Changing responsiveness to growth hormone releasing factor in the perinatal sheep

Synthetic human pancreatic growth hormone releasing factor 1-44-amide was administered (8 micrograms/kg iv bolus) to chronically catheterised fetal sheep between 77 and 135 days of gestation and to infant sheep. At all ages human pancreatic growth hormone releasing factor induced a significant growth hormone response. In fetuses less than 120 days the integrated growth hormone response to human pancreatic growth hormone releasing factor (n = 5) was 250 +/- (SE) 50 ng X hr X ml-1 compared (p less than 0.001) to -22.8 +/- 8.6 ng X hr X ml-1 in saline treated controls (n = 7). In fetuses older than 120 days (n = 5), the response to human pancreatic growth hormone releasing factor was 110.8 +/- 15.6 ng X hr X ml-1 compared to -12.0 +/- 17.6 ng X hr X ml-1 in saline treated controls (n = 4 p less than 0.001). In 4 infant lambs (4-12 days) the response to human pancreatic growth hormone releasing factor (56.5 +/- 14.5 ng X hr X ml-1) was greater than in 6 control injected lambs (0.95 +/- 1.5 ng X hr X ml-1). The magnitude of the response to growth releasing factor decreased progressively with increasing postconceptual age (r = -0.80, p less than 0.001). These observations demonstrate that the fetal somatotrope can respond to exogenous growth releasing factor from at least 77 days of gestation. The progressive decrease in responsiveness may reflect the gradual development of somatostatin mediated inhibitory control or altered responsiveness of the somatotrope.     

Cellular origin and release of intrinsic factor from isolated rat gastric mucosal cells

The cellular content and secretion of intrinsic factor was measured by [57Co]cyanocobalamin binding using isolated rat gastric mucosal cells. The intrinsic factor/R-protein ratio was above 9:1 as evaluated by specific anti-intrinsic factor antibodies. In unfractionized cells with 23 +/- 1.3% parietal cells the intrinsic factor content of 148 +/- 47 fmol/10(6) cells remained almost unchanged over 3 h, whereas basal secretion rose up to 57 +/- 10. In fractionized cells (Percoll) with 3-85% parietal cells most intrinsic factor was found in the parietal cell-depleted fraction (content: 441 +/- 30, secretion/3 h: 139 +/- 16, mean formation/h: 50 +/- 12 fmol/10(6) cells). The intrinsic factor content of the different cell fractions correlated with that of pepsin. [14C]Aminopyrine uptake, an indirect measure of parietal cell H+ production, was inversely related. Carbachol (1 X 10(-6)-10(-3) mol/l) stimulated intrinsic factor secretion, 1 X 10(-3) mol/l being maximally effective (90 +/- 8% above basal). This response was inhibited by atropine and pirenzepine, but not by prostaglandin E2 (PGE2) and somatostatin. Dibutyryl cyclic adenosine monophosphate (dibutyryl cAMP, 43 +/- 7%) and hexoprenaline (24 +/- 5%) enhanced intrinsic factor secretion less effectively and pentagastrin like histamine lacked any stimulatory effect. We conclude that in the rat intrinsic factor is produced and released from chief cells mainly under cholinergic control.     

IgG4 levels in polyvalent immunoglobulins and immunoglobulins prepared by precipitation with caprylic acid

Levels of IgG4 in immunoglobulin preparations obtained either by conventional ethanol fractionation or by ethanol and caprylic acid fractionation (Allergam) were measured by an enzyme-immunoassay (competitive Elisa). In 9 ethanol preparations, the mean percentage of IgG4 was 2.1% +/- 0.5. In 10 preparations of Allergam, the mean percentage of IgG4 was 4.6% +/- 1. The concentration of IgG4 in Allergam preparations is about twice the concentration found in conventional ethanol preparations.     

