilane self-assembled monolayer (SAM) films by a photocleavage mechanism, which renders the surface amenable to further SAM modification. Patterned UV exposure creates alternating regions of intact SAM film and hydrophilic, reactive sites. The exposed regions can undergo a second chemisorption reaction to produce an assembly of SAMs in the same molecular plane with similar substrate attachment chemistry. The UV-patterned films are used as a template for selective buildup of fluorophores, metals, and biological cells.

After total-body exposure to various doses of ionizing radiation, the ability of lymphocytes to interact specifically with high endothelial venules of rat cervical and mesenteric lymph nodes was analyzed in frozen sections. Following a radiation dose of 1.5 Gy, high endothelial venules remained intact and the binding of unirradiated lymphocytes to the venules was enhanced relative to unirradiated controls. At radiation doses above 5.0 Gy, damage to high endothelial venules was observed histologically as well as assessed functionally. There was a significant decrease in specific lymphocyte-venule binding and a significant increase in nonspecific binding. These findings suggest that radiation-induced damage to high endothelial venules might play a role in radiation-induced immunosuppression by interfering with the normal passage of lymphocytes from the blood into lymph nodes via a specific interaction between lymphocytes and high endothelial venules.

In order to elucidate the mechanism of cell adhesion, the size of the functional site, both in the fibronectin molecule and in the mouse fibroblast cell, responsible for cell adhesion activity, was determined. The size was assumed to be equivalent to the target size, that can be determined from the X-ray inactivation dose. The target size of the cell-binding site in the fibronectin molecule was 32 kdalton. The molecular weight was much larger than that of the tripeptide, which has been reported to be the minimum peptides having a cell-binding activity. This suggests that submolecular regions in fibronectin other than the tripeptide are necessary for cell adhesion. The target size in the cell responsible for the adhesion to the fibronectin-coated surface was 4300 kdalton. The large molecular weight of the target could be explained by assuming that a complex protein system is involved in the cell-adhesion process in the cell.

In 24 hours adherent marrow cell cultures (AMCC) were represented by single stretched fibroblasts. In non-feeder-supplemented AMCC most of the CFU-f remained single fibroblasts or passed through 1-3 cell doublings [correction of dudlings]. The colony stimulating activity of irradiated marrow cells was found to be diffuse across the Millipore filter, which seems to indicate that haemopoietic marrow cells produce a colony stimulating factor which is required for triggering the CFU-f from the Go-period of the cell cycle into cell proliferation.

CFU-f-derived stromal colony formation was accomplished in adherent marrow cell cultures (AMCC) with serum-rich medium. It turned out to require additional stimulation by hemopoietic feeder cells: by irradiated marrow cells and spleen cells if they possess megakaryocytes and platelets or by platelets from the blood. PDGF, EGF and IL-3 did not substitute the colony stimulating activity of feeder cells. Thymus, lymph node cells and blood leucocytes had no colony stimulating activity. At low oxygen concentrations which improve colony formation the stimulating activity of hemopoietic feeder cells was expressed, as well. Thus, CFU-f colony formation depends on stimulation by hemopoietic cells in addition to serum growth factors. In full populations of marrow cells the CFU-f colony formation is stimulated by marrow cells which accompany the CFU-f.

Exposure of cultured rabbit lens epithelial cells to repetitive doses of UV-B radiation delays their growth and alters the synthesis of specific proteins. Irradiated cells on the shoulder of the survival curve exhibited a dose-dependent decrease in growth when subcultured in serum-supplemented medium. UV-B irradiation did not affect the subsequent attachment efficiency of the cells. Control and UV-B irradiated cells were incubated with [35S]methionine and the pattern of protein synthesis in the cells was analyzed by SDS-PAGE and autoradiography. Analysis of the labeled proteins from cells exposed to UV-B radiation showed the induction of a 32 kD polypeptide and the loss of a 26 kD polypeptide compared with controls. Analysis of the proteins released by the UV-B irradiated cells into the culture medium revealed the 50% loss of a 37 kD radiolabeled protein compared with controls. The alteration of protein synthesis in lens epithelial cells by UV-B radiation may contribute to cataract formation.

