A conditionally immortalized epithelial cell line for studies of intestinal drug transport.

A new cell culture model that better mimics the permeability of the human small intestine was developed for studies of passive drug transport. The intestinal epithelial cell line, 2/4/A1, conditionally immortalized with a temperature-sensitive mutant of the growth-promoting oncogene simian virus 40 (SV40) large T, was grown on permeable supports. The cells grew at 33 degrees C, where the oncogene is fully active, but stopped growing and entered a differentiation program at 39 degrees C, where the oncogene is inactive. Significant cell death was observed at 39 degrees C and, therefore, growth conditions under which 2/4/A1 cells survive during the differentiation process were developed. Cells grown on extracellular matrices which contained laminin at an intermediate temperature of 37 degrees C formed viable differentiated monolayers with tight junctions, an increased expression of brush border enzymes, and a paracellular permeability that was comparable to that of the human small intestine. The permeability of 17 structurally diverse drugs gave a sigmoidal relationship with the absorbed fraction of the drugs after oral administration to humans. The relationship was compared with those obtained with the well established Caco-2 model and after in vivo perfusion of the human jejunum. The transport of drugs with low permeability in 2/4/A1 monolayers was comparable to that in the human jejunum, and up to 300 times faster than that in Caco-2 monolayers. The transport of drugs with high permeability was comparable in all models. These results indicate that 2/4/A1 monolayers are promising alternatives to Caco-2 monolayers for studies of passive drug transport.

Chitosans as absorption enhancers of poorly absorbable drugs. 3: Influence of mucus on absorption enhancement.

Chitosans are potent nontoxic absorption enhancers after nasal administration but their effects on the intestinal epithelium in vivo has not been studied in detail. In this study, the effects of chitosans with varying molecular weights and degrees of acetylation on the absorption of a poorly absorbed model drug (atenolol) were studied in intestinal epithelial cell layers with or without a mucus layer and in an in situ perfusion model of rat ileum. The effects of the chitosans on epithelial morphology and release of lactate dehydrogenase (LDH) into the perfusate were investigated in the in situ model. The chitosans had pronounced effects on the permeability of mucus-free Caco-2 layers and enhanced the permeation of atenolol 10- to 15-fold, with different absorption kinetics for different chitosans, in accordance with previous results. In contrast, enhancement of atenolol absorption through rat ileum was modest. LDH release from the tissues perfused with chitosans did not increase, indicating that the chitosans were used at nontoxic concentrations. Morphological examination of the perfused ileal tissues revealed more mucus discharge from the tissues exposed to chitosans than from controls, which suggested that the discharged mucus may inhibit the binding of chitosan to the epithelial surface and hence decrease the absorption-enhancing effect. This hypothesis was supported by studies with intestinal epithelial HT29-H goblet cells covered with a mucus layer. The binding of chitosan to the epithelial cell surface and subsequent absorption-enhancing effects were significantly reduced in mucus-covered HT29-H cultures. When the mucus layer was removed prior to the addition of chitosan, the cell surface binding and absorption-enhancing effects of the chitosans were increased. We conclude that the modest absorption-enhancing effects of unformulated chitosan solutions in the perfused rat ileum are a result of the mucus barrier in this tissue. This effect may be overcome by increasing the local concentrations of both chitosan and drug, i.e,. through formulation of the chitosan into a particulate dosage form.

Freeze-thaw immobilization of liposomes in chromatographic gel beads: evaluation by confocal microscopy and effects of freezing rate.

Biological membranes immobilized in chromatographic gel beads constitute a multifunctional affinity matrix. Membrane protein-solute interactions and drug partitioning into the lipid bilayers can conveniently be studied. By the use of confocal laser-scanning microscopy (CLSM) the distribution of immobilized model membranes in the beads has been visualized for the first time. Freeze-thaw-immobilized liposomes in Superdex 200 gel beads were situated in a thick shell surrounding a liposome-free core. The amount of phospholipids immobilized by freeze-thawing was dependent on the temperature in the cooling bath and the type of test tube used. A bath temperature of -25 degrees C gave higher immobilization yield than freezing at -75 or -8 degrees C did. Freeze-thawing in the presence of liposomes did not affect the gel bead shape or the refractive index homogeneity of the agarose network of the beads, as shown by confocal microscopy.

Mechanism of absorption enhancement in humans after rectal administration of ampicillin in suppositories containing sodium caprate.

