The in vitro effect of toluene diisocyanate on lymphocyte cyclic adenosine monophosphate production by isoproterenol, prostaglandin, and histamine: a possible mode of action.

Toluene diisocyanate (TDI) significantly inhibits the rise in intracellular cyclic 3',5'-adenosine monophosphate (cAMP) that follows in vitro incubation of human lymphocytes with 6.7 x 10(-3) M isoproterenol and 1 x 10(-6) M prostagladin E1 (p less than 0.05). TDI has no significant effect on th production of lymphocyte cAMP following incubation with histamine (1 x 10(-3) M). The inhibitory action of TDI is greatest at a concentration of 3.3 x 10(-4) M and diminishes as the TDI concentration is increased or decreased. TDI also caused four- to fivefold stimulation of lymphocyte cAMP, an effect that is maximal at 1 x 10(-3) M, a concentration which has no significant inhibitory effect on stimulation of cAMP by isoproterenol or prostagladin E1. Conversely, 3.3 x 10(-4) M TDI, which inhibits cAMP production by isoproterenol and prostaglandin, has little stimulatory effect itself on cAMP production. This evidence suggests that TDI might induce obstructive airways disease through pharmacologic mechanisms and that TDI may be acting as a partial agonist.     

T lymphocyte activation is associated with viral replication in chronic hepatitis B virus infection of childhood.

Activated circulating T lymphocytes expressing HLA-DR (mean +/- s.d. 11.0 +/- 5.2%) or interleukin-2 receptor (IL-2R) (2.1 +/- 1.7%) were significantly increased in 63 children with chronic hepatitis B virus (HBV) infection when compared with 33 age-matched healthy controls (3.0 +/- 1.3%, P less than 0.01, and 0.1 +/- 0.4%, P less than 0.01). HBeAg-positive patients had higher percentage of DR (11.9 +/- 5.1%) or IL-2R (2.4 +/- 1.7%) positive T lymphocytes than anti-HBe-positive children (7.4 +/- 3.6% and 1.1 +/- 1.5%, P less than 0.01 and P = 0.02 respectively). Similarly, HBV DNA-positive patients had higher percentage of DR (10.5 +/- 3.3) or IL-2R (3.2 +/- 1.7%) positive T cells than HBV DNA-negative children (6.6 +/- 2.8% and 1.2 +/- 1.5%, P less than 0.01 for both). There was a positive correlation between percentage of DR positive T lymphocytes and levels of HBV DNA. Sixty-two per cent of the DR-positive T lymphocytes were cytotoxic/suppressor and 35% helper/inducer. The relationship between viral replication and T lymphocyte activation is discussed.     

Reduced sensitivity of lymphocyte beta-adrenergic receptors in patients with endogenous depression and psychomotor agitation

It has been suggested that there are altered levels of norepinephrine or other neurotransmitters at functionally important receptors in patients with depressive disorders. This hypothesis is difficult to study in the human central nervous system. However, noradrenergic function can be assessed indirectly with peripheral-blood lymphocytes used as a model of the beta-adrenergic receptor complex. We found that drug-free inpatients with endogenous depression had lower isoproterenol-stimulated cyclic AMP levels in intact lymphocytes than did healthy control subjects (3.9 +/- 0.5 vs. 7.4 +/- 1.0 pmol per 10(6) cells, P less than 0.01). The density and affinity of beta-adrenergic receptors were similar in controls and depressed subjects (beta-receptor number, 5.4 +/- 0.7 and 5.3 +/- 0.8 fmol per 10(6) cells; binding affinity, 106 +/- 7.6 vs. 99.2 +/- 11.4 pM, respectively). When the depressed patients were subdivided by psychomotor manifestations, binding characteristics were indistinguishable among the subgroups. However, a significant reduction in beta-adrenergic responsiveness was observed in patients with psychomotor agitation, as compared with controls (2.6 +/- 0.5 vs. 7.4 +/- 1.0 pmol per 10(6) cells, P less than 0.01), but not in patients with psychomotor retardation (5.8 +/- 1.1 pmol per 10(6) cells, P less than 0.05). Thus, the desensitization of beta-adrenergic receptors was correlated more closely with the severity of psychomotor agitation than with the overall severity of depression.     