The potential photodynamic therapy photosensitizers Methylene Blue, Azure C, Methylene Violet, Thionine, Methylene Green, Haematoporphyrin, Nile Blue A, chloroaluminium phthalocyanine and bis-aluminium phthalocyanine were examined for their photoeffects and dark toxicity against a human superficial bladder carcinoma cell-line. By examination of [3H]thymidine uptake into dye-treated cells after irradiation with a copper-vapour pumped dye laser, it was found that Methylene Blue was the most phototoxic and dark toxic of all the dyes tested, suggesting that the dye might be of some use as a topically applied photodrug for use in photodynamic therapy of superficial or early-recurring carcinomas.

The interactions between HeLa S3 tumoral cells and human fibroblasts after nitrogen-laser irradiation (337.1 nm) have been studied by using an in vitro cell invasion model. For the quantitative and morphological evaluation of nitrogen-laser radiation action upon tumoral adhesion to the fibroblast monostrate, we used: a) 3H-thymidine labelling of HeLa S3 tumoral cells; b) morphological modifications studies by phase contrast and scanning electron microscopy. The results emphasized the following aspects: 1. In non-irradiated cell cultures we noticed three interaction stages: adhesion, tumoral spreading and displacement with fibroblasts destruction; on the other side, we found a reduced adhesion to non-irradiated human fibroblasts of laser irradiated tumoral cells. 2. Significant percent increasing of non-irradiated tumoral cells adhesion to fibroblast monostrate, irradiated with various laser fluences (e.g. 0.2 kJ/m2--48.1%; 0.8 kJ/m2--63.8% and for 1.6 kJ/m2--79.5%). This phenomenon evidenced the close interrelation between irradiation fluences and tumoral adhesion rates. 3. The importance of numerical ratio between tumoral cells and fibroblasts in tumoral adhesion and invasion processes (e.g. ratio 1:10 tumoral adhesion reached 8.1%; in 1:5--25.9%; in 1:1--59.4% and for 2:1--83.9%). 4. Marked cytotoxic effects for both cell types after exposure to high and very high laser fluences (1.6--6.4 kJ/m2). Our results emphasize near UV-laser irradiation effects upon some of tumoral adhesion and invasion mechanisms and demonstrate the interrelations between cell populations manifesting a different vital potential.

The influence of lidocaine (L) and procainamide (P) on the chemotactic ability and adherence to nylon wool of In-111-labeled human polymorphonuclear leukocytes (PMNs) was investigated. At the normal therapeutic levels of L (0.022 mM whole blood) or P (0.03 mM whole blood) no change in PMN function was observed. However, at and above five times the aforementioned blood levels of L, significant reduction in the chemotactic ability of PMNs was noted (p less than 0.005). The adverse effects of In-111 radiation appeared insignificant at all L or P concentrations during the 3-hr observation period. The labeled PMNs were resistant to the toxic effects of a higher concentration of P than that of L, and the reduction in PMN chemotaxis and adherence to nylon wool was not apparent until the P concentration reached 1.5 mM.

The in vitro efficacy of 20 Gy (2000 rad) of external beam irradiation delivered to patients with choroidal melanomas prior to enucleation was investigated in 11 patients whose tumors were grown in cell culture. Phase-contrast microscopy was used to compare growth patterns between irradiated and nonirradiated tumors. Cell types were determined by histologic stains, and electron microscopy identified intracytoplasmic melanin. Irradiated melanomas did not grow and did not attach to culture flasks, thus demonstrating that preenucleation irradiation alters the in vitro growth of melanoma cells.

Forty seven individual haemopoietic cell clones bearing unique radiation markers were studied in long-term bone marrow cultures. Throughout cultivation, clones appeared at different times, 1 to 12 weeks after explanation, survived during 1-10 weeks and markedly varied in size. Usually, the number of metaphases characteristic of an individual clone rapidly increased, achieved a maximum and declined. The cells of disappeared clones were never seen again. The experimental results provide further evidence for the model of haemopoiesis by clonal succession. The data obtained are discussed with respect to the functioning of haemopoietic stem cell population.

