The consequences of K recycling and accumulation in the renal medulla were examined by measuring the effect of elevated K concentration on ion transport by the medullary thick ascending limb of Henle's loop. Perfused and bathed in vitro, thick limbs from both mouse and rabbit displayed a graded, reversible reduction of transepithelial voltage after increasing K concentration from 5 to 10, 15, or 25 mM. The effect was reproducible whether osmolality was 328 or 445 mosmol/kg H2O, and whether K replaced Na or choline. Net chloride absorption and transepithelial voltage were reduced by almost 90% when ambient K concentration was 25 mM. When either lumen or bath K was increased to 25 mM, net Na absorption was reduced. There was spontaneous net K absorption when perfusate and bath K concentration was 5 mM. Analysis of transepithelial K transfer after imposition of chemical gradients demonstrated rectification in the absorptive direction. Absorption of K by this segment provides a means to maintain high medullary interstitial concentration. Accumulation of K in the outer medulla, by reducing NaCl absorption, would increase volume flow through the loop of Henle and increase Na and water delivery to the distal nephron. K recycling thus might provide optimum conditions for K secretion by the distal nephron.
J B Stokes
In 17 adults, serum, hepatic bile, and saliva samples were analyzed for their sedimentation profile of IgA and secretory component (SC), and for their concentrations of albumin, orosomucoid, transferrin, IgG, IgA, alpha 2-macroglobulin (alpha 2M), IgM, and SC. Polymeric IgA(p-IgA) averaged 13% (50-700 micrograms/ml) of total IgA in serum, 70% (43-88%) in bile, and 93% (74-98%) in saliva. Most of the p-IgA in bile sedimented with SC, which also occurred free (8-44%), and with IgM. In bile, albumin (155-1,485 micrograms/ml) was the predominant protein, followed by IgG (32-480 micrograms/ml), and total IgA (37-209 micrograms/ml). In saliva, p-IgA (72-902 micrograms/ml) predominated, followed by albumin (16-385 micrograms/ml) and IgG (9-178 micrograms/ml). Secretion-to-serum albumin-relative concentration ratios (S/S-ARCR = 1 for albumin) in bile averaged 22 for p-IgA, 1.91 for IgM, 1.28 for monomeric IgA (m-IgA), 0.70 for IgG, and 0.57 for alpha 2M, indicating for p-IgA, IgM, and to a lesser extent for m-IgA, a selective excretion into bile. In saliva, a 16-fold greater selective excretion of p-IgA (mean S/S-ARCR = 354) was found. Labeled m- and p-IgA were injected intravenously into five patients. Specific activities indicated that for p-IgA 50% was serum derived in bile, as compared with 2% in saliva, and to 85% for m-IgA in bile. In the patient with the highest excretion of 125I-p-IgA in bile, only 2.8% of the injected dose was recovered in bile within 24 h after injection. Compared with rats and rabbits, the serum-to-bile transport of p-IgA in humans is much smaller.
D L Delacroix, H J Hodgson, A McPherson, C Dive, J P Vaerman
This study was designed to approach two primary questions concerning hematopoietic stem cells (HSC) in mice: what is the concentration of HSC with extensive proliferative potential in marrow, and how long can an HSC continue to function in an intact animal? The assay system was the W/Wv mouse, a mouse with an inherited HSC defect, reflected in a reduction in all myeloid tissue and most particularly in a macrocytic anemia.
Dane R. Boggs, Sallie S. Boggs, Debra F. Saxe, Lora A. Gress, Don R. Canfield
Hairy cell leukemia is a well described clinical entity, but the cell of origin for this leukemic cell and its function are still unknown. There are no totally specific markers for this cell, although tartrate-resistant acid phosphatase staining has been used extensively as a diagnostic test. This study describes three monoclonal murine antibodies with variable specificity for hairy cells. Antibody 1 was highly specific for hairy cells and was not found to react with normal or leukemic cells in this limited study. It did not react with the cells of all patients. It also did not react with all of the hairy cells of some of the positive cases. Antibodies 2 and 3 reacted with virtually all hairy cells but not with normal peripheral blood cells. However, reactions were obtained with certain leukemic myelomonoblasts and some activated B cells. The most obvious use for these three antibodies is for diagnostic purposes. They should also be helpful reagents to investigate the origin of the leukemic hairy cell. The possibility that antibody 1 detects a tumor-specific antigen is discussed.
D N Posnett, N Chiorazzi, H G Kunkel
Free fatty acid (FFA) transport was measured in 11 and glycerol turnover in 5 newborns with continuous tracer infusion of [1-13C]palmitate or [2-13C]glycerol, respectively. In addition, simultaneous determination of glucose production in the latter group with [6,6-2H2]glucose tracer and measurement of the appearance rate of [13C]glucose derived from [13C]glycerol allowed calculation of gluconeogenesis from glycerol.