Comparison of adherence of Pseudomonas aeruginosa to respiratory epithelial cells from cystic fibrosis patients and healthy subjects.

The adherence of Pseudomonas aeruginosa PAO1 to primary cultures of cystic fibrosis nasal polyp (CFNP), normal human nasal polyp (NHNP), and immortalized CF and normal cell lines was studied. PAO1 bound significantly more to primary CFNP cells than to NHNP cells as the mean adherence +/- standard deviation of 5 x 10(7) CFU of 35S-labeled bacteria per ml per well was 15.09 x 10(6) +/- 4.25 x 10(6) CFU/ml per well and 7.62 x 10(6) +/- 2.11 x 10(6) CFU/ml per well, respectively (Mann-Whitney U test, P less than 0.0001). There was no significant difference in PAO1 adherence to the immortalized CF and normal cell lines. The primary CFNP cells had more receptors (115 per cell) than did NHNP cells (34 per cell). P. aeruginosa binding to CFNP was blocked by GlcNAc, NeuAc, L-Fuc, and D-Gal, while binding to NHNP was blocked only by GlcNAc, suggesting that receptors on the two cell types were qualitatively different. Pseudomonas supernatants containing protease, phospholipase C, and neuraminidase activity increased adherence to CFNP and NHNP cells. The Pseudomonas exoproducts modified epithelial cell glycoconjugates, as characterized by binding of fluorescein isothiocyanate-labeled lectins and the release of sialic acid. There was minimal release of fibronectin by the bacterial supernatants. The affinity of P. aeruginosa for CF epithelial cells appeared to be due to an increased number of receptors and modification of the epithelial cell surface by P. aeruginosa exoproducts that exposed asialoganglioside binding sites.     

Decreased adrenergic responses in lymphocytes and granulocytes in atopic eczema.

The physiologic and cyclic adenosine monophosphate (cAMP) response to beta adrenergic stimulation in lymphocytes and granulocytes was examined in atopic eczema. These cells were isolated by Ficoll-Hypaque gradient from 10 patients with atopic eczema, and their responses were compared to 10 normal subjects. In eczema, basal concentrations of cAMP were normal in both lymphocytes and granulocytes. Lymphocyte cAMP response in eczema was decreased both to epinephrine (10-5 M) and to isoproterenol (10-5 M) but normal to prostaglandin E1 (PGE1). It was also noted that the glycogenolysis response to isoproterenol was significantly less at 10-5 M in eczema, but the fall in glycogen was normal with PGE (10-5 M and 10-7 M). The inhibition of lysosomal enzyme release from granulocytes after zymosan stimulation was significantly less (p less than 0.01) in eczema with all concentrations of isoproterenol tested. There was also a decrease in cyclic AMP response to isoproterenol in the polymorphonuclear leukocytes. PGE1 inhibited lysosomal enzyme release and stimulated cAMP normally. In eczema, both lymphocytes and polymorphonuclear leukocytes have a decreased beta adrenergic response.     

Platelet serotonin release by human polymorphonuclear leucocytes stimulated by cotton dust bacteria