Alveolar macrophages lavaged from mice at various times after 1800 rads of thoracic x-irradiation were compared to control mice. Determination of their numbers, size distribution, glass adherence, latex particle uptake, cytochemistry, morphology, protein, phospholipid, and hydrolytic enzyme composition showed that defective bacterial uptake and clearance, which has been demonstrated by other workers, may be related to a transient decrease in their numbers rather than activity. A decrease in numbers is explainable on the basis of a population of radiosensitive precursors in the lung interstitium. The work load in the alveolar space may prolong the duration of their residence in the alveoli as well as increase their size and content of hydrolytic enzymes and ingested surfactant components.

The ATP content of human fibroblasts increases or decreases depending on the mode of exposure (local and whole-body irradiation at 180 and 8 mW/cm2, respectively). Irradiation gives rise to conjugative changes in the monolayer density of cells and ATP accumulation in them.

The effect of nonionizing electromagnetic radiation in the microwave range was studied on the adherence, opsonization, phagocytosis, nitroblue tetrazolium reduction and antibody-dependent cell cytotoxicity of peritoneal macrophages from BALB/c mice. After 15 min of irradiation with microwaves modulated in pulses at a frequency of 2,450 MHz and a power density of 32 mW/cm2, an increase in all the parameters studied was observed. The use of microwaves as a possible tool for the stimulation of the immune system is discussed.

Cell proliferation in long-term suspension cultures of mouse bone marrow was studied. Bone marrow cells were maintained on irradiated or conventional non-irradiated semiallogeneic adherent feeder layers and the origin of the proliferating cells was investigated by using the unique T6 chromosome marker. In cultures containing irradiated feeder layers total cell numbers were less at the first week after "recharging," but the levels soon reached those of conventional non-irradiated cultures. At the first weeks after recharging, proliferating cells, maintained on conventional non-irradiated feeder layers, originated mainly from the adherent layer. The contribution of the recharging population to the proliferating cell population increased with time. In contrast, the proliferating cells grown on irradiated adherent layers originated mainly from recharging population from the first week after recharging. The origin of the nonadherent CFUs and their development in F1 hybrid mice and mice of syngeneic strains was also examined. The pluripotent stem cells grown on non-irradiated feeder layers were derived mainly from the adherent layer.

Exposure of the macrophage-like cell line J774.1 to 20 gray of cobalt-60 gamma radiation resulted in a block of tritiated thymidine incorporation, along with an increase in cell "activation," as assessed by increases in lysosomal enzyme and ectoenzyme content, PMA-induced H2O2 production, and NBT staining, ingestion of E(IgG), spreading, and membrane ruffling. These changes are evident within 1 day postradiation and peak at 4 days postradiation.

Marrow stromal cells are generally thought to be radioresistant. However, when the marrow was irradiated in vivo or in vitro before its use for the continuous long-term marrow culture, doses of radiation as low as 500 rad interfered with the establishment of the adherent stromal layer. Moreover, when the stromal layer was permitted to establish, similar doses of radiation interfered with its potential to support the proliferation and maintenance of the hemopoietic stem cell. Thus, marrow stromal cells appear to be more radiosensitive than hitherto thought. The type of damage may vary, however, according to the dose of radiation. Small doses may interfere with such functions as adhesion or cell division while larger doses may completely destroy the cell.

The immobilization of erythrocyte as the whole cell without hemolysis was studied. It found that erythrocyte could be treated and immobilized stably by radiation polymerization of specific monomers having high viscous and long oxyethylene units chain such as methoxypolyethyleneglycol methacrylate (M-23G) and polyethyleneglycol dimethacrylate (14G). Irradiation dose without hemolysis was limited less than 1 X 10(5) r and a comonomer system consisting of M-23 G-14G, 1:1 and small quantity of glutaraldehyde (GA) was the optimum carrier composition. The functional properties of the immobilized erythrocyte was also investigated. It was found that the immobilized cell could be carried out carbon monoxide-oxygen gas exchange effectively and reversibly so as in the intact cell. The immobilized erythrocyte also showed the catalase activity just as in the intact cell. The stability of erythrocyte increased greatly by the immobilization for standing at low and room temperatures and hardly hemolyzed in non-isotonic medium such as pure water. It was observed in scanning electron microscope that the immobilized erythrocyte had a hollow disk shape same as in intact cell and covered with a thin polymer layer.