PURPOSE: The medium chain fatty acid sodium caprate (C10) is approved as an absorption enhancer but its mechanism of action has not been studied in humans. The aim of this study was to investigate the mechanism of action of C10 in human subjects after rectal administration. METHODS: Twelve healthy human subjects were randomised to receive ampicillin suppositories with (AM-C10) or without (AM) C10. Serum and urine samples were collected and analysed for ampicillin by HPLC. Rectal biopsies were taken before and 25 min (approximate time of maximum serum concentration, Cmax, for ampicillin) and 185 min (during the final part of the elimination phase) after rectal administration of the suppositories. The osmolality of the rectal fluid was also measured. RESULTS: AM-C10 administration increased Cmax, area under the serum concentration-time curve (AUC) and urinary recovery of ampicillin 2.6-, 2.3- and 1.8-fold, respectively, compared to AM. Histological examination of the biopsies showed that AM-C10 exposure resulted in reversible mucosal damage that occurred at the same time as the Cmax for ampicillin while AM prolonged mucosal damage. A reversible increase in rectal fluid osmolality was observed with both treatments. CONCLUSIONS: AM-C10-enhanced absorption of ampicillin coincides with non-specific damage to the rectal mucosa. C10 itself as well as the suppository base and the hyperosmolality of the rectal fluid contributed to this effect. However, the histological damage was reversible with AM-C10, suggesting that C10 also has a protective effect on the rectal mucosa.

Expression of tenascin in developing and adult mouse lymphoid organs.

The extracellular matrix (ECM) is essential in regulating many cell functions in non-lymphoid cells, and the ECM may also play a role in the function of the immune system. Tenascin is a hexameric glycoprotein of the ECM. In mouse, two major polypeptides of MW 210 KD and 260 KD are formed by differential splicing. Northern blot screening of various mouse tissues showed that the short 6 KB tenascin message was strongly expressed in the adult thymus, whereas very little or no tenascin mRNA could be detected in spleen. In addition, immunoblotting and histological analysis with monoclonal anti-tenascin antibodies revealed the presence of tenascin in lymph nodes and spleen. In thymus, only a short-splice variant of tenascin was detected by immunoblotting, which supported the Northern blot results. Immunohistology showed that the epithelial reticular stroma in both embryonic and adult mouse thymus expressed tenascin, as did the postnatal mesenchymal reticular stroma in lymph nodes and spleen. The distribution of tenascin in the thymus was more restricted than that of fibronectin and laminin.

Induction of mouse tenascin expression by a human sarcomatoid Wilms' tumor cell line growing in nude mice.

A new cell line from a sporadic Wilms' tumor was established and extensively characterized. In nude mice, the tumor cells rapidly formed tumors which, in histological characteristics and extracellular-matrix (ECM) composition resembled sarcomatoid Wilms' tumor. The tumor cells produced B chains of laminin, but no A chain, and laminin was deposited into the ECM in a punctate pattern typical of sarcomatoid tumors. Strong expression of tenascin was detected within the stromal ECM of the tumors. Species-specific antibodies reacting either with human or with mouse tenascin showed that tenascin was exclusively derived from mouse host cells. The human Wilms' cell line thus induced a strong stromal response with increased deposition of tenascin. The cell line may be useful for studying the behavior of sarcomatoid Wilms' tumor cells and for identifying factors that stimulate synthesis of tenascin.

Human blood dendritic cells exhibit a distinct T-cell-stimulating mechanism and differentiation pattern.

In this study, the mechanisms underlying stimulation of T-cell proliferation by human blood dendritic cells (BDC) and their differentiation have been defined with a panel of monoclonal antibodies (MoAbs). It was found that the MoAbs against LFA-1 (CD11a), CD11c, LFA-3 (CD58), ICAM-1 (CD54) or HLA-DR could significantly suppress T-cell proliferation in an allogeneic mixed lymphocyte reaction (P < 0.05), while being unable to inhibit clustering of BDC with T cells. Addition of anti-CD18 or CD45 MoAbs increased the size of clusters after 18 h of culture, but had no effect on the proliferation of T cells (P < 0.05). The suppressive effect of the MoAbs may be viewed not as an inhibition of contact between BDC and T cells, but rather as a blocking of co-stimulatory signals for T-cell activation, which are mediated by interaction of the adhesion molecules. After depleting the BDC preparations of monocytes, we used a double staining in FACS analysis to demonstrate that BDC do not express specific T (CD3), B (CD20 and CD21) and myeloid cell markers (CD11b, CD13 and CD14), but abundant class II antigens. This pattern remained unaltered after 8 days of culture in the presence of 100 U/ml GM-CSF, although a threefold increase of HLA-DQ and ICAM-1 molecules on the cultured cells was observed.