Spontaneous cytotoxicity of human lymphocytes for tumours is increased by interferon (IFN) without change in the overall fraction of cells binding to targets. We developed an indirect immunofluorescent technique to stain lymphocytes conjugated to K-562 tumour cells in agarose with monoclonal antibodies. This allowed assessment of lymphocyte subpopulations binding to tumour cells without disruption of conjugates. Overall binding of non-adherent (NA) lymphocytes to tumour targets following incubation at 37 degrees C for 6 h was 13.3 +/- 0.3% compared to 12.5 +/- 0.7% with inclusion of IFN at 100 u/ml. When NA lymphocytes were incubated with K-562 tumour cells without IFN, OKM1 and OKT3 staining lymphocytes comprised 16.8 +/- 3.5% and 83.0 +/- 1.3% of the total lymphocyte population and 32.5 +/- 1.3% and 70.2 +/- 2.6% of lymphocytes conjugated to tumours. Incubation with IFN significantly increased OKM1 staining cells in the total NA population to 57.2 +/- 5.6% (P less than 0.01) and within tumour conjugates to 59.2 +/- 2.7% (P less than 0.01) while OKT3 staining cells decreased to 58.3 +/- 5.2% (P less than 0.02) and 45.3 +/- 1.2% (P less than 0.01), respectively. IFN increased cytotoxicity of NA cells for 51Cr-labelled K-562 by 66% at an effector to target ratio of 30:1 (P less than 0.001). These results demonstrate that OKM1 staining cells bind more avidly to tumour targets in the absence of IFN. IFN selectively increases the proportion of OKM1 staining lymphocytes with a concomitant increase in their binding to tumour cells. Therefore, enhancement of cytotoxicity by IFN in the NK system may result, in part, from conversion of OKT3 to OKM1 staining cells which are more efficient killers.     

Pilus-mediated adherence of Escherichia coli K1 to human oral epithelial cells.

We examined the effect of host age and health status on the adherence of mannose-sensitive piliated Escherichia coli K1 to human oral epithelial cells. Mannose-sensitive piliated bacteria adhered in comparable numbers to newborn, older infant, and adult cells (125 +/- 61, 198 +/- 54, and 139 +/- 69 bacteria per cell, respectively). Prematurity and serious illness did not alter adherence in newborns. The increased susceptibility of premature newborns to E. coli K1 cannot be explained by enhanced epithelial cell adherence.     

Enzyme replacement therapy of exocrine pancreatic insufficiency in man. Relations between in vitro enzyme activities and in vivo potency in commercial pancreatic extracts.

I assayed 16 commercially available pancreatic extracts (representing capsules, tablets and enteric-coated tablets) for enzyme activities and the relation between the in vitro activities and in vivo potency evaluated in man. Lipase activity ranged from 10 to 3600 units per unit, with 11 preparations containing less than 600 units per unit. The preparations with the highest lipase activities were llozyme, 3600, Kuzyme HP, 2330, Festal, 2073, Cotazym, 2014, and Viokase, 1636 units. Lipase activity in vitro correlated with potency in vivo for tablets and capsules, with tablets and capsules being effective in reducing steatorrhea by 56.1 +/- 9 per cent and 48.6 +/- 10 per cent (mean +/- S.E.M.) respectively, P less than 0.001. Enteric-coated tablets were less effective (20 +/- 13 per cent reduction, P greater than 0.02). The longer the gastric pH remained greater than or equal to 4 (r = 0.915, P less than 0.01) and the higher the average duodenal pH (r = 0.966, P less than 0.01), the more marked the reduction in steatorrhea.     

Halothane hepatitis. Detection of a constitutional susceptibility factor

We studied susceptibility to halothane hepatitis with an in vitro test that detects cell damage from electrophilic drug intermediates. Metabolites of phenytoin were generated by incubation of phenytoin with rat hepatic microsomes in the presence of the epoxide hydrolase inhibitor 1,1,1-trichloropropene oxide (TCPO), which prevents the further metabolism of phenytoin to an inert metabolite. In lymphocytes exposed to this system, cytotoxicity was measured by trypan blue dye exclusion and was expressed as the percentage increase in trypan blue-positive cells after the addition of TCPO. In the presence of TCPO, lymphocytes from 11 patients with halothane hepatitis exhibited an increase in cytotoxicity at 0.06 mM phenytoin that was eight times greater than the increase in healthy controls (54 +/- 10 per cent [mean +/- S.E.M.] vs. 7.1 +/- 2.2 per cent, P less than 0.0001). Patients with other liver diseases and persons recently exposed to halothane without adverse effects did not differ from healthy controls. In three patients with halothane hepatitis who were studied serially, the lymphocyte abnormality was still present after 13 months. Family studies revealed abnormal results on 10 cytotoxicity tests among 19 members of four families. We propose that there is a familial, constitutional susceptibility factor that predisposes persons to halothane hepatitis.     