P. F. Bougnères, I. E. Karl, L. S. Hillman, D. M. Bier
The presence of circulating immune complexes (IC) in patients with infective endocarditis has been well documented but the contributions of host and bacterial components to these IC have not been defined. To study this question, IC were isolated from serum of a patient with Streptococcus faecalis endocarditis by differential polyethylene glycol precipitation and competitive binding to staphylococcal protein A. A rabbit antiserum raised against the purified IC had reactivity by crossed immunoelectrophoresis primarily with an antigen derived from the cytoplasm of the infective organism. The antigen was a protein with a 12,000-dalton molecular mass. In situ radiolabeling of the IC bound to the protein A demonstrated a component of the same molecular mass as the bacterial antigen recognized by the antiserum. The patient serum had multiple antibody specificities reactive with bacterial antigens, including the antigen recognized by the rabbit anti-IC antiserum. These techniques for isolation and characterization of circulating IC may have value in the study of IC diseases in which the inciting antigens are not known.
R D Inman, P B Redecha, S J Knechtle, E S Schned, I van de Rijn, C L Christian
Pemphigus is an antibody-mediated autoimmune skin disease in which loss of cell-to-cell contacts in the epidermis results in blister formation. Patients with pemphigus develop antibodies that bind to the keratinocyte cell surface, the site of primary pathology. The purpose of this study was to characterize the antigen(s) to which pemphigus antibodies bind. Because we could detect pemphigus antigen by indirect immunofluorescence on the surface of multiply-passaged cells in cultures of both a spontaneously transformed mouse keratinocyte cell line (Pam) and normal human epidermal cells, we used these cells as a source of antigen. In order to demonstrate biosynthesis of antigen and to characterize the antigen(s), we radiolabeled cell cultures with [14C]glucosamine or d-[2-3H]mannose and used different pemphigus sera to immunoprecipitate antigen from nonionic detergent extracts of these labeled cells. Specifically precipitated radiolabeled molecules were identified using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and fluorography. Sera from five of seven pemphigus patients specifically precipitated (from extracts of both Pam cells and human epidermal cells) a molecule that, when reduced, was ∼130 kD, whereas seven normal human sera and two pemphigoid sera did not precipitate this molecule. The findings that (a) these precipitated molecules comigrated on SDS-PAGE and that (b) the 130-kD molecule could no longer be precipitated from cell extracts that had been previously reacted with a pemphigus serum, indicate that reactive pemphigus sera bind the same molecule. The molecule was not detected in the culture medium of these cells. This finding, along with the cell surface immunofluorescence pattern, suggests that the antigen is bound to the cell surface. Cultured mouse and human fibroblasts do not synthesize the antigen. The antigen contains protein because it was degraded by V8 protease and chymotrypsin, and it could also be labeled with [14C]amino acids. It is probably not a sulfated proteoglycan because it did not label with 35SO4. Taken together, these data indicate that some, but not all, pemphigus sera bind a specific cell surface glycoprotein that is synthesized by keratinocytes.
John R. Stanley, Mina Yaar, Pamela Hawley-Nelson, Stephen I. Katz
Entry of proteins into the cerebrospinal (CSF) from the blood is partially determined by the size of the protein. To determine whether other characteristics of proteins influence CSF entry, proteins or protein fragments were iodinated, inoculated intravenously, and serum and CSF were sampled at later times. The Fc fragment of immunoglobulin G (IgG) did not enter the CSF significantly better than the Fab fragment suggesting that choroidal Fc receptors are not of importance for selective immunoglobulin entry. To determine the role of protein charge on entry, bovine serum albumin [isoelectric point (pI) = 3.9] was chemically altered to provide an albumin with an average pI of 6 (A-6) and another with a pI of 8.5 (A-8). All albumins were of the same size on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A-8 entered the CSF approximately 10-fold better than the native albumin. A-6 was intermediate, entering approximately twofold better. At the time of increased CSF protein concentration during an acute viral encephalitis these differences were narrowed but not eliminated. It is concluded that charge is an important determinant of protein entry into the CSF.
D E Griffin, J Giffels
Antibodies to different components of the extractable nuclear antigen (ENA) have been thought to be serological markers for clinical subsets of rheumatic diseases. However, incomplete characterization and standardization of antigenic components such as ribonucleoprotein (RNP), Sm, and SS-B (Ha), and the multiplicity of autoantibodies produced by different patients have confounded correlations between autoantibody specificity and disease subsets. This study describes the preparative separation of the antigens Sm, RNP, and Ss-B (Ha) by electrofocusing and their use in a rocket electrophoretic assay that in one step identifies and quantifies the multiple reactivities of patient sera exhibiting the speckled FANA pattern. Preparative electrofocusing generates milligram quantities of these antigens with retention of their immunologic and biochemical characteristics, facilitating further study of their biological properties and relationships to disease subsets.
T B Okarma, J A Krueger, H R Holman
The platelets from three patients with Bernard-Soulier syndrome have been analyzed by surface-labeling coupled with two-dimensional gel electrophoresis and compared with normals. As well as the previously described absence or deficiency in glycoprotein (GP) Ib(alpha) it could be shown that GP Ib beta and an additional low molecular weight glycoprotein GP17 were not detectable using carbohydrate-labeling methods or deficient to the same extent as the GPIb alpha subunit. In addition, the thrombin cleavable glycoprotein could not be detected using carbohydrate-labeling methods in two patients and was deficient in a third. This finding was confirmed in a fourth patient by one-dimensional gel electrophoresis. Thus, the changes in the membrane of Bernard-Soulier platelets are more complex than previously thought.
