Sensitization to Low Dose 5-Fluorouracil: SUBSEQUENT ENHANCEMENT OF ITS SYSTEMIC ANTITUMOR EFFECT IN THE RAT

• Text
• PDF

Abstract

This report describes a novel method of immunochemotherapy; the active immunization to the drug 5-fluorouracil (5-FU) with enhanced antitumor activity resulting from its subsequent systemic administration. Two metastasizing carcinomas in the Fischer strain (F344) rat have been used: a chemically induced bladder carcinoma (FBCa) and a spontaneous mammary adenocarcinoma (MACa). Both tumors grow rapidly and result in 100% mortality within 10 wk of implantation. Neither tumor is sensitive to systemic 5-FU alone. Intradermal sensitization to 5-FU before FBCa tumor implantation, followed by 5-FU administered systemically, resulted in significant tumor regression and improvement in survival with eradication of all tumor and cure in 20% of animals. A similar antitumor effect was observed with the MACa. A comparable drug effect was observed when methotrexate sensitization was given before FBCa implantation followed by systemic MTX. Specificity to the sensitizing drug was demonstrated by the lack of effect of sensitization with either 5-FU or MTX unless followed by systemic therapy with the requisite sensitizing agent. Sensitization to 5-FU has also been assessed after FBCa implantation followed by resection of the local tumor. Resection was performed after distant tumor metastases had occurred, and was followed by systemic 5-FU therapy. Whereas tumor resection alone failed to cure any animal, sensitization to 5-FU increased cure rate fourfold over animals receiving systemic 5-FU alone. Antibody to 5-FU in the sera of sensitized animals has been suggested by an immunoenzymatic staining technique and its specificity confirmed in a radioimmunoassay. It is postulated that a combination of the systemic agent and the antibody elicited to it by sensitization produces the significant antitumor effect observed. The antitumor effect observed with this new approach to immunochemotherapy warrants further experimental and clinical study.

Authors

Rudolf E. Falk, Mark Hardy, Leonard Makowka, Julita Teodorczyk-Injeyan, Judith A. Falk

Chlorination of Taurine by Human Neutrophils: EVIDENCE FOR HYPOCHLOROUS ACID GENERATION

• Text
• PDF

Abstract

The model hydrogen peroxide-myeloperoxidase-chloride system is capable of generating the powerful oxidant hypochlorous acid, which can be quantitated by trapping the generated species with the β-amino acid, taurine. The resultant stable product, taurine chloramine, can be quantitated by its ability to oxidize the sulfhydryl compound, 5-thio-2-nitro-benzoic acid to the disulfide, 5,5′-dithiobis(2-nitroben-zoic acid) or to oxidize iodide to iodine. Using this system, purified myeloperoxidase in the presence of chloride and taurine converted stoichiometric quantities of hydrogen peroxide to taurine chloramine. Chloramine generation was absolutely dependent on hydrogen peroxide, myeloperoxidase, and chloride and could be inhibited by catalase, myeloperoxidase inhibitors, or chloride-free conditions. In the presence of taurine, intact human neutrophils stimulated with either phorbol myristate acetate or opsonized zymosan particles generated a stable species capable of oxidizing 5-thio-2-nitrobenzoic acid or iodide. Resting cells did not form this species. The oxidant formed by the stimulated neutrophils was identified as taurine chloramine by both ultraviolet spectrophotometry and electrophoresis. Taurine chloramine formation by the neutrophil was dependent on the taurine concentration, time, and cell number. Neutrophil-dependent chloramine generation was inhibited by catalase, the myeloperoxidase inhibitors, azide, cyanide, or aminotriazole and by chloride-free conditions, but not by superoxide dismutase or hydroxyl radical scavengers. Thus, it appears that stimulated human neutrophils can utilize the hydrogen peroxide-myeloperoxidase-chloride system to generate taurine chloramine. Based on the demonstrated ability of the myeloperoxidase system to generate free hypochlorous acid we conclude that neutrophils chlorinate taurine by producing this powerful oxidant. The biologic reactivity and cytotoxic potential of hypochlorous acid and its chloramine derivatives suggest that these oxidants play an important role in the inflammatory response and host defense.

Authors

Stephen J. Weiss, Roger Klein, Adam Slivka, Maria Wei

Hematopoietic Stem Cells with High Proliferative Potential: ASSAY OF THEIR CONCENTRATION IN MARROW BY THE FREQUENCY AND DURATION OF CURE OF W/W^v MICE

• Text
• PDF

Abstract

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.

Authors

Dane R. Boggs, Sallie S. Boggs, Debra F. Saxe, Lora A. Gress, Don R. Canfield

Lipid Transport in the Human Newborn: PALMITATE AND GLYCEROL TURNOVER AND THE CONTRIBUTION OF GLYCEROL TO NEONATAL HEPATIC GLUCOSE OUTPUT

• Text
• PDF

Abstract

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.