Endogenous prostaglandins, adenosine 3':5'-monophosphate and sodium transport across isolated frog skin.

1. Sodium transport across isolated frog skin, as measured by the short-circuit current, was decreased by acetylsalicylic acid, mefenamic acid, paracetamol and phenylbutazone. Indomethacin (6 X 10(-6) M) had a biphasic effect on the short-circuit current: a transient increase followed by a sustained decrease. 2. The release of prostaglandin-like material from the skin was reduced by acetylsalicylic acid and indomethacin. Paracetamol caused a significant reduction in the short-circuit current response of the skin to low doses of arachidonic acid, but the response to the highest dose tested was not significantly altered. 3. Indomethacin (6 X 10(-6) M) increased the sensitivity of the skin to applied prostaglandin E1. The other prostaglandin synthetase inhibitors did not have this effect. Indomethacin (6 X 10(-6) M) also enhanced the effect of antidiuretic hormone on the short-circuit current. 4. Indomethacin (30 X 10(-6) M) increased the short-circuit current and diminished the response to applied prostaglandin E1. 5. In sulphate Ringer, theophylline increased the short-circuit current and diminished the response to prostaglandin E1. 6. Prostaglandin E1 increased the levels of cyclic AMP in frog skin and these increases preceded the increases in short-circuit current. There was a seasonal variation in the level of cyclic AMP in the skin: the levels in winter exceeded those in summer. There was also a seasonal variation in the cyclic AMP response to prostaglandin E1: the winter response was greater than that in summer. 7. Indomethacin (6 X 10(-6) M) had a biphasic effect on cyclic AMP levels in the skin, an initial increase followed by a decrease. Indomethacin also potentiated prostaglandin E1 stimulated cyclic AMP accumulation. 8. Theophylline increased cyclic AMP levels in the skin and potentiated prostaglandin E1 stimulated cyclic AMP accumulation. 9. Pre-treatment of the skin with theophylline reversed the effects of cyclic AMP on the short-circuit current and open-circuit potential. 10. It is concluded that endogenous prostaglandins help to maintain sodium transport across isolated frog skin and that the effects of E-type prostaglandins on the short-circuit current are mediated by increased cyclic AMP levels. The transient increase in short-circuit current and the increased skin sensitivity caused by indomethacin (6 X 10(-6) M) are attributed to inhibition of phosphodiesterase activity. The failure of theophylline to potentiate the short-circuit current response of the skin to prostaglandin E1 is attributed to alteration of cyclic AMP action on the skin by theophylline.     

Different mechanisms underlying reduced beta 2-adrenoceptor responsiveness in lymphocytes from neonates and old subjects

To investigate the mechanism underlying age-dependent changes in beta-adrenoceptor function we have determined beta 2-adrenoceptor density (by (+/-)-125iodocyanopindolol (ICYP) binding) and beta 2-responsiveness (cyclic AMP responses to isoprenaline stimulation) in lymphocytes derived from 20 neonates, 54 young adults (19-30 years) and 15 old subjects (60-86 years). In young adults the mean number of lymphocyte beta 2-adrenoceptors amounted to 862 +/- 36 (range 500-1560) ICYP binding sites/cell (N = 54); it was slightly higher in old subjects with 1230 +/- 94 (698-1980) ICYP binding sites/cell (N = 15). In contrast, lymphocytes derived from neonatal blood had a significantly lower mean beta 2-adrenoceptor number (385 +/- 35 (130-608) ICYP binding sites/cell, N = 20, P less than 0.01). (-)-Isoprenaline (0.01-100 microM)-induced increases in lymphocyte cyclic AMP content were significantly lower in neonates and old subjects than in young adults. While for neonates and young adults significant positive correlations between beta 2-adrenoceptor density and 10 microM (-)-isoprenaline-induced cyclic AMP increases exist, in old subjects cyclic AMP increases were much lower than could be expected from the beta 2-adrenoceptor number. It is concluded that the mechanism underlying reduced beta 2-adrenoceptor responsiveness in neonates and old subjects is different: while in neonates it seems to be due to the reduced beta-adrenoceptor number, in old subjects it is caused by a post-receptor defect--presumably by a decreased activity of the adenylate cyclase.     