K J Clemetson, J L McGregor, E James, M Dechavanne, E F Lüscher
In this investigation we found that little of intravenously infused [14C]deoxycorticosterone (DOC) was converted to [14C]DOC-SO4 that entered plasma. Moreover little of intravenously infused [3H]DOC-SO4 was metabolized by way of DOC except by intestinal bacterial enzymes. However evidence was obtained that plasma DOC is converted to DOC-SO4 in liver, but little of the DOC-SO4 formed in liver escapes into blood; rather the DOC-SO4 enters bile and in the intestine is converted, in part, to progesterone (or metabolites thereof) by the action of bacterial enzymes. The estimated intrahepatic fractional conversion of DOC to DOC-SO4 was significantly greater in premenopausal women (0.72 +/- 0.118, mean +/- SEM) than in men (0.28 +/- 0.036, P less than 0.005).
M L Casey, P C MacDonald
Several aspects of the myocardial O2 supply/consumption relationship were determined after coronary artery occlusion and subsequent β-adrenergic blockade in 16 anesthetized open-chest dogs. Small artery and vein O2 saturations, and hence extraction, were obtained microspectrophotometrically and combined with radioactive microsphere blood flow determinations to calculate regional myocardial O2 consumption. Eight dogs remained untreated after coronary artery ligation while another group was given 2 mg/kg propranolol, 10 min after occlusion. Untreated occlusion resulted in decreased arterial and especially venous O2 saturations, indicating an increased O2 extraction. Ischemic O2 consumption was reduced and the subendocardial/subepicardial consumption ratio was reversed (1.26 vs. 0.37) due to the pattern of occluded area flow. Calculated O2 supply/consumption also decreased. Propranolol produced no significant changes in volume or distribution of flow within the ischemic region while reducing flow, extraction, and consumption in the unoccluded region. The heterogeneity of arterial and particularly venous O2 saturations within the ischemic region decreased dramatically. Venous O2 saturations were elevated relative to the control group resulting in a reduced O2 extraction. The decrease in heterogeneity of arterial and venous O2 saturations suggest that propranolol eliminates microregions of relatively high O2 extraction, consumption, and/or a majority of vessels with extremely low flow. This leads to a significant improvement in the O2 supply/consumption ratio in the ischemic myocardium of the dog. This may be due to a reduction in the heterogeneity and level of β1-adrenergic receptor activity within the heart.
Robert S. Conway, Harvey R. Weiss
Peripheral blood T lymphocytes from 29 of 31 patients with rheumatoid arthritis incorporated significant quantities of thymidine when cultured with pooled human immunoglobulin G (IgG). In contrast to the observation of general reactivity to pooled Igg, responses to pooled IgM were rare (3 of 26 patients). None of 11 controls responded to either IgG or IgM. Response to IgG is maximal on day 6 of culture and is dependent on concentration of IgG. The responding cells recognize determinants on monoclonal light chains and/or Fab fragments. Response to light chains follows one of three patterns: preferential response to lambda chains, preferential response to kappa chains, and essentially equal response to either kappa of lambda chains.
N Radoiu, R P Cleveland, M A Leon
The suppression of renin release by angiotensin II (AII) (the so-called short feedback loop) is blunted in essential hypertension. To determine whether this abnormality is reversible, renin release was assessed in sodium-restricted essential hypertensives and normal controls: (a) during the administration of captopril for varying intervals and (b) following the infusion of graded doses of AII (0.3-3 ng/kg per min) before and after plasma levels of AII had been chronically reduced with captopril (25-50 mg every 6 h) for 70 h.
Meryl S. Leboff, Robert G. Dluhy, Norman K. Hollenberg, Thomas J. Moore, Richard J. Koletsky, Gordon H. Williams
The oxidant damage of lung tissue during in vivo hyperoxic exposure appears to be amplified by neutrophils that release toxic amounts of oxygen metabolites. In our studies cloned lung epithelial cells (L2 cells), lung fibroblasts, and pulmonary artery endothelial cells were cultured under either ambient (Po2 ∼ 140 torr) or hyperoxic (Po2 ∼ 630 torr) conditions for 48 h (24 h for endothelial cells). After cultivation, phorbol myristate acetate- or opsonized zymosan-stimulated neutrophils were added to the cultivated monolayers for 4 h, and lung cell damage was quantitated using 51Cr release as an index. The data show that stimulated neutrophils are able to injure the three lung cell lines tested, with endothelial cells being highly susceptible to this injury and L2 cells being slightly more susceptible than lung fibroblasts. The studies also demonstrate that all three lung cell lines exposed to sustained hyperoxia are more susceptible to neutrophil-mediated cytotoxicity than their time-matched air controls. Hydrogen peroxide was the main toxic oxygen metabolite because catalase (2,500 U/ml) completely protected the target cells. Equivalent quantities of hydrogen peroxide generated by glucose oxidase instead of by neutrophils gave a similar degree of target cell injury. Superoxide dismutase at high concentrations (250 μg/ml) provided some protection. Other systems that detoxify oxygen metabolites were without protective effect. These findings indicate that the increase in susceptibility of lung cells to neutrophil-mediated oxidant damage is a toxic effect of hyperoxia on lung cells. This specific manifestation of oxygen damage provides insight into the integration between primary mechanisms (oxygen exposure) and secondary mechanisms (release of oxygen metabolites by neutrophils) with respect to the cellular basis for pulmonary oxygen toxicity.