Studies of the mechanism of passive anaphylaxis in human airway smooth muscle

This investigation was carried out to study allergic contraction of passively sensitized human airway smooth muscle in response to specific antigen challenge. We attempted to determine the role played by histamine, slow reaction substances (SRSs), and cyclooxygenase products in the mediation of this response in tracheal smooth muscle. Tissues were passively sensitized with serum from ragweed-sensitive patients (15 h, 4 degrees C). Subsequent challenge with ragweed antigen produced a slowly developing contraction. The peak contraction to a dose producing a maximal response was 37 +/- 6% of the carbachol maximum. Mepyramine (5 X 10(-6) M) did not alter the contraction. Methylprednisolone (2 X 10(-5) M) attenuated the response to antigen but had no significant effect on the contractile response to arachidonic acid. Indomethacin (5.6-28 X 10(-6) M) enhanced the peak antigen-induced contractions by 25 +/- 11% whereas 5,8,11,14-eicosatetraynoic acid (6.4 X 10(-5) M) selectively attenuated the antigen-induced contraction by 86 +/- 12%. Nordihydroguarietic acid (6-12 X 10(-6) M) attenuated both the antigen plus arachidonate induced responses. FPL-55712 (1-2 X 10(-6) M) antagonized the contractions to antigen. Compound 48/80 and goat antihuman immunoglobulin E produced similar slowly developing contractions in sensitized and in some nonsensitized tissues. These responses, except for an early component of the response to 48/80, were independent of histamine and were reversed by FPL-55712. These findings suggest that arachidonic acid metabolites mediate (slow reacting substances) and modulate (prostaglandins) allergic contraction of human airway smooth muscle while any histamine released contributes little or nothing to the contraction in the larger airways.     

Reversibility of decreased insulin-stimulated glucose transport capacity in diabetic muscle with in vitro incubation. Insulin is not required

The mechanisms by which insulin deficiency affects muscle glucose transport were investigated. Epitrochlearis muscles from rats with streptozotocin-induced diabetes and from controls were incubated in vitro for 0.5-14 h. The incubation was shown not to impair muscle energy stores or tissue oxygenation. Diabetes decreased basal 3-O-methylglucose transport by 40% (p less than 0.01), and insulin-stimulated (20 milli-units/ml) glucose transport capacity by 70% (p less than 0.001). In vitro incubation gradually normalized insulin responsiveness (3.77 +/- 0.38 before versus 8.97 +/- 0.65 mumol X ml-1 X h-1 after 12 h of incubation). Basal glucose transport remained significantly reduced. The reversal of the insulin responsiveness did not require the presence of rat serum and, furthermore, took place even in the absence of insulin. In fact, insulin responsiveness was higher after incubation (14 h) with no insulin than with 100 microunits/ml insulin (9.85 +/- 0.59 versus 8.06 +/- 0.59 mumol X ml-1 X h-1, p less than 0.05). Glucose at 30 mM did not affect the normalization of the insulin-stimulated glucose transport capacity, whereas incubation in serum from diabetic rats resulted in a slightly (26%) blunted reversal (7.60 +/- 0.39 versus 8.89 +/- 0.45 mumol X ml-1 X h-1 with diabetic versus control serum for 14 h, p less than 0.05; before incubation the value was 3.87 +/- 0.40). Inhibition of protein synthesis by cycloheximide blocked the normalization by 80%. These results suggest the presence in diabetic serum of some labile factor that might inhibit the glucose transport system. The results indicate that the decreased insulin-stimulated glucose transport capacity, in the insulin-deficient diabetic muscle, is not a direct consequence of the lack of insulin or of high glucose concentrations.     

An ultrarapid filtration method adapted to the measurements of water and solute permeability of synthetic and biological vesicles.

