Experimental Biology and Medicine

Plasma disappearance of substances taken up largely by the liver, such as bromosul-fophthalein (BSP) (1-3) or indocyanine green (4, 5), has been used widely as a test for liver function. Recently, the plasma disappearance of a bile acid, cholylglycine, has been proposed as a test for hepatic injury (6, 7). This bile acid clearance is based on the assumption that the plasma disappearance of cholylglycine may be equated with hepatic uptake. To test this assumption, we have defined the tissue distribution of intravenously injected cholylglycine in rats and hamsters; we have used animals with an acute bile fistula, as well as animals with a bile duct obstruction.

Materials and methods. Experimental design. A mixture of [l-¹⁴C]cholylglycine and [⁹⁹Tc]albumin was injected intravenously as a bolus. Ten minutes later, animals were killed and tissues were removed for determination of radioactivity. The⁹⁹Tc was used to estimate the amount of blood contaminating each tissue sample. The sampling time of 10 min was chosen since this time interval corresponds to about three half-lives for plasma disappearance of cholylglycine (in man) (8) and was judged to be a sufficiently long interval for nearly complete tissue uptake to occur.

Chemicals. [1-¹⁴C]Cholylglycine (sp act 51.7 mCi/mmole