Norbert Suttorp, Lawrence M. Simon
To determine the pharmacologic importance of methotrexate (MTX) polyglutamates, we examined the formation, retention, and effect of these metabolites in cultured human breast cancer cells. Two cell lines (MCF-7 and ZR-75-B) converted the drug to γ-polyglutamate derivatives in a dose- and time-dependent reaction. After 24-h incubations with 2 μM MTX, polyglutamates of two to five amino acids in length accounted for 55.4% (51.9 nmol/g) of intracellular drug in the MCF-7 cells and 87.6% (62.4 nmol/g) of drug in ZR-75-B cells. In contrast, MDA-231 cells showed lesser accumulation of MTX, and only 32% (4.06 nmol/g) of the intracellular drug was in the form of polyglutamates, a difference that could only partially be explained by decreased ability of these cells to take up free drug from the medium. When MCF-7 and ZR-75-B cells containing polyglutamates were transferred to drug-free medium for 24 h, 22 and 51% of the total intracellular drug were, respectively, retained in each cell line. The loss of intracellular drug was primarily accounted for by disappearance of parent compound and polyglutamates containing 1-3 additional glutamyl residues. The rates of disappearance from cells decreased with increasing glutamyl chain length. All of the 4-NH2-10-CH3-PteGlu5 and 47 and 38% of the 4-NH2-10-CH3-PteGlu4 remained in the MCF-7 and ZR-75-B cells, respectively, and could be identified in the cytosol after 24 h in drug-free medium. The retention of MTX polyglutamates in these two cell lines in excess of dihydrofolate reductase binding capacity led to prolonged inhibition of thymidylate synthesis and loss of cell viability after removal of extracellular MTX. After 24-h incubation with 2 μM MTX and an additional 24 h in drug-free medium, [3H]deoxyuridine incorporation was still inhibited to 30% of control in the MCF-7 cells and 34.7% of control in ZR-75-B cells; this persistent inhibition was associated with a 30% reduction in cell numbers in each cell line during the 24-h period in drug-free medium. In contrast, [3H]deoxyuridine incorporation and cell growth quickly recovered to normal in the MDA-231 cells following removal of 2 μM MTX from the medium after a 24-h incubation. Prolonged inhibition of both thymidylate synthesis and cell growth was observed in this cell line in drug-free medium only after a 24-h incubation with 10 μM MTX, a condition that leads to the synthesis of 11.3 nmol/g of MTX polyglutamates.
Jacques Jolivet, Richard L. Schilsky, Brenda D. Bailey, James C. Drake, Bruce A. Chabner
Humoral factors released from platelets during pulmonary embolism may be the cause of several attendant cardiopulmonary abnormalities. This study examines the role of thromboxanes (Tx) after experimental embolism induced with 0.5 g/kg autologous clot in four groups of five dogs: (a) untreated embolized controls; (b) pretreatment with the Tx synthetase inhibitor, imidazole 25 mg/kg · h i.v., starting 30 min before embolization; (c) pretreatment with the cyclooxygenase inhibitor indomethacin, 5 mg/kg, 12 h per os and 1 mg/kg, 1 h i.v. before the experiment; (d) treatment with prostacyclin (PGI2) 100 ηg/kg · min i.v. for 1 h, 1 h after embolization. Within 30 min, embolization led to increases of 6-keto-PGF1α, the stable hydrolysis product of PGI2, from 0.11±0.08 ηg/ml (mean±SD) to 0.33±0.10 ηg/ml (P < 0.005) and TxB2, the stable product of TxA2, from 0.10±0.04 ηg/ml to 0.38±0.06 ηg/ml (P < 0.001). Increases were observed in total dead space (VD/VT) from 0.46±0.03 to 0.61±0.08 (P < 0.025, physiologic shunting (Q̇S/Q̇T) from 16±4% to 38±9% (P < 0.01), pulmonary vascular resistance (PVR) from 2.27±0.59 mm Hg·min/liter to 9.21±1.90 mm Hg·min/liter (P < 0.005) and mean pulmonary arterial pressure from 14±6 mm Hg to 34±1 mm Hg (P < 0.001). Cardiac index (CI) fell from 139±11 ml/kg·min to 95±17 ml/kg·min in 4 h (P < 0.025). Imidazole pretreatment prevented a rise of TxB2, but not 6-keto-PGF1α; indomethacin blocked both. Both agents maintained VD/VT at base line and limited increases in Q̇S/Q̇T and PVR. CI was higher after imidazole pretreatment compared with controls (P < 0.025). Indomethacin led to intermediate levels of CI. PGI2 lowered TxB2 (P < 0.025), VD/VT (P < 0.025), Q̇S/Q̇T (P < 0.025) and PVR (P < 0.05) within 30 min. During PGI2 infusion, CI was higher than controls. Concentrations of TxB2 correlated with VD/VT, r = 0.79 and Q̇S/Q̇T, r = 0.69 (P < 0.001). Treatment of three dogs with the imidazole derivative ketoconazole, 10 mg/kg IV, 30 min after 0.75 g/kg autologous clot resulted in a lowering of physiologic dead space, but no other improvement of cardiopulmonary function. These results show that a number of cardiopulmonary abnormalities induced by pulmonary embolism are related directly or indirectly to platelet secretions and that VD/VT is closely allied to TxA2 levels.