Expression of CD54, CD58, CD14, and HLA-DR on macrophages and macrophage-derived accessory cells and their accessory capacity.

Human peripheral monocytes can differentiate in vitro into macrophages (Mph) possessing a low accessory activity in T cell stimulation. Mph can be converted into a state of high accessory activity by treatment with dibutyryl cyclic AMP. This finding was used in this study to achieve Mph-derived AC (MphAC). Among the surface antigens on AC which have been shown to participate in accessory events leading to T cell proliferation, MHC class II antigens, CD58 (LFA-3) and CD54 (ICAM-1) seem to be especially important. We show here that the high accessory capacity of MphAC was not correlated with a high level of the surface antigens HLA-DR, CD58, and CD54. The amount of CD54 molecules was, in fact, lower on the MphAC than on the Mph.

All CD2-positive human lymphocytes rosette with sheep erythrocytes in the presence of polyethylene glycol.

The efficiency of rosette formation between human peripheral resting lymphocytes and sheep erythrocytes (E) is remarkably increased in the presence of 1-4% polyethylene glycol of MW 10,000 (PEG). In fact, all CD2 ('sheep E receptor')-positive lymphocytes formed rosettes. CD2 positivity was assayed by immunofluorescence staining with the monoclonal antibody (mAb) Leu 5b. PEG also induced autologous E rosetting which otherwise did not take place under the usual conditions. The specificity of the rosettes was assessed by blocking CD2 with mAb Leu 5b and by blocking the CD2 target structure (T11TS) on the sheep E with the mAb L180/1. The mAb Leu 5b abolished all rosette formation, but mAb L180/1 only abolished the sheep E rosettes as this mAb is specific for sheep E and does not cross-react with human E. The results indicate that sheep E in the presence of PEG specifically rosette with all CD2-positive lymphocytes.

Activation of human T cells by neuraminidase-galactose oxidase-treated erythrocytes involving CD2 (T11) and its complementary structure.

Human T lymphocyte proliferation induced by neuraminidase-galactose oxidase (NAGO)-treated autologous erythrocytes (HENAGO) plus polyethylene glycol (PEG) has previously been shown to be independent of accessory cells. Here, we show that the response to HENAGO + PEG was accompanied by interleukin 2 (IL-2) release and was inhibited by anti-IL-2 and anti-IL-2 receptor antibodies. HENAGO alone initiated DNA synthesis together with phorbol ester (12-O-tetradecanoyl-phorbol-13-acetate; TPA). To elucidate the nature of the stimulatory signals NAGO-treated sheep erythrocytes (SENAGO) were used in additional experiments. In parallel to the superior rosetting capacity of SE compared to HE. SENAGO were by themselves stimulatory, and the response was further enhanced by PEG or TPA. Antibody L180/1, specific for the T11 (CD2) target structure (T11TS) on SE, homologous to the human CD2 ligand LFA-3, abolished the response to SENAGO alone or when combined with PEG or TPA. The results suggest that ENAGO induce T-cell response through CD2-LFA-3-T11TS interaction, and via other surface antigens bound by the oxidatively induced aldehyde groups on ENAGO.

Activation of human T lymphocytes through CD3 and CD2 (T11) with anti-CD3-coupled sheep erythrocytes.

Proliferative activation of T lymphocytes depends on cell-cell cooperation, i.e. interactions between specific cell surface receptors and their ligands. My co-workers and I have recently shown that an activating signal is mediated by interaction between the T cell surface antigen CD2 (T11) and its ligand on sheep erythrocytes (SE), the T11 target structure (T11TS), which is the homologue to the human LFA-3 antigen. Here I demonstrate that the anti-CD3 monoclonal antibody (MoAb) UCHT1 coupled to SE (SE-UCHT1) most efficiently induces accessory cell (AC)-independent T cell proliferation, and that SE can substitute for AC in stiumulation with phytohaemagglutinin (PHA). Inhibition studies with MoAb suggest that (1) concurrent stimulation of CD3 and CD2 is essential for interleukin 2 production and proliferation, since SE-UCHT1 treated with the anti-T11TS MoAb L180/1 are not mitogenic; (2) proximity of CD3 and CD2 is required during the stimulation, since a mixture of SE exposing either anti-CD3 or T11TS is not mitogenic; and (3) that T cell-AC interactions involving LFA-1 can be replaced by LFA-3-CD2 interactions, since the anti-LFA-1 MoAb 60.3 does not inhibit the SE-UCHT1 response, and only partly the SE + PHA response. These results demonstrate a functional linkage between the CD3 and CD2 structures, making accessory LFA-1 signals superfluous in proliferative activation of human resting peripheral T cell.