An ultrarapid filtration method was adapted to the determination of water and solute permeability of membrane vesicles. This method consisted of measuring substance washout from vesicles first loaded with 3H2O or labeled solutes, placed on filters, and rinsed at high rates for short periods. The retention of the vesicles on the filters was analyzed and was found to be a function of the nature and porosity of the filters as well as of the vesicle origin. Washing buffer flow rate and washing duration did not affect vesicle retention. The diffusional water permeability of cholesterol-free liposomes was determined at 16 degrees C. Its value was reduced by a factor of 2.5 when the liposomes were prepared with 20% cholesterol and a threefold increase was noted when the liposomes were preincubated with gramicidin (6 mg/g lipid). Water permeability of liposomes was strongly temperature-dependent: Ea = 15.3 kcal/mol. Diffusional water permeability of pink ghosts was also measured: a value of (4.4 +/- 0.2) X 10(-3) cm/s (n = 3) was obtained at 13 degrees C. This permeability was reduced by 45.2% with 0.4 mM HgCl2. The urea permeability of intestinal and renal brush-border membrane vesicles was (1.15 +/- 0.18) X 10(-6) cm/s (n = 7) and (1.67 +/- 0.08) X 10(-6) cm/s (n = 9), respectively. The renal value was reduced by a factor of 4.4 by 100 mM thiourea. This ultrarapid filtration technique provides an accurate method of transport measurement in sealed membranes such as liposomes and plasma membrane vesicles.     

Colony-forming fibroblast precursors of the circulating blood

Colony-forming fibroblast precursors were detected in circulating blood of adult guinea pigs by CFUf in vitro colony assay. SFU amount to 0.9 +/- 0.2 (M +/- m) per 10(5) explanted leucocytes ranging from 0.04 X 10(-5) to 3.0 X 10(-5) for individual donors. The presence of collagen type I and lack of factor VIII antigen and of FC-receptors proved that CFU-derived colonies in the blood cultures were composed of fibroblasts.     

Shortening of bleeding time after intranasal administration of 1-deamino-8-D-arginine vasopressin to patients with chronic uremia

1-deamino-8-D-arginine vasopressin (DDAVP) was administered intranasally in a dose of 2 micrograms/kg BW to 17 uremic patients (16 maintained on chronic hemodialysis and 1 treated conservatively). The bleeding time was significantly shortened 120 minutes after DDAVP administration (from 18.1 +/- 7.5 minutes to 12.3 +/- 6.4 minutes p less than 0.001). Factor VIII related antigen (VIII: Ag) did not change. Factor VIII ristocetin cofactor activity (VIII: RCof) significantly increased (from 251.2 +/- 162.0 to 336.5 +/- 167.2 p less than 0.025). Platelet count decreased significantly after DDAVP (from 174.9 +/- 43.8 X 10(9)/l to 155.6 +/- 45.9 X 10(9)/l 30 minutes p less than 0.01 and 129.8 +/- 45.2 X 10(9)/l p less than 0.005 120 minutes after DDAVP). Antithrombin III concentration, and hematocrit did not change. Our data indicate that further clinical studies of intranasal DDAVP in uremic patients during episodes of bleeding are warranted.     

Calcium-induced conformational change in fragment 1-86 of factor X

Time-resolved fluorescence of the single tryptophan residue Trp41 in fragment 1-86 of factor X (FX F1-86) is studied using a time-correlated single photon counting technique with synchrotron radiation as the excitation source. Calcium ions are believed to induce a conformational change in the N-termini of the activated factor X and other vitamin K dependent proteins, which is accompanied by a decrease in fluorescence intensity. The titration with calcium yields a sigmoidal fluorescence titration curve with a transition midpoint concentration of 0.44 mM. The wavelength-dependent tryptophan fluorescence decays of the apo-FX F1-86 (in the absence of calcium) and Ca-FX F1-86 are characterized by conventional multiexponential analysis and fluorescence lifetime distribution analysis. In the absence of calcium there are three significant classes of fluorescence lifetimes (ns) that are nearly wavelength independent: 0.55 +/- 0.08 (component A), 2.6 +/- 0.1 (component B), and 5.3 +/- 0.3 (component C). However, their preexponential amplitudes vary with wavelength. The decay associated emission spectra of the individual components show that components B and C contribute over 85% to the total fluorescence for all examined wavelengths. However, in the presence of calcium, the analysis of the time-resolved fluorescence data of Ca-FX F1-86 yields four wavelength-independent lifetimes (ns) of 0.30 +/- 0.09 (component D), 0.65 +/- 0.10 (component A), 2.7 +/- 0.2 (component B), and 5.4 +/- 0.3 (component C). Calcium addition to the apo-FX F1-86 leads to a decrease in the fluorescence intensities of components B and C while their decay times remain unaffected. In Ca-FX F1-86 an additional component D arises that has a decay time of 0.30 ns and that contributes up to 35% to the total fluorescence intensity. A comparison with a previous investigation of prothrombin fragment 1 demonstrates the extensive structural and functional homology between the N termini of prothrombin and factor X(a).     