Takayoshi Utsunomiya, Michael M. Krausz, Lawrence Levine, David Shepro, Herbert B. Hechtman
The role of cytotoxic T lymphocytes in host defenses against infectious agents is unknown as these cells have not previously been demonstrated to kill microorganisms directly. We studied the cytotoxicity of T lymphocytes purified from peripheral blood mononuclear cells of healthy subjects for the multicellular schistosomula of Schistosoma mansoni. Unstimulated and phytohemagglutinin (PHA)-stimulated T cells were cultured with schistosomula at a 5,000:1 effector/target (E:T) ratio for 18 h at 37 degrees C. Unstimulated T cells killed 2.1 +/- 0.6% of schistosomula as judged by dye uptake and did not change their infectivity for mice. In contrast, PHA-stimulated T cells killed 41.3 +/- 3.1% of schistosomula by dye uptake and 56.7 +/- 7.7% of these organisms could not mature to adult worms in vivo. Killing was associated with and dependent on increased binding of PHA-stimulated T lymphocytes to schistosomula. Significant schistosomula killing first was noted after 2 h of exposure to T cells to PHA and peaked at 24; enhanced killing by PHA-stimulated cells was observed at an E:T ratio of 500:1 and was maximal at 5,000:1. Exposure of T lymphocytes to oxidative mitogens, soluble antigens, and alloantigens also resulted in enhanced killing of schistomula. These studies show that T lymphocytes activated by a variety of stimuli develop the capacity to kill schistosomula of Schistoma mansoni. Direct killing of infectious agents by cytotoxic T cells may contribute to host resistance to infections.
J J Ellner, G R Olds, C W Lee, M E Kleinhenz, K L Edmonds
Erythrocytes infected with Plasmodium falciparum trophozoites and schizonts are not seen in the peripheral circulation because they attach to venular endothelium via knoblike structures on the infected erythrocyte membrane. We have recently shown that erythrocytes containing P. falciparum trophozoites and schizonts likewise attach to cultured human venous endothelial cells via knobs. In search of a more practical target cell for large scale binding studies designed to characterize and isolate the knob ligand, we tested various normal cells and continuous cell lines for their ability to bind P. falciparum-infected erythrocytes. Of the 18 cell types tested, binding of infected erythrocytes was observed to a human amelanotic melanoma cell line and amnion epithelial cells as well as to human aortic and umbilical vein endothelial cells. 96-100% of amelanotic melanoma cells bound 17±4 (±1 SEM) infected erythrocytes per positive cell, whereas fewer endothelial cells (4-59%) and amnion epithelial cells (8-19%) were capable of binding 12±5 and 4±1 infected erythrocytes per positive cell, respectively. Further studies designed to compare the mechanism of binding to the amelanotic melanoma cell line and endothelial cells showed the following results. First, that adhesion of infected erythrocytes to these two cell types was parasite stage-specific in that only erythrocytes containing late ring forms, trophozoites, and schizonts bound. Erythrocytes containing early ring forms, which do not attach to venular endothelium in vivo, did not bind to either cell type. Second, erythrocytes infected with trophozoites and schizonts of P. vivax or a knobless strain of P. falciparum, both of which continue to circulate in vivo, did not bind to either target cell type. Third, transmission electron microscopy showed that infected erythrocytes attached to the amelanotic melanoma cells via knobs. We conclude that cultured human endothelial cells and an amelanotic melanoma cell line share common determinants on their surface and that the mechanism of binding to these two different cell types is similar. The amelanotic melanoma cell line offers a useful substitute for endothelial cells in binding studies requiring large numbers of target cells.