An action of disodium cromoglycate: inhibition of cyclic 3',5'-AMP phosphodiesterase.

Disodium cromoglycate (DSCG) prevents allergic asthma by inhibiting the release of chemical mediators of immediate-type allergic reactions. The mechanism of this action is unclear and prompted us to examine the effect of DSCG on cyclic adenosine 3',5'-monophosphate (cAMP), the implicated regulator of IgE-mediated reactions. We used the peripheral blood lymphocyte as a model to mirror the biochemical events occurring in the allergic shock organs. Isolated peripheral blood lymphocytes from perennial allergic asthmatic children receiving only DSCG had significantly (p less than 0.005) lower phosphodiesterase (PDE) activity (mean 1.05 +/- 0.17 SE per 10(6) cells) than normal individuals (2.93 +/- 0.14) and allergic children receiving methylxanthines (4.08 +/- 0.28) or no medications (3.58 +/- 0.2). DSCG (10 mug/ml) significantly lowered PDE activity in normal lymphocytes (p less than 0.005) in a beef heart extract (p less than 0.001), and 100 mug/ml lowered PDE activity in fetal rabbit lung homogenates (p less than 0.001). DSCG (10 mug/ml) significantly elevated (p less than 0.01) cAMP concentration in normal human lymphocytes (118 +/- 38 vs 30 +/- 10 picomoles cAMP/10(6) lymphocytes). Thus, DSCG appears to inhibit chemical mediator release by increasing intracellular cAMP through the inhibition of cAMP PDE.     

Endotoxin enhancement of lymphocyte adherence to cultured sheep lung microvascular endothelial cells.

The most common predisposing factor for development of the adult respiratory distress syndrome is gram-negative sepsis. Our previous studies have shown that a single infusion of Escherichia coli endotoxin into sheep causes early sequestration of lymphocytes in the lungs' microcirculation. In this report, we examined the effects of endotoxin on sheep lymphocyte adherence to sheep pulmonary microvascular endothelial cells in vitro. Endothelial cells were exposed to endotoxin, and subsequent adherence of 51Cr-labeled lymphocytes was measured in a monolayer adhesion assay. Endotoxin enhanced adherence of lymphocytes isolated from blood and caudal mediastinal node (CMN) lymph in a time- and dose-dependent manner. Adherence of CMN lymphocytes increased from a control value of 13.6 +/- 1.6% to 29.9 +/- 3.1% after 4 h of treatment with 1 microgram/ml endotoxin. Both B and T lymphocytes contributed to the increased adherence. Pretreatment of the endothelial cells with cycloheximide revealed that the endotoxin-enhanced adherence was partially dependent upon protein synthesis. Morphologic studies revealed that enhanced adherence was accompanied by a 5-fold increase in migration of lymphocytes between endothelial cells. In contrast to human umbilical vein endothelial cells, antibodies to the known lymphocyte adherence molecules, lymphocyte function-associated antigen (LFA-1), CD-44, and the lymphocyte homing receptor (LECAM-1), were ineffective in blocking adherence to the sheep pulmonary endothelial cells. We conclude that the acute sequestration of lymphocytes in the pulmonary microcirculation of sheep after endotoxin administration is due to increased adhesive properties of the endothelial cells. Our data suggest that this adherence is mediated by as yet undescribed mechanisms that may be unique to pulmonary microvascular endothelium.     

Induction of aggregation of Raji human B-lymphoblastic cells by vasoactive intestinal peptide.