Determination of steady state and nonsteady-state glycerol kinetics in humans using deuterium-labeled tracer

Using deuterium-labeled glycerol as tracer and gas-liquid chromatography-mass spectrometry techniques for the determination of isotopic enrichment, we have developed a simple and ethically acceptable method of determining glycerol appearance rate in humans under steady-state and nonsteady-state conditions. In normal subjects, the appearance rate of glycerol in the post-absorptive state was 2.22 +/- 0.20 mumol X kg-1 X min-1, a value in agreement with those reported in studies with radioactively labeled tracers. The ratio nonesterified fatty acid (NEFA) appearance rate/glycerol appearance rate ranged from 1.95 to 3.40. In insulin-dependent diabetic patients with a mild degree of metabolic control, the appearance rate of glycerol was 2.48 +/- 0.29 mumol X kg-1 X min-1. The volume of distribution of glycerol, determined by the bolus injection technique, was (mean) 0.306 l X kg-1 in normal subjects and 0.308 l X kg-1 in insulin-independent diabetic patients. To evaluate the usefulness of the method for determination of glycerol kinetics in nonsteady-state conditions, we infused six normal subjects with natural glycerol and calculated the isotopically determined glycerol appearance rate using a single compartment model (volume of distribution 0.31 l X kg-1). During these tests, the expected glycerol appearance rates were successively 5.03 +/- 0.33, 7.48 +/- 0.39, 9.94 +/- 0.34, 7.48 +/- 0.39, and 5.03 +/- 0.33 mumol +/- kg-1 X min-1, whereas the corresponding isotopically determined appearance rates were 4.62 +/- 0.45, 6.95 +/- 0.56, 10.85 +/- 0.51, 7.35 +/- 0.34, and 5.28 +/- 0.12 mumol X kg-1 X min-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative interactions of factor IX and factor IXa with human platelets

Both factor IX and factor IXa were bound to gel filtered platelets in the presence of CaCl2 (2-20 mM) and human alpha-thrombin (0.06-0.2 units/ml) with maximal binding occurring in 10-20 min at 37 degrees C, and rapid reversibility was observed when unlabeled ligands were added in 100-fold molar excess. Competition studies with various coagulation proteins revealed that neither factor XI nor high molecular weight kininogen, at 300-fold molar excess, could compete with 125I-labeled factor IXa for binding sites on thrombin-activated platelets, whereas prothrombin and factor X, in 450-fold molar excess, could displace approximately 15 and 35%, respectively, of bound factor IXa in the absence of added factor VIII. Analysis of saturation binding data in the presence of CaCl2 and thrombin without factors VIII and X indicated the presence of 306 (+/- 57) binding sites per platelet for factor IX (Kd(app) = 2.68 +/- 0.25 nM) and 515 (+/- 39) sites per platelet for factor IXa (Kd = 2.57 +/- 0.14 nM). In the presence of thrombin-activated factor VIII (1-5 units/ml) and factor X (0.15-1.5 microM), the number of sites for factor IX was 316 (+/- 50) with Kd = 2.44 (+/- 0.30) nM and for factor IXa 551 (+/- 48) sites per platelet (Kd = 0.56 +/- 0.05 nM). Studies of competition for bound factor IXa by excess unlabeled factor IX or factor IXa, and direct 125I-labeled factor IXa binding studies in the presence of large molar excesses of factor IX, confirmed the conclusion from these studies that factor IX and factor IXa share approximately 300 low-affinity binding sites per thrombin-activated platelet in the presence of Ca2+ and in the absence of factor VIII and factor X, with an additional 200-250 sites for factor IXa with Kd(app) similar to that for factor IX. The presence of factor VIII and factor X increases by 5-fold the affinity of receptors on thrombin-activated platelets for factor IXa that participate in factor X activation.     