A study was made of adherence to plastic Petri dishes and viability (as non-pyknotic cell counts) of rat bone marrow cells cultured for 5 to 22 h in 199 medium containing 15 per cent fresh isologous serum. An overall decrease in the number of viable myelokaryocytes, including mature neutrophils, was observed in the cultures irradiated with doses of 3 to 12 Gy. In addition, gamma-irradiation increased substantially the adherence of neutrophils and, to a lesser extent, of other myelokaryocytes. A possibility of early radiation-induced disturbances in granulocytic maturation is discussed.

Polymorphonuclear leucocytes (PMN) from normal human subjects were labelled with [111In]oxine (20 muCi 10(8) cells). In the presence of 20% autologous serum (AS), dissociation of 111In from the cells resulted in mean losses of radioactivity of 13% at 3 h and 30% at 24 h. Adherence of 111In-labelled PMN to cultured porcine endothelial monolayers was increased by 40.7 +/- 31.6% after 60 min incubation in 20% AS at 37 degrees C when compared with unlabelled cells. Phagocytosis and intracellular killing of Candida albicans were unaltered by labelling. Elution of 111In from labelled PMN together with enhanced adhesiveness may have important implications for the study of PMN kinetics and the investigation of inflammatory disease.

The formation of rosettes between spleen cells from nonimmune mice and sheep red blood cells (SRBC) has been investigated. The number of splenic rosette-forming cells (RFC) in the intact spleen was very low but greatly increased after sublethal whole body x-irradiation. These cells were inhibited by pretreatment of the cells with AKR anti-C3H- antigen antiserum, in the presence of fresh guinea pig serum, but not with rabbit anti-mouse immunoglobulin antiserum. The splenic RFC also increased after cortisone acetate administration into mice. These results suggest that the RFC are a T cell sub-population having x-ray resistant and cortisone-insensitive characteristics. The x-irradiation of mice in vivo selectively increased the number of splenic RFC in consequence of reduction in the number of cells having non-T cell characteristics. These RFC are presumably antigen-specific sheep red blood cell rosettes, a quite different situation as in the case of human T lymphocyte rosette formation.

Among the various parameters that are supposed to play a role in aging at the cellular level, the "free radical theory" involves biochemical modifications that can be induced by radiation. Human embryonic lung fibroblasts were serially subcultivated at low density under chronic low dose rate irradiation (40 mrad/day) and in a normal environment. Irradiation increases cell attachment and the population doubling/day throughout their entire in vitro lifespan. Consequently, the doubling potential reached by irradiated cells was higher than that of control cultures. Finally, the total number of cells produced under chronic irradiation was 8-14 times higher than in a normal environment. These results are discussed with respect to the increased enzymatic activities (superoxidismutase, catalase, glutathion-reductase, G6PD) found in some irradiated organisms.

The role of chemotherapy, X-irradiation and a combination of both on the phenomenon of adherence of yeast to buccal epithelial cells (BEC) was investigated in vitro. Growth of three Candida spp. in the presence of eight of eleven antineoplastic agents led to reduction of adherence of the isolates tested (reduction between 30% and 61% of the control value), and this effect was observed whether exponential or stationary phase Candida cells were used. Exposure of C. albicans to various doses of radiation also led to a reduction in adherence of this yeast to BEC between 31% and 53% of the control value. This reduction was shown to be dose related. Similar results were obtained when BEC were exposed to radiation, and the effects of radiation treatment was accentuated when both yeast and BEC were irradiated simultaneously. Furthermore, treating C. albicans with a combination of chemotherapy and radiation led to the greatest reduction in adherence of yeast to BEC compared to when the yeast was treated with either chemotherapy or radiation alone (reduction between 63% to 74% as compared with control). The possible mechanism/s involved in reduction of adherence of yeast to BEC are discussed.