Subsets of neurons in the thymic cortex, Peyer's patches and lymphoid tissues of the respiratory system deliver vasoactive intestinal peptide (VIP) at nanomolar concentrations. The possible effects of VIP on B-cell adhesiveness in these tissues were examined in studies of the homotypic aggregation (HA) of human B-lymphoblastoid cells of the Raji line, which express a mean of 27,950 VIP receptors/cell with a mean Kd of 0.8 nM. Mean HA, assessed microscopically, attained a maximum of 54% after 8 hr with 0.1 microgram/ml of phorbol 12-myristate 13-acetate (PMA) (P < 0.01) and 31% after 24 hr with 10(-8) M VIP (P < 0.05), as contrasted with 13% and 20% at the respective times in medium alone, and both stimuli also increased the mean size of aggregates. The presence of the phosphodiesterase inhibitor Ro 20-1724 permitted 10(-9) M VIP, which had no effect alone, to raise the mean cyclic AMP content of Raji cells by more than 10-fold and concurrently to elevate mean HA from 55% in medium alone at 48 hr to 70% and from 55% at 72 hr to 68% (P < 0.05 for both). Monoclonal antibodies to lymphocyte function-associated (LFA-1) adhesive protein and to intercellular adherence molecule-1 (ICAM-1) suppressed significantly the HA of Raji cells induced by VIP and PMA. The effects of VIP on compartmental immunity in the lungs and intestines thus may be mediated in part by increases in lymphocyte adhesiveness, which could contribute to the regional accumulation of specifically immunocompetent cells.     

Angiotensin II stimulates airway ciliary motility in rabbit cultured tracheal epithelium

We studied the effect of angiotensin II on ciliary activity in cultured rabbit tracheal epithelium in vitro. Administration of angiotensin II (10(-6) M) elicited an increase in ciliary beat frequency (CBF), as assessed by a photoelectric method, from the baseline value of 906 +/- 21 to 1260 +/- 33 beats min-1 (mean +/- SE, P less than 0.001). This ciliostimulatory effect was dose-dependent, with the maximal increase and EC50 value being 35.6 +/- 5.2% (P less than 0.001) and 5 x 10(-12) M respectively. Nifedipine, Ca2(+)-free medium, indomethacin and the phospholipase A2 inhibitor mepacrine, but not nordihydroguaiaretic acid, reduced the change in CBF. The ciliostimulation induced by angiotensin II was abolished by pretreatment of tissues with [Sar1-Ile8]angiotensin II, an angiotensin II receptor antagonist. Angiotensin II did not increase cyclic AMP levels in epithelial cells. These results suggest that angiotensin II interacts with its specific receptors and stimulates airway ciliary activity through a Ca2(+)-dependent prostaglandin release, without affecting intracellular cyclic AMP levels. Thus, angiotensin II may modulate mucociliary transport function in the respiratory tract.     

Cell-mediated immunity to Epstein-Barr-virus-transformed lymphoblastoid cells in acute infectious mononucleosis.

Mononuclear peripheral blood leukocytes from 21 patients with infectious mononucleosis and 16 healthy controls were tested in a 51Cr-release assay for cytotoxicity against two human lymphoblastoid cell lines derived from the same donor. One line contained the Epstein-Barr virus (EBV); the other did not. Acute-phase leukocytes were significantly more cytotoxic against the EBV-infected cell line than were control leukocytes. Mean (+/- S.E.) lysis at a leukocyte-target-cell ratio of 100:1 was 10.6 +/- 1.6 per cent for patients and 3.4 +/- 0.6 per cent for controls (P less than 0.0005). Cytotoxicity correlated with the percentage of atypical lymphocytes. Cells of three patients with acute mononucleosis-like illnesses failed to show killing activity above those of normal controls. Cytotoxicity against the EBV-negative line was not significantly different for each group. The finding of cytotoxic cells in infectious-mononucleosis patients with atypical lymphocytes suggests that these cells operate in vivo to limit the proliferation of altered EBV-transformed B lymphoblasts.     