Purification of adenylosuccinate lyase from rat skeletal muscle by a novel affinity column. Stabilization of the enzyme, and effects of anions and fluoro analogues of the substrate.

The turnover of prothrombin and of factor X was investigated in rabbits fed on a 1%-cholesterol-supplemented or a standard diet by studying the evolution of radioactivity in blood and in plasma from these animals after the intravenous injection of either 125I-rabbit factor X or 125I-bovine prothrombin. For factor X, half-lives and fractional pool sizes were similar for the two groups of rabbits in the extravascular, intravascular and plasma compartments. However, the equivalent plasma fractional pool size for the two groups of rabbits was only 73% of that in the intravascular compartment. The fractional catabolic rate for the hypercholesterolaemic rabbits [0.064 +/- 0.007 (of the intravascular pool)/h] was not significantly different from that in the rabbits fed on the standard diet (0.074 +/- 0.008/h). However, the absolute catabolic rate, and therefore the rate of synthesis, was significantly higher (1.261 +/- 0.141 mg/day per kg body wt. of rabbit) in the rabbits fed on the cholesterol-supplemented than that in the rabbits fed on the standard diet (0.705 +/- 0.019 mg/day per kg). The prothrombin half-lives and fractional pool sizes were similar for the two groups of rabbits in the extravascular and the intravascular compartments. The fractional catabolic rate for the hypercholesterolaemic rabbits [0.041 +/- 0.003 (of the plasma pool)/h] was not significantly different from that in the rabbits fed on the standard diet (0.035 +/- 0.003/h). However, the absolute catabolic rate and therefore the rate of prothrombin synthesis was significantly higher (3.96 +/- 0.48 mg/day per kg body wt.) in the rabbits fed on the cholesterol-supplemented than that in the rabbits fed on the standard diet (2.24 +/- 0.12 mg/day per kg).     

Further quantification of human spermatogenesis: germ cell loss during postprophase of meiosis and its relationship to daily sperm production

Potential daily sperm production per gram of testicular parenchyma (PDSP/g) based on pachytene plus diplotene primary spermatocytes was determined by two methods for 10 men aged 26 to 53 years and compared with published daily sperm production per gram parenchyma (DSP/g) based on round spermatid nuclei for these same men. The PDSP/g based on primary spermatocytes was similar (P greater than 0.05) whether determined by histometric analysis (10.0 +/- 1.1 X 10(6] or from homogenates of parenchyma (10.4 +/- 1.0 X 10(6]. When PDSP/g values were compared to DSP/g (5.9 +/- 0.9 X 10(6) for the histometric or 5.5 +/- 0.8 X 10(6) for the homogenate method), 44.9 +/- 6.7% loss of potential sperm production during postprophase of meiosis was detected by the histometric approach and 48.3 +/- 7.9% loss by the homogenate method. Similar results were obtained when testes of 15 additional men aged 26 to 53 years were evaluated by the homogenate method; PDSP/g was 10.6 +/- 0.6 X 10(6), DSP/g was 5.8 +/- 0.5 X 10(6), and the loss during postprophase of meiosis was 45.3 +/- 4.4%. For all 25 men, DSP/g was significantly (P less than 0.01) correlated with PDSP/g (r = + 0.70) and with the percentage loss of potential sperm production during postprophase of meiosis (r = 0.86). Over 73% of the variation in DSP/g could be attributed to variation in the percentage loss during postprophase of meiosis. Whether faulty meiotic divisions and/or degenerating secondary spermatocytes were responsible, almost half of the potential sperm production in these 25 men was lost during postprophase of meiosis.     