Changes in total number, differentials, cell protein, adherence properties, acetyltransferase and acetylhydrolase activities, prostaglandin E2 and leukotriene C4 production, as well as Ca2+ ionophore A23187 stimulation were examined in resident peritoneal cells isolated from mice 2 h to 10 days postexposure to a single dose (7, 10 or 12 Gy) of gamma-radiation. Radiation dose-related reductions in macrophage and lymphocyte numbers and increases in cellular protein and capacity to adhere to plastic surfaces were evident. In vivo irradiation also elevated the activities of acetyltransferase and acetylhydrolase (catalysing platelet-activating factor biosynthesis and inactivation, respectively) in adherent and nonadherent peritoneal cells, particularly 3-4 days postexposure. Blood plasma from irradiated animals did not reflect the increased cellular acetylhydrolase activity. Prostaglandin E2 and leukotriene C4 synthesis were elevated postexposure, suggesting increased substrate (arachidonate) availability and increased cyclooxygenase and lipoxygenase activities. Ionophore stimulation of enzyme activities and eicosanoid release also differed in irradiated peritoneal cells. While the properties of adherence, platelet-activating factor synthesis/inactivation-associated enzyme activities, and eicosanoid production are generally characterized as those of macrophages, lymphocytes or their products may influence or contribute to the observed radiation-induced changes.

UVB induced changes of blood cell properties were investigated in 12 MS patients and in 10 healthy volunteers serving as normal controls. The mean cell volume (MCV) was determined by electronic sizing, the granulocyte and lymphocyte adherence was estimated in a capillary assay, and the phagocytic activity of granulocytes was measured in a test system based on the incorporation of opsonized baker's yeast (Saccharomyces cerevisiae). In MS patients the MCV of red cells and lymphocytes decreased rapidly within 6 UVB treatments. In contrast, the reduction of the granulocyte volume was delayed (between the 6th and 12th UVB). In the control group the mean value of the red cell and lymphocyte MCV remained rather unaffected. There was a slight rise of the granulocyte volume after the 6th UVB. The only significant change of adherence was an increase of granulocyte adherence in MS patients. Untreated patients had a significantly enhanced phagocytic activity in comparison to the control group. 6 UVB treatments included a significant reduction of the phagocytic activity in MS patients. However, subsequently the percentage of phagocytizing cells increased again, whereas the particle uptake per cell continued to decrease. In the control group only minor UVB induced changes of phagocytosis were observed. The in vitro UV irradiation caused an enhanced phagocytosis in the majority of cases in both controls and MS patients. In general, under the UVB treatment all parameters examined changed in the sense of a normalisation, in that the measured values reached a new level lying between the extreme pretreatment values accompanied by a reduced standard deviation. The effect of UVB was more pronounced in MS patients when compared with normal controls. This could result from an enhanced sensitivity to the influence of UVB of pathologically altered cells in MS patients. The monitoring of the MCV of red cells and lymphocytes as well as the repeated testing of granulocyte phagocytosis are recommended for supportion of therapy planning and follow-up of MS patients.

Adherence of granulocytes to the endothelium of small veins in different microvascular beds was quantified by visual counting of the adherent cells. Increased adherence of granulocytes induced by the stress of preparation or by ultraviolet light irradiation of the microvasculature was greatly reduced by i.v. administration of sialic acid. In the rabbit ear chamber laser irradiation induced increased granulocyte stickiness which was similarly prevented by intra-arterial infusion of sialic acid. In normal rats, sialic acid treatment induced granulocytosis and the effect was more striking on Busulphan-induced leucopenia in rats. Glucuronic acid applied under identical conditions had no effect on granulocyte adhesiveness in vivo or on the peripheral blood granulocyte count. According to these findings exogenous sialic acid greatly reduces the effect different pathogenic stimuli have of inducing adherence of granulocytes to blood vessel walls and induces granulocytosis by mobilizing cells from the "marginating" and reserve pools.