In vivo activated T lymphocytes in the peripheral blood and cerebrospinal fluid of patients with multiple sclerosis

We found an increase in peripheral-blood lymphocytes bearing the T-cell-specific activation antigen Ta1 in 20 of 35 patients with progressive multiple sclerosis, 4 of 18 patients with stable or improving multiple sclerosis, 1 of 17 patients with other neurologic diseases, and 1 of 14 normal controls (P less than 0.0002, Fisher's exact test). No increases in two other markers of T-cell activation, T113 and the interleukin-2 receptor, were found. In the cerebrospinal fluid, patients with progressive multiple sclerosis (pleocytosis, 3.9 +/- 1.6 cells per cubic millimeter) had 42 +/- 3.0 per cent Ta1+ cells. In contrast, patients with other inflammatory central nervous system diseases (36 +/- 13 cells per cubic millimeter) had 9.6 +/- 1.8 per cent Ta1+ cells (P less than 0.01). In patients with other neurologic diseases without inflammation (0.7 +/- 0.16 cells per cubic millimeter), the percentage of Ta1+ cells was equivalent to that in patients with multiple sclerosis (39 +/- 5.4 per cent), although the absolute number was lower. There was a positive correlation between the presence of Ta1+ cells in the spinal fluid and blood of patients with other neurologic diseases, but not patients with multiple sclerosis. Less than 1 per cent of lymphocytes from the spinal fluid of patients with multiple sclerosis expressed interleukin-2 receptors, as compared with 9.8 per cent of cells from subjects with other inflammatory neurologic diseases (P less than 0.01). These results suggest that the T cells in the spinal fluid of patients with multiple sclerosis may be activated by a different mechanism or in a different temporal sequence from that in patients with other nervous system diseases. Furthermore, the increase in Ta1+ cells in the peripheral blood of patients with multiple sclerosis demonstrates systemic immune activation in the disease; monitoring such cells may provide an objective measure of abnormal immunologic activity.     

Protection of human respiratory epithelium from Pseudomonas aeruginosa adherence by phosphatidylglycerol liposomes.

The ability of phosphatidylglycerol (DSPG) liposomes to prevent adherence of Pseudomonas aeruginosa to primary cultures of non-cystic fibrosis (CF) and delta F508 homozygous CF human respiratory epithelium was studied. The culture model was characterized by the simultaneous presence of various cellular phenotypes: well-differentiated respiratory epithelial cells, ciliated and nonciliated cells, and migrating cells which can be assimilated into a regenerating epithelium after injury. DSPG liposomes significantly decreased the binding of P. aeruginosa to migrating cells of both non-CF and delta F508 homozygous CF cultures compared with control cultures (35.5 x 10(-3) +/- 8.1 x 10(-3) bacteria per micron 2 versus 23.9 x 10(-3) +/- 2.5 x 10(-3); P < 0.01 for non-CF cultures and 88.8 x 10(-3) +/- 17.2 x 10(-3) bacteria per micron 2 versus 29.1 x 10(-3) +/- 0.6 x 10(-3), P < 0.001 for CF cultures). After treatment with DSPG liposomes, the size of P. aeruginosa aggregates bound to migrating cells in both non-CF cultures and delta F508 homozygous CF cultures was significantly decreased (14.4 +/- 3 bacteria per aggregate versus 11.9 +/- 2.5 bacteria per aggregate [P < 0.05] and 29.9 +/- 8.4 bacteria per aggregate versus 17.3 +/- 2.3 bacteria per aggregate [P < 0.01], respectively). Moreover, the control cultures were characterized by a differential P. aeruginosa adherence according to both the cellular phenotype and the mutation. The migrating cells bound more bacteria than the stationary cells of both non-CF and delta F508 homozygous CF cultures. The CF migrating cells bound significantly more bacteria than the non-CF migrating cells (88.8 x 10(-3) +/- 17.2 x 10(-3) bacteria per microns 2 versus 35.5 x 10(-3) +/- 8.1 x 10(-3) bacteria per micron 2, P < 0.001). These results suggest that DSPG liposomes are able to decrease P. aeruginosa adherence to CF and non-CF respiratory epithelium, particularly to migrating cells, which mimic a regenerating epithelium after injury. DSPG liposomes could also represent a hydrophobic barrier limiting the deleterious action of P. aeruginosa exoproducts.     