Cooperative interaction between factor VII and cell surface-expressed tissue factor

Assembly of the extrinsic pathway on cell surfaces was investigated by studying the binding and activity of factor VII on the bladder carcinoma cell line J82 which expressed 18,800 milliunits of tissue factor activity/10(6) cells. In binding studies, the association of factor VII to monolayers of cells was time-, temperature-, and calcium-dependent. The ligand binding was specific, reversible, and saturable. This interaction was inhibited by a monoclonal antibody to human brain tissue factor. Factor VII added to the cells was recovered as factor VII rather than factor VIIa when incubated in the presence of factor X neutralizing antibodies, suggesting that these cells produced factor X. Specific factor VII binding to the cell revealed a sigmoidal binding isotherm with half-maximal binding occurring at 314 +/- 145 pM to 38,300 +/- 14,300 sites/cell. Hill plots of the binding data indicated an average slope of 2.1. Binding parameters were also determined kinetically. At maximal factor VII-tissue factor complex formation the apparent Km for factor X was 274 nM, the Vmax was 4.15 nM/min, and the kcat was estimated to be 14 s-1. In the presence of excess tissue factor and factor X, increasing amounts of factor VII added to the J82 cells demonstrated a sigmoidal relationship with the rate of factor Xa formation. Hill plots indicated a slope of 2.0 at the lower factor VII concentrations which changed to 1.0 at the higher input amounts of factor VII. Hanes plots were used to determine the apparent dissociation constant of the interaction (222 +/- 85 pM). The Vmax was 5.54 +/- 1.04 nM/min for the cleavage of factor X. These data are consistent with factor VII binding to at least two sites on tissue factor (receptor) with positive cooperativity. Because at saturation the stoichiometry of the factor VII-tissue factor complex is 1:1, tissue factor must be expressed as a dimer on the surface of the J82 cells.     

Respiratory impedance measured with head generator to minimize upper airway shunt

A new method for measuring total respiratory input impedance (Zrs), which ensures minimal motion of extrathoracic airway walls, was tested over frequencies of 4-30 Hz in 14 normal subjects and 10 patients with airway obstruction. It consists of applying pressure variations around the head, rather than at the mouth, so that transmural pressure across upper airway walls is equal to the small pressure drop across the pneumotachograph. Compared with reference Zrs values obtained by directly measuring airway wall motion with a head plethysmograph and correcting the data for it, the investigated method provided similar values for respiratory resistance at all frequencies (30 Hz, 3.67 +/- 2.24 cmH2O X 1(-1) X s compared with 3.55 +/- 2.00) but slightly overestimated respiratory reactance at the largest frequencies (30 Hz, 2.82 +/- 1.28 cmH2O X 1(-1) X s compared with 2.52 +/- 1.22, P less than 0.01). In contrast, when the data were not corrected for airway wall motion, resistance was largely underestimated, especially in patients (-48% at 30 Hz, P less than 0.001), and the reactance-frequency curve was shifted to the right. The investigated method is almost as accurate as the reference method, provides equally reproducible data, and is much simpler.     