Epidermotropic lymphocytes are an essential cellular component of the skin-associated lymphoid tissues (SALT). Dermal lymphocytic infiltrates are also characteristics of inflammatory dermatoses, such as psoriasis, and may be involved in the pathogenesis of the disease, although the mechanisms by which lymphocytes are recruited into these sites are not known. We have used an in vitro lymphocyte adherence assay to test the hypothesis that specialized endothelial cells are present in inflamed skin, and are capable of supporting lymphocyte adherence and promoting lymphocyte emigration. In this assay, we assessed the binding of lymphocytes overlaid onto frozen sections of normal and psoriatic skin. Peripheral blood mononuclear cells (PBMC) from patients, from healthy volunteers, and from rat thoracic duct bound specifically to dermal endothelia in psoriatic plaques, in steroid-resistant areas of plaques, but not in uninvolved skin or skin from healthy individuals. Analysis of the binding properties of lymphocyte subsets revealed preferential adherence by CD4+ T cells as compared with CD8+ T cells and to B cells. Interestingly, PBMC from patients undergoing ultraviolet light therapy failed to adhere to autologous skin or to lesion-containing skin sections from untreated patients. Additional studies indicate that the lymphocyte-endothelial interaction is an energy- and calcium-dependent process and involves surface glycoprotein and carbohydrate moieties, requirements similar to those found in specific lymphocyte interactions with high endothelial venules in lymph nodes during the homing process. Pretreatment of lymphocytes with antibodies directed against homing receptors mediating migration into lymph nodes and into gut-associated lymphoid tissues, however, did not interfere with lymphocyte adherence to psoriatic endothelium. In contrast, anti-lymphocyte function associated antigen (LFA)-1 antibody partially inhibited lymphocyte binding to chronic plaques. We conclude that a tissue-specific receptor/ligand interaction independent of LFA-1 directs lymphocyte emigration from the dermal microvasculature into the psoriatic dermis and that LFA-1 plays only an accessory role in the adhesion process.

The ability of human tumour cells to form colonies in soft agar is enhanced by the presence of autologous phagocytic/adherent cells. We investigated the effect of irradiation on the ability of the adherent cells to support human tumour colony formation. Relatively low doses of irradiation significantly increased the growth enhancing ability of adherent cells in 17/19 cases. The possibility that the enhancement was mediated by inactivation of radiosensitive contaminating lymphocytes was explored. Depletion of T lymphocytes from unirradiated adherent cells by a monoclonal antibody and complement resulted in little overall change in tumour colony growth. However, elimination of only the suppressor subset (OKT8+) of T lymphocytes resulted in increased colony growth relative to control values obtained with unirradiated adherent cells. In contrast, depletion of T lymphocytes from irradiated adherent cells by a pan T monoclonal antibody and complement decreased colony formation. Thus, the ability of irradiated macrophages to enhance tumour colony growth appeared to be mediated by a T lymphocyte. The effect of irradiation on isolated populations of macrophages and T lymphocytes was also examined. The enhanced ability of irradiated adherent cells to support tumor colony growth appeared to have been due to treatment of T lymphocytes alone. The results indicate that both adherent macrophages and lymphocytes may influence the growth of clonogenic human tumour cells.

The right hemithorax of albino rats was subjected to irradiation of 14.3 Gy at a single dose of 2.2 Gy/min. Local macrophages (BM) were lavaged and studied at different time-points (1 day to 6 mos.) following irradiation. During the phase of radiation pneumonitis, BM activation was revealed, according to an increase in the spreading index, DNA relaxation, enhanced phagocytic capacity of isologous thymocytes. Six months after treatment (the phase of lung fibrosis), the yield of BM from affected lungs was increased along with a diminished percentage of cells capable of phagocytizing dead thymocytes. A phasic time-dependence of BM changes in the postirradiation period was discussed.