John A. Schmidt, Iroka J. Udeinya, James H. Leech, Robert J. Hay, Masamichi Aikawa, John Barnwell, Ira Green, Louis H. Miller
To determine if previously reported peripheral blood suppressor cell defects are also found in the central nervous system (CNS) of patients with multiple sclerosis (MS), we studied cerebrospinal fluid (CSF) and peripheral blood lymphocytes from 40 MS patients and 15 patients with other neurological diseases. With an indirect immunofluorescence technique using the OKT series of monoclonal antibodies (OKT4, marking helper/inducer cells, OKT5 and OKT8 marking suppressor/cytotoxic cells, and OKT3 marking all peripheral T cells) we found that MS patients tested in the first 2 wk of exacerbation had invariably diminished CSF suppressor/cytotoxic cells, which was followed by an elevation of these cells in the 3rd wk of exacerbation. Repeat studies of three patients showed that perturbations of CSF suppressor/cytotoxic cells were dependent on clinical status. These observations add to the accumulating data that suggest altered immunity in the pathogenesis of MS.
N Cashman, C Martin, J F Eizenbaum, J D Degos, M A Bach
We have analyzed the cellular interactions required for the generation of histamine- and concanavalin A (Con A)-induced suppressor T cells by employing a co-culture assay and techniques for fractionation of human blood mononuclear cells (PBMC). PBMC cultured in the presence of histamine (0.1 mM-1 mM) or Con A (20 micrograms/ml) for 24 h, mitomycin treated and subsequently combined with autologous mitogen-stimulated mononuclear cells, significantly suppressed a subsequent blastogenic response. PBMC fractionated over nylon wool columns and depleted of adherent cells and enriched for T cells (NWNA-T) were unable to generate suppressor activity. However, suppressor cell function by NWNA-T cells was reconstituted by the addition of autologous monocytes. In both the histamine and ConA suppressor systems, the requirement for monocytes in the activation process was enhanced by suspending the NWNA-T population in supernatants derived from allogeneic monocytes stimulated with heat-killed Staphylococcus albus. These crude supernatants contained leukocytic pyrogen (LP) and lymphocyte activating factor (LAF). Sequential purification and separation of the crude supernatants using gel-filtration, immunoadsorption, and isoelectric focusing demonstrated that only those fractions containing LP and LAF were capable to reconstituting NWNA-T cell histamine and Con A-induced suppressor activity. Thus, these studies suggest that the accessory role of supernatants derived from activated monocytes in the generation of suppressor cells may be mediated by LP/LAF. Further studies are in progress to explore the mechanism by which soluble factors stimulate suppressor T cells.
D J Beer, C A Dinarello, L J Rosenwasser, R E Rocklin
Expansion of the bile salt pool size in rats increases maximum excretory capacity for taurocholate. We examined whether increased bile salt transport is due to recruitment of centrolobular transport units or rather to adaptive changes in the hepatocyte. Daily sodium cholate (100 mg/100 g body wt) was administered orally to rats. This treatment was well tolerated for at least 4 d and produced an 8.2-fold expansion of the bile salt pool. This expanded pool consisted predominently (99%) of cholic and deoxycholic acids. Significantly increased bile salt transport was not observed until 16 h after bile acid loading, and maximum elevations of transport capacity to 2.3-fold of control required ∼2 d. In contrast, maximum sulfobromophthalein excretion rates increased 2.2-fold as early as 4 h and actually fell to 1.5-fold increase at 4 d. We studied the possibility that this adaptive increase in bile salt secretory transport was due to changes in canalicular surface membrane area, lipid composition, or increased number of putative carriers. Canalicular membrane protein recovery and the specific activities of leucine aminopeptidase, Mg++-ATPase and 5′-nucleotidase activities were unaltered by bile salt pool expansion. The content of free and esterified cholesterol and total phospholipids was unchanged in liver surface membrane fractions compared with control values. In contrast, sodium cholate administration selectively increased specific [14C]cholic acid binding sites twofold in liver surface membrane fractions. Increased numbers of [14C]cholic acid receptors (a) was associated with the time-dependent increase in bile salt transport, and (b) was selective for the taurine conjugate of cholate and (c) was reduced by chenodeoxycholate. Changes in bile acid binding sites 16 h following taurocholate and chenodeoxycholate and the lack of change with glycocholate was associated with comparable changes in bile salt transport. In conclusion, selective bile salts increase bile salt transport in the liver through an adaptive increase in the density of putative bile acid carriers in liver surface membrane.
Francis R. Simon, Eileen M. Sutherland, Manuel Gonzalez
The catabolic pathways of streptokinase, plasmin, and activator complex prepared with human plasminogen were studied in mice. 125I-streptokinase clearance occurred in the liver and was 50% complete in 15 min. Incubation with mouse plasma had no effect on the streptokinase clearance rate. Complexes of plasmin and α2-plasmin inhibitor were eliminated from the plasma by a specific and saturable pathway. Competition experiments demonstrated that this pathway is responsible for the clearance of injected plasmin. Streptokinase-plasminogen activator complex formed with either 125I-plasminogen or 125I-streptokinase cleared in the liver at a significantly faster rate than either of the uncomplexed proteins (50% clearance in <3 min). Streptokinase incubated with human plasma also demonstrated this accelerated clearance. p-Nitrophenyl-p′-guanidinobenzoate-HCl or pancreatic trypsin inhibitor-treated complex cleared slowly compared with untreated complex independent of which protein was radiolabeled. Significant competition for clearance was demonstrated between α2-macroglobulin-trypsin and activator complex only when the plasmin(ogen) was the radiolabeled moiety. Large molar excesses of α2-plasmin inhibitor-plasmin failed to retard the clearance of activator complex. Hepatic binding of streptokinase-plasmin, in liver perfusion experiments, was dependent upon prior incubation with plasma (8-10% uptake compared to a background of ∼ 2.5%). Substitution of human α2-macroglobulin for plasma also resulted in binding when the incubation was performed for 10 min at 37°C (7.5%). Electrophoresis experiments confirmed the transfer of 0.8 mol plasmin/mol α2-macroglobulin when activator complex was incubated at 37°C with α2-macroglobulin for 40 min. Streptokinase transfer from activator complex to α2-macroglobulin was negligible. The in vivo clearance of activator complex is proposed to involve active attack of the complex on the α2-macroglobulin “bait region,” resulting in facilitated plasmin transfer. Dissociated streptokinase is rapidly bound and cleared by sites in the liver.
Steven L. Gonias, Monica Einarsson, Salvatore V. Pizzo
Studies were conducted in anesthetized, paralyzed dogs on the effect of aerosolized histamine on phrenic nerve activity. The paralyzed dogs were ventilated in phase with their recorded phrenic nerve activity at a constant inspiratory flow-rate, using a cycle-triggered ventilator. Phrenic nerve activity was measured before and during administration of aerosolized histamine while the inspiratory flow-rate and arterial blood gases were kept constant. In addition, before and after histamine, phrenic nerve activity was recorded for single bursts during which the ventilator was switched off. The effects of histamine on respiratory resistance were prevented by prior administration of isoproterenol and atropine. Although no changes occurred in respiratory resistance, histamine increased the instantaneous magnitude of phrenic nerve activity. The effect was evident early in the inspiratory period and was found even when the lungs were not inflated. Inflation of the lungs excited phrenic nerve activity; this effect increased after histamine. All of these actions of histamine were abolished by vagotomy. We conclude that histamine increased phrenic nerve activity during inspiration by a vagal reflex.
A I Pack, B C Hertz, J F Ledlie, A P Fishman
Leukemic cells from 70% of patients with Ia+CALLA+ non-T cell acute lymphoblastic leukemia (ALL) express an antigen (B1) found on all normal B lymphocytes. In this study, ALL cells that do not express the B1 antigen were studied in an attempt to further elucidate the cellular lineage of these tumors. Non-T cell ALL lines and tumor cells isolated from patients with non-T cell ALL that are Ia + CALLA + B1- were studied in vitro with a variety of agents known to promote cellular differentiation. Phorbol diester (TPA) or phytohemagglutinin conditioned leukocyte culture media were capable of inducing the expression of B1 on all four non-T cell ALL lines tested. In contrast, B1 could not be induced under the identical conditions on a promyelocytic leukemia line or a T cell lymphoblastic leukemia line. With the induction of B1 on non-T cell ALL lines, cytoplasmic mu-heavy chain (c mu) became undetectable, whereas the expression of CALLA and Ia were unchanged. The expression of B1 was accompanied by a decrease of cellular proliferation and DNA synthesis, but not significant morphologic changes were noted. In addition, no other B or T cell antigens were detected. The cellular origin of non-T cell ALL was further investigated using tumor cells isolated from leukemic patients. Tumor cells from eight patients with Ia + CALLA + B1-c mu- ALL could be induced in vitro with TPA to express both B1 and c mu. In contrast, cells from five patients with Ia + CALLA-B1-c mu- non-T cell ALL could not be induced with TPA to express CALLA, B1 or c mu. These studies suggest that the non-T cell ALL are heterogeneous and represent a spectrum of early B cell differentiation including the pre- pre-B cell (Ia + CALLA + B1-c mu-), the intermediate pre-B cell (Ia + CALLA +B1 + c mu-), and finally the "true" pre-B cell (Ia + CALLA + B1 + c mu+). The cellular origin of the remaining Ia + CALLA-B1-c mu- form of non-T cell ALL (20%) is still unknown.
L M Nadler, J Ritz, M P Bates, E K Park, K C Anderson, S E Sallan, S F Schlossman
The effects of adrenal steroids on proopiomelanocortin (POMC) levels in rat pituitary have been studied by two-dimensional gel electrophoresis. In intact rats the relative abundance of POMC was much higher in the neurointermediate lobe (N-IL) than in anterior pituitary (AP); in both tissues the predominant species appeared to be of 29,000-dalton (29K) molecular mass, with lesser amounts of a 31K form. In both tissues, the 31K and 29K forms showed multiple spots, consistent with different degrees of sialoglycosylation. Adrenalectomy was followed by a marked increase in AP levels of POMC, and a marked decrease in N-IL levels. In adrenalectomized rats, dexamethasone administration did not affect N-IL levels of POMC, but suppressed 35S incorporation into POMC in AP in a dose-related manner; deoxycorticosterone showed minimal effects on AP levels of POMC, but progressively elevated N-IL levels; 9 alpha fluorocortisol (9 alpha fF) progressively both suppressed AP levels, and raised N-IL levels of POMC. Estimation of immunoreactive (ir) ACTH and ir-beta-endorphin in parallel samples showed an elevation of N-IL levels in response to mineralocorticoids (deoxycorticosterone, 9 alpha fF), and a paradoxical elevation of AP levels in response to glucocorticoids (dexamethasone, 9 alpha fF) compared with oil-injected adrenalectomized controls. We conclude (a) that glucocorticoids suppress the secretion of ir-ACTH and ir-beta-endorphin to a greater extent than they inhibit the synthesis of POMC; (b) that mineralocorticoids specifically elevate the N-IL levels of both POMC and its immunoreactive product (beta-endorphin).