The subpopulation structure of human T-lymphocytes studied by using a monoclonal antibody (MCA) to CD27]

The obtained murine mAb LT27 (IgG2a) assigned to the cluster of differentiation CD27 was used to study the distribution of antigen CD27 among human lymphocytes scbpopulations in normal state and immunopathology. In normal donors the antigen CD27 was found to be expressed most frequently on CD4+ cells (90 +/- 8% of which coexpressed antigen CD27) and to the lesser extent on- CD8+ cells (only 77 +/- 28% of CD8+ cells carried antigen CD27). 79 +/- 12% of double negative lymphocytes (CD3+CD4-CD8-) expressed antigen CD27. In patients with hypogammaglobulinemia the proportion of CD4+CD27+/CD4+ and CD8+CD27+/CD8+ was significantly reduced to 80 +/- 11% (p < 0.01) and 45 +/- 19% (p < 0.001), respectively. The ratio CD4+CD27+/CD4+ varied insignificantly with the increase of CD4+ population, but the increase of the CD8+ population was accompanied by the definite tendency to a decrease of the ratio CD8+CD27+/CD8+. The distribution of CD27 antigen inside CD4+ and CD8+ subpopulations was found to be different from the distribution of CD29 and CD45RA antigens.     

31P NMR measurement of ATP synthesis rate in perfused intact rat hearts

Using 31P NMR and the saturation-transfer method, the unidirectional rate of ATP synthesis was measured in isolated, Langendorff-perfused, isovolumic rat hearts operating at a rate pressure product of 25.6 +/- 2.5 (SE) X 10(3) mmHg X min-1 and consuming O2 at a rate of 35 +/- 2 mumol O2 X min-1 X (g dry wt)-1, at 37 degrees C. This rate was 7.2 +/- 0.9 mumol X s-1 X (g dry wt)-1 and was related to the rate of oxygen atom consumption by a ratio of 6.3 +/- 0.9. These data show that in the intact heart the unidirectional rate of ATP synthesis exceeds the net rate of ATP synthesis and consumption by approximately a factor of 2.     

IgE immune complexes induce immediate and prolonged release of leukotriene C4 (LTC4) from rat alveolar macrophages

Alveolar macrophages obtained by lung lavage from rats were incubated with monoclonal mouse anti-DNP IgE and specific antigen (DNP-HSA) and were found to release a slow reacting substance (SRS), which was characterized by high performance liquid chromatography as leukotriene C4 (LTC)4. Alveolar macrophages incubated with 1 microM A23187 (calcium ionophore) released similar amounts of SRS (6.0 +/- 2.2 and 5.7 +/- 3.7 X 10(-10) mol of LTC4 per 5 X 10(6) alveolar macrophages, respectively). The optimal conditions and mechanism of LTC release by IgE and antigen were examined. LTC4 release was maximal when freshly retrieved alveolar macrophages were incubated for 20 min with 10 micrograms/ml IgE and then for 20 min with 100 ng/ml antigen or for 20 min with IgE and antigen that had been preincubated together for 30 min at room temperature. In addition, LTC4 release was maximal when cells were challenged with IgE and antigen in a protein-free balanced salt solution and when the cells were tumbled to prevent adherence. Dose response experiments revealed that macrophages released LTC4 when stimulated with as little as 10 ng IgE and 100 ng DNP-HSA. Alveolar macrophages did not release LTC when challenged with IgE or DNP-HSA alone. Activation of LTC4 release by IgE and antigen was rapid in onset (2.5 to 5 min), and washing to remove fluid phase IgE and antigen revealed that once activated, alveolar macrophages were capable of prolonged and continuous release of LTC4. Peritoneal lavage cells stimulated with IgE and antigen did not release SRS but could release SRS when incubated with A23187 (5.7 +/- 1.3 X 10(-10) mol LTC4/5 X 10(6) macrophages). A large variability existed between individual rats in the ability of their alveolar macrophages to be activated by IgE and antigen to release LTC4. DNP-HSA labeled with 125I was used to show formation of immune complexes of IgE and antigen when IgE and antigen were incubated together before macrophage challenge. IgE immune complexes containing as little as 2 ng of antigen elicited the release of LTC4 from alveolar macrophages. These data indicate that rat alveolar macrophages release primarily LTC4 when challenged with IgE immune complexes, and that the alveolar macrophage may differ in this respect from peritoneal macrophages that do not release detectable quantities of LTC4 when challenged under identical conditions.     