The mechanism of physiologic alteration by high (HDR) or low dose rate (LDR) (5 or 120 cGy/min) irradiation of plateau-phase bone marrow stromal cell cultures was investigated using a technique of in vitro bone marrow transplantation. Purified stromal cell cultures from C57BL/6J, C3H/HeJ, or (C57BL/6J X DBA2/J)F1 (B6D2F1) mouse marrow were irradiated to doses of 2.5 to 10 Gy at LDR or 10-100 Gy at HDR and were then engrafted in vitro with nonadherent hematopoietic cells from murine continuous bone marrow cultures. Parameters of engraftment quantitated included: (1) numbers of adherent proliferating hematopoietic cell colonies, "cobblestone islands" (2) cumulative production of nonadherent hematopoietic cells over 8 weeks after engraftment, (3) M-CSF, GM-CSF and multi-CSF (IL-3) dependent hematopoietic progenitor cells forming greater than or equal to 50 cell colonies in semisolid medium, (4) cumulative production of CFUs, and (5) number of adherent stromal cells positive for detectable extracellular laminin or collagen type IV (markers of endothelial cells, reticular adventitial cells, or sinus lining cells). There was a decrease in cobblestone island formation between 5 and 10 Gy and this parameter possibly increased at doses of 50 and 100 Gy. There was no difference between HDR and LDR irradiation to 10 Gy. Irradiation to doses above 10 Gy decreased support of engrafted cells forming CFU-GM and CFU-GEMM. Measures of CFUs after 10 Gy were variable but indicated a possible increase with HDR and no effect of LDR at 1 week and a decrease in both HDR and LDR groups at 3 weeks after engraftment. Thus, LDR and HDR irradiation in vitro alter several specific parameters of marrow stromal cell support for engrafted hematopoietic stem cells.

The effect of cytochalasin B (CB), nocodazole, and irradiation on the adherence and internalization of 3H-labeled EMT6 spheroid-derived single cells and inert microspheres in unlabeled, intact EMT6 multicellular spheroids has been examined. CB inhibited adhesion and internal migration, whereas nocodazole did not stop adhesion but did prevent later internalization. Treatment of labeled cells with 5, 15 and 25 Gy 250 kV X-rays before adherence did not effect their adherence or later internalization. The same radiation treatments administered to the spheroids either immediately before or after the introduction of unirradiated single cells did not affect adherence, but the depths reached by labeled cells and microspheres were reduced largely because of the consequent reduction in spheroid growth. Microsphere size (9, 15, or 25 microns) and surface charge (negative, or non-ionic) had minimal, if any, effect on the adherence and internalization of these particles.

The requirement of B cells activated by mitogen (dextran sulfate plus lipopolysaccharide) for accessory cells was studied by partition analysis. Small numbers of splenic B cells were activated to clonal growth, as determined by visual inspection, and to immunoglobulin (Ig) synthesis, as determined by release of Ig into the culture fluid. By placing irradiated adherent cells in the periphery of the microculture wells and forcing responding cells to different areas of the well (slant experiments), it was observed that no cell contact was necessary for B cell activation, and that "promoted" contact ("Rock and Roll" experiments) does not increase the efficiency of activation. Sequential microcultures suggest that only some irradiated adherent cells act as accessory cells, but they can perform this function to more than one B cell. Attempts to perform limiting dilution analysis by varying irradiated adherent cell input showed non-single-hit behavior. When the data were rearranged, taking into account the distribution of irradiated adherent cells, then single-hit behavior with about 1 to 5% of irradiated adherent cells acting as an accessory cells for B cell clonal activation was observed. The evidence suggests that an uncommon irradiated adherent cell releases a soluble factor necessary for B cell activation and/or clonal proliferation.