Response of 1alpha,25-dihydroxyvitamin D3 to hypocalcemia in human subjects.

We determined the response of 1alpha,25-dihydroxyvitamin D3 after mithramycin-induced hypocalcemia in eight subjects with polyostotic Paget's disease of bone. Thirty-six hours after infusion of mithramycin (25 microgram per kilogram), the average calcium declined from 9.9 +/- 0.14 (S.E.M.) to 8.0 +/- 0.19 mg per deciliter (P less than 0.005). Serum parathyroid hormone increased from 122 +/- 6 to 226 +/- 36 microliter eq per milliliter (P less than 0.05), serum phosphate decreased from 3.8 +/- 0.11 to 2.9 +/- 0.14 mg per deciliter (P less than 0.005), and urinary cyclic 3,5'-adenosine monophosphate increased from 4.6 +/- 0.35 to 7.5 +/- 0.80 mumol per gram of creatinine (P less than 0.005). Serum 1alpha25-dihydroxyvitamin D3 rose from 98 +/- 12 to 332 +/- 61 pM (P less than 0.05), the increase following the changes in parathyroid hormone and phosphate by 12 to 24 hours. When this lag period was taken into account, there was a significant relation (P less than 0.01) between the increase in 1alpha,25-dihydroxyvitamin D3 and changes in parathyroid hormone (correlation coefficient, r = +0.91) and phosphate (r = -0.96). The relatively rapid response of 1alpha,25-dihydroxyvitamin D3 to hypocalcemia occurs with a time course consistent with regulation by parathyroid hormone and phosphate.     

Heterogeneity of human lymphocyte subpopulations to pharmacologic stimulation : I. Lymphocyte responsiveness to beta adrenergic agents

Measurement of lymphocyte cyclic 3′5′-adenosine monophosphate (cyclic AMP) has been used in man to study the potential beta adrenergic defect in asthma. In this study, we evaluated the possibility of heterogeneity of beta adrenergic response with the use of the E rosette technique for thymic-derived (T) lymphocytes to compare a rosette-forming cell (RFC)-rich T cell population with a RFC-depleted (predominately bursal derived B lymphocyte) population. Lymphocytes were isolated and categorized into 4 populations: whole (unseparated) lymphocytes, RFC-rich, and RFC-depleted populations and a recombination population consisting of E-rosetting cells (T lymphocytes) + immunofluorescent-staining cells (primarily B lymphocytes) recombined in their initial proportion in the whole population. Kinetics and dose responses of isoproterenol-induced cyclic AMP were evaluated for each population. Maximal responses for all populations except the RFC-depleted population occurred at 5 and 12 min; the RFC-depleted population showed equal response at all incubation periods. Significant differences (p < 0.01) between the RFC-rich + -depleted population occurred at 5 and 12 min but not at 30 min. The RFC-rich population showed consistently greater cyclic AMP stimulation than the RFC-depleted population (p < 0.025) at 10⁻⁵ M (377% compared with 74% above baseline), 10⁻⁴ M (347% compared with 56%), and 10⁻³ M (400% compared with 80%) isoproterenol. No consistent differences were found between the whole and recombination populations at any dose of isoproterenol, suggesting no marked effect of the separation procedure itself. Propranolol at 1 × 10⁻⁴ M inhibited the isoproterenol stimulation of each population. The use of a phosphodiesterase inhibitor did not change the differences observed between RFC-rich and -depleted populations. These data suggest that striking heterogeneity of beta adrenergic responses exist between T lymphocytes and a B cell-enriched lymphocyte population. Studies evaluating beta adrenergic function in man must consider the suitability of this cell type because of the heterogeneity of peripheral blood lymphocyte subpopulations.