Morphometric analysis of cell types in the ovine corpus luteum throughout the estrous cycle

The cellular composition of ovine corpora lutea obtained during the early (Day 4), mid (Days 8 and 12), and late (Day 16) stages of the estrous cycle was determined by morphometric analysis. Individual corpora lutea were collected via midventral laparotomy from a total of 19 ewes. A center slice from each corpus luteum was processed for electron microscopy and subsequent morphometric analysis of the numbers and sizes of steroidogenic and nonsteroidogenic cells. Luteal weight progressively increased throughout the estrous cycle (p less than 0.05). Corpora lutea collected on Day 16 were assigned to one of two subgroups on the basis of gross appearance and weight: nonregressed (NR, 542 +/- 25 mg) or regressed (R, 260 +/- 2 mg). There were no significant changes in the proportion of the corpus luteum occupied by small luteal cells (19 +/- 2%) or large luteal cells (36 +/- 1%) throughout the estrous cycle. The total number of steroidogenic cells per corpus luteum increased from 21.8 +/- 3.7 (X 10(6)) on Day 4 to 61.7 +/- 5.4 (X 10(6)) on Day 8 (p less than 0.05) and remained elevated thereafter. The number of small luteal cells was 10.0 +/- 2.7 (X 10(6)), 39.7 +/- 1.4 (X 10(6)), 46.1 +/- 5.8 (X 10(6)), 49.0 +/- 13.7 (X 10(6)), and 29.9 +/- 8.6 (X 10(6)) on Days 4, 8, 12, 16 (NR), and 16 (R), respectively (p less than 0.05, Day 4 vs. Days 8, 12, 16 NR). In contrast, the number of large luteal cells was 11.8 +/- 1.5 (X 10(6)) on Day 4 and did not vary significantly during the remainder of the estrous cycle. The numbers of nonsteroidogenic cell types increased (p less than 0.05) from Day 4 to Day 16 (NR) but were decreased in regressed corpora lutea (Day 16 R). Regression was characterized by a 50% decrease (p less than 0.05) in the total number of cells per corpus luteum from 243 +/- 57 ( X 10(6)) on Day 16 (NR) to 125 +/- 14 ( X 10(6)) on Day 16 (R) (p less than 0.05). Small luteal cells remained constant in volume throughout the entire estrous cycle (2520 +/- 270 microns 3), whereas large luteal cells increased in size from 5300 +/- 800 microns 3 on Day 4 to 16,900 +/- 3300 microns 3 on Day 16 (NR) (p less than 0.05). In summary, small luteal cells increased in number but not size throughout the estrous cycle, whereas large luteal cells increased in size but not number.     

Limiting dilution analysis of the frequency of antigen-reactive lymphocytes isolated from the central nervous system of Lewis rats with experimental allergic encephalomyelitis

Lymphocytes were isolated from the spinal cord and draining lymph nodes of Lewis rats with acute experimental allergic encephalomyelitis (EAE) 12 days after immunization with myelin basic protein (MBP) and tetanus toxoid (TT). An average of 8.0 +/- 2.0 X 10(6) cells was obtained from the spinal cord. Of these 71.1 +/- 8.6% expressed the helper-T-cell marker W3/25 and 14.8 +/- 6.2% expressed the killer/suppressor-T-cell marker OX8. By limiting dilution analysis of cells exhibiting an antigen-specific proliferative response, the average frequencies of cells reactive to MBP and TT were 3.36 +/- 2.4 and 7.60 +/- 4.1 per 10(4), respectively. In the draining lymph nodes, the frequencies of cells reactive to MBP and TT were 2.24 +/- 1.7 and 2.69 +/- 2.5 per 10(4). At a relatively early stage of clinical EAE, MBP-reactive T cells comprise only a small minority of the cells which can be isolated from the spinal cord; lymphocytes reactive to a protein antigen irrelevant to EAE pathogenesis are present in comparable numbers. This finding suggests that most of these cells accumulate as a result of mechanisms not specific for MBP-reactive lymphocytes.