The autoimmune hemolytic anemia of NZB mice is pathogenetically mediated by a genetically prescribed anti-erythrocyte autoantibody response directed to the X erythrocyte autoantigen. The cellular locus of the immunoregulatory defect underlying the anti-X response was explored by adoptively transferring bone marrow cells (BMC) from NZB mice to lethally irradiated histocompatible recipients. Before adoptive transfer, BMC from donor mice were assayed for antigen-binding lymphocytes with receptors for the X autoantigen (X-ABL) by immunocytoadherence assays and for anti-X autoantibody-secreting cells (X-PFC) by plaque-forming cell assays. Twelve weeks after adoptive transfer, splenic lymphocytes from recipient mice were assayed for X-PFC and humoral anti-X autoantibody by Coombs' tests. Transfer of 15 to 30 x 10(6) BMC containing 6 to 12 x 10(3) X-ABL but no X-PFC from 6- to 8-week-old NZB mice to lethally irradiated BALB/c, B10.D2, C57BL/Ks, and DBA/2 mice produced X-PFC in 70% of the recipients. Development of X-PFC was not simply dependent upon available X-ABL since transfer of 15-30 x 10(6) BMC, containing comparable numbers of X-ABL, from BALB/c, B10.D2, C57BL/Ks, or DBA/2 mice to NZB or syngeneic recipients did not produce X-PFC. Transfer of BMC from NZB mice to BALB/c, B10.D2, and DBA/2 mice with weekly administrations of AKR anti-theta antiserum had no effect on the development of X-PFC; Tlymphocyte ablation was evidenced by the absence of theta+ spleen cells. These results suggest that the pathogenetic anti-X response is not genetically prescribed at the level of macrophages, humoral factors, or T cells, but rather appears to be a phenotypic expression of a primary B lymphocyte defect permitting or promoting differentiation of NZB X-ABL.

Monolayer cell colonies normally are pushing off single cells, which go into suspension. A dose-dependent increase of these cell detachments followed by readhesion and colony forming would influence the colony survival curve. Detached cells being in suspension can be verified by means of the colony forming test after decantation and inoculation of the medium. The amount of colony forming cells in the late phase of synthesis does scarcely change following 300 rds of 60Co gamma irradiation. The diminution of cell adhesion expected as a result of radiation damage could not be observed.

The aim of this study was to investigate the adherence in vitro of Candida albicans to various parts of the gastrointestinal (GI) mucosa from irradiated and non-irradiated mice and to attempt to inhibit this adhesion with a chitin derivative. Adhesion was assayed using 3H-leucine labelled yeast, to which GI tissue-disks removed from irradiated (400R Cobalt) and non-irradiated animals were exposed at various time intervals post-irradiation. In non-irradiated mice differences in adherence of C. albicans to various parts of the GI tract were observed, the highest adherence being to duodenal tissues. In irradiated mice, an increase in adherence to all parts of the GI mucosa was observed. Based on findings from previous studies that a chitin derivative (CSE) inhibits adhesion of C. albicans to various tissues in vitro and in vivo, we tested the effect of CSE on the adhesion of C. albicans to GI tissues. The results show that CSE inhibited the adhesion of C. albicans to GI tissues from both irradiated and non-irradiated mice by 75-85%. The relevance of the findings to the pathogenesis of candidiasis is discussed.

The colony-formation was stimulated by X-irradiation (0.03-0.30 Gy) of cultured Chinese hamster cells. The stimulation was only noted in the nonadhered cells which was perhaps associated with a change in their adhesive properties. It was shown that low-level radiation induced changes in the structural organization of the cell membrane.

The number of fibroblast colonies in bone marrow cultures depends on FCFC concentration in explanted cells and FCFC cloning efficiency. For mouse bone marrow the efficiency of fibroblast colony formation increases in the presence of the feeder (irradiated bone marrow of spleen cells). Colony-stimulating feeder activity does not depend on the presence of phagocytic and stromal cells in the feeder cell population. Trypsinization of the bone marrow leads to the release of additional FCFC and the increase of their concentration in bone marrow cell suspensions.

The adherence of peripheral blood leukocyte to a hydrophilic substrate (cotton wool) was studied in healthy donors and patients with Hodgkin's disease and solid tumors. It was shown that neutrophils, eosinophils and lymphocytes contribute to the adhesive ability. Patients with primary solid neoplasms revealed an increased leukocyte adherence. Following a single therapeutic irradiation, this index appeared significantly enhanced in those with Hodgkin's disease. Possible causes of the disturbances are discussed.