Accessory cell-independent activation of T cells by oxidized red blood cells plus polyethylene glycol.

We have previously shown that the combination of neuraminidase-galactose oxidase (NAGO)-treated autologous erythrocytes (EOX) and polyethylene glycol (PEG) is highly mitogenic for human peripheral blood lymphocytes (PBL). In this report, we show that EOX plus PEG-induced T lymphocyte proliferation is independent of HLA-DR and Leu M3-positive accessory cells (AC). Purified T (pT) cells and PBL were equally stimulated by EOX + PEG, while pT cells were unresponsive to the mitogens phytohemagglutinin (PHA), NAGO, and the anti-CD3 antibody UCHT1, even in the presence of PEG. These findings indicate that specific signals from AC may be replaced by unspecific stimuli in T cell activation.

Optically eliminating the visible outlines of pores in intact polycarbonate (Nuclepore) filters.

Under the microscope, Nuclepore filters display pore outlines. The polycarbonate material has two refractive indices (n = 1.584 and 1.616), which polarize transmitted light into two sets of rays at right angles to one another. This birefringence was used to eliminate the image of the pore outlines by the use of a specially made mounting medium with n20D = 1.584 in combination with polarized light: in a filter preparation of human leukocytes mounted in a medium with one matching refractive index and focused in polarized light, pore outlines were not visible. The preparation of the matching mounting medium, its use and its properties are described and discussed.

Stimulation of human lymphocytes by oxidized red blood cells (RBC) in the presence of polyethylene glycol (PEG).

In the present work, it is shown that oxidized red blood cells (RBC) in the presence of polyethylene glycol (PEG) is a powerful cellular class-II MHC-free mitogen for human lymphocytes. Human lymphocytes were stimulated with oxidized autologous human RBC or sheep RBC in the absence or presence of PEG, M.W. 10,000. The RBC were oxidized with the enzyme combination neuraminidase-galactose oxidase or with sodium periodate. The presence of PEG strongly potentiated the response. Optimal stimulatory conditions were obtained with a 10:1 ratio of oxidized RBC to lymphocytes and a PEG concentration of 25 +/- 5%.

Stimulation of human lymphocytes by phytohemagglutinin (PHA) in a new ultra-microtest plate.

The requirements for monocytes in lymphocyte proliferation were studied in ultra-microcultures. For this purpose, an ultra-microtest plate was developed which comprises 121 wells, each having 0.23 microliter volume and 0.28 mm2 culture area. Human peripheral lymphocytes were seeded in the wells in numbers ranging from 1-57 cells/well and subsequently stimulated with phytohemagglutinin (PHA). Proliferation, assessed by microscopy in situ, was established in 46% of the wells where adherent non-specific, esterase-positive cells were present and in 6% where such cells were absent. The results indicate that PHA-stimulated human lymphocytes can proliferate in the absence of monocytes. The new microplate should be a valuable tool for dissecting the early events in T cell activation, especially if combined with various analytical methods such as time-lapse video, autoradiography and surface-marker techniques.

Splenic macrophages during pregnancy in the mouse.

Pregnant CBA/Ca mice at mid-gestation showed a two-fold increase in the relative content of splenic macrophages, a change which was suggested to be due to enhanced migration. The proportion of spleen cells carrying a high density of Fc-receptors (assayed by EA-rosetting) was similarly increased in pregnant mice. The splenic content of non-lymphoid strongly esterase-positive cells was dramatically elevated at mid-gestation, but this increase was shown basically to be due to elevated numbers of esterase-staining erythroid blast cells. The pattern of the increase of Ig-secreting spleen cells was compared with that of macrophages and erythroid blast cells, and possible unspecific interaction at tissue-level is discussed.