B A Khalid, A T Lim, D R Fraillon, J W Funder
An antiserum prepared against a lambda-Bence Jones protein from a patient (SUT) who had multiple myeloma and amyloidosis had specificity for lambda-light chains of the chemically defined variable (V) region lambda-chain subgroup lambda VI. Sequence analyses of protein SUT and of five other lambda-light chains recognized immunologically as of the V lambda VI subgroup revealed that all six proteins had the N-terminal sequence characteristic for prototype lambda VI proteins. The isotypic nature of the V lambda VI subgroup was demonstrated immunochemically: lambda VI molecules were detected among light chains isolated from the IgG proteins of each of 12 normal individuals and lambda VI antigenic determinants were also detectable on the intact IgG proteins. The frequency of lambda VI molecules among lambda-type light chains is estimated to be approximately 5% based on the finding that 5 of 91 lambda Bence Jones proteins were of the V lambda VI subgroup. Proteins of the V lambda VI subgroup, in contrast to those of the other five chemically-classified lambda chain subgroup, appear to be preferentially associated with the amyloid process as evidenced by the fact that all six lambda VI proteins were obtained from patients with amyloidosis AL and, in addition, 5 of 42 lambda-type monoclonal immunoglobulins from patients with primary or myeloma-associated amyloidosis were classified by immunodiffusion analyses as having lambda VI-type light chains.
A Solomon, B Frangione, E C Franklin
Human malaria parasites (Plasmodium falciparum) grown in continuous erythrocyte culture utilize hypoxanthine for synthesis of both guanosine and adenosine nucleotides. Unlike the mature human erythrocyte, the malaria parasite depends on a constant supply of guanylates, primarily for synthesis of nucleic acids. This parasite specific requirement for guanylates led us to predict that a block in the hypoxanthine to guanosine monophosphate pathway would be selectively lethal to the parasite. Bredinin (4-carbamoyl-1-beta-D-ribofuranyosyl-imidazolium-5-olate) inhibited the synthesis of guanosine monophosphate from inosine monophosphate by parasitized erythrocytes. This block in guanylate synthesis was fatal to both a drug-sensitive (FCR-3) and a drug-resistant (VNS) strain of the malaria parasite at a bredinin concentration of 50 microM, arresting growth of the parasite at the trophozoite stage of development. These studies emphasize the essential role of guanylates and their synthesis from hypoxanthine in the metabolism of malaria parasite. They further suggest that bredinin or similar agents that selectively interfere with parasite guanylate metabolism may have potential for antimalarial chemotherapy.
H K Webster, J M Whaun
In this study, we demonstrate that both highly purified T4+ and T8+ lymphocytes can produce substantial amounts of Interleukin 2(IL 2) when stimulated with the combination of concanavalin A (Con A) and phorbol myristate acetate. Furthermore, addition of IL 1 to macrophage-depleted T lymphocytes significantly increased IL 2 production by lymphocytes of either the T4+ or T8+ phenotype. These findings provide a basis for further studies of the molecular mechanisms involved in human immune cell interactions.
T A Luger, J S Smolen, T M Chused, A D Steinberg, J J Oppenheim
Type V hyperlipoproteinemia (HLP) is characterized clinically by hepatosplenomegaly, occasional eruptive xanthomas, and an increased incidence of pancreatitis. These patients have striking hypertriglyceridemia due to increased plasma chylomicron and very low density lipoprotein concentrations in the fasting state, without a deficiency of lipoprotein lipase or its activator protein, apolipoprotein (apo) C-II. ApoE, a protein constituent of triglyceride-rich lipoproteins, has been implicated in the receptor-mediated hepatic uptake of these particles. ApoE has three major alleles: E2, E3, and E4, and the products of these alleles are apoE2, apoE3, and apoE4, respectively. ApoE phenotypes were determined in 30 type V HLP patients as well as in 37 normal volunteers. Among the type V patients, 33.3% were noted to be homozygous, and 40.0% heterozygous for E4 (normal, 2.7 and 21.6%, respectively). These data suggest that apoE4 may play a role in the etiology of the hyperlipidemia in a significant number of type V HLP patients.
G Ghiselli, E J Schaefer, L A Zech, R E Gregg, H B Brewer Jr