Authors

P. F. Bougnères, I. E. Karl, L. S. Hillman, D. M. Bier

Lung Cell Oxidant Injury: ENHANCEMENT OF POLYMORPHONUCLEAR LEUKOCYTE-MEDIATED CYTOTOXICITY IN LUNG CELLS EXPOSED TO SUSTAINED IN VITRO HYPEROXIA

• Text
• PDF

Abstract

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.

Authors

Norbert Suttorp, Lawrence M. Simon

Plasmodium Falciparum Malaria: AN AMELANOTIC MELANOMA CELL LINE BEARS RECEPTORS FOR THE KNOB LIGAND ON INFECTED ERYTHROCYTES

• Text
• PDF

Abstract

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.

Authors

John A. Schmidt, Iroka J. Udeinya, James H. Leech, Robert J. Hay, Masamichi Aikawa, John Barnwell, Ira Green, Louis H. Miller

Regulation of Bile Salt Transport in Rat Liver: EVIDENCE THAT INCREASED MAXIMUM BILE SALT SECRETORY CAPACITY IS DUE TO INCREASED CHOLIC ACID RECEPTORS

• Text
• PDF

Abstract

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.

Authors

Francis R. Simon, Eileen M. Sutherland, Manuel Gonzalez

Catabolic Pathways for Streptokinase, Plasmin, and Streptokinase Activator Complex in Mice: IN VIVO REACTION OF PLASMINOGEN ACTIVATOR WITH α₂-MACROGLOBULIN

• Text
• PDF

Abstract

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.

Authors

Steven L. Gonias, Monica Einarsson, Salvatore V. Pizzo

Ammonium Handling by Superficial and Juxtamedullary Nephrons in the Rat: EVIDENCE FOR AN AMMONIA SHUNT BETWEEN THE LOOP OF HENLE AND THE COLLECTING DUCT

• Text
• PDF

Abstract

Papillary and surface micropuncture was used to assess the effects of a chronic metabolic acidosis on the renal tubular handling of ammonium by surface nephrons, juxtamedullary nephrons, and the terminal segment of collecting duct. Rats chronically fed ammonium chloride had an expected decline in arterial pH and bicarbonate concentration associated with a doubling in the amount of ammonium excreted and a decline in urine pH. The glomerular filtration rate and absolute delivery of water and sodium to micropuncture sites of surface and deep nephrons was not measurably altered. Ammonium delivery to the end of the proximal tubule increased from 853±102% to 1,197±142% (SE) of the filtered load of ammonium after the induction of metabolic acidosis. This increase was due to a rise in tubular fluid ammonium content from 2.31±0.23 to 4.06±0.28 mM/liter. After the induction of acidosis, absolute and fractional delivery of ammonium ion to the end of the distal tubule was less than to the end of the accessible portion of the proximal tubule. These findings indicate that ammonium is lost in the intervening segment.

Authors

John Buerkert, Daniel Martin, David Trigg

Metabolic Acidosis Suppresses 25-Hydroxyvitamin D₃-1α-Hydroxylase in the Rat Kidney: DISTINCT SITE AND MECHANISM OF ACTION

• Text
• PDF

Abstract

Effect of metabolic acidosis on two distinct 25-hydroxyvitamin D3-1α-hydroxylase (1α-hydroxylase) systems was studied in the kidneys of vitamin D-deficient rats; one is localized in the proximal convoluted tubule (PCT), is activated in vitamin D deficiency, and is regulated primarily by parathyroid hormone (PTH) via cyclic AMP; the other is localized in the proximal straight tubule (PST), is latent in vitamin D deficiency, and is selectively stimulated by calcitonin via a cyclic AMP-independent mechanism. The 1α-hydroxylase activities were measured in the PCT and PST microdissected from the kidney of vitamin D-deficient rats with or without metabolic acidosis of varying duration. The 1α-hydroxylase activity decreased in the PCT from 0.74±0.07 fmol/mm per h to 0.24±0.02 at day 3 of metabolic acidosis without a further decline at day 7. Neither metabolic acidosis of 16 h duration nor reduction of the incubation medium pH from 7.4 to 7.0 affected the enzyme activity in the PCT. To examine the underlying mechanism for the suppression of 1α-hydroxylase activity, PTH, cyclic AMP, or calcitonin was given to rats with metabolic acidosis of 3 d duration. Although PTH failed to augment the suppressed 1α-hydroxylase activity in the PCT, cyclic AMP restored it to the level of control rats. The 1α-hydroxylase activity in the PST remained undetectable in control rats and in acidotic rats with or without PTH or cyclic AMP treatments. However, calcitonin stimulated the 1α-hydroxylase activity in the PST equally from undetectable to 0.75±0.09 fmol/mm per h in control and to 0.78±0.10 in acidotic rats. The data suggests that metabolic acidosis suppresses 1α-hydroxylase only in the PCT by inhibiting PTH-dependent adenylate cyclase, and that cellular events beyond cyclic AMP in the PCT and the events responsive to calcitonin in the PST are unaffected. The results show the definite advantage of using defined single nephron segments to study the hormonal and ionic control of the 1α-hydroxylase system in the kidney.

Authors

Hiroyuki Kawashima, Jeffrey A. Kraut, Kiyoshi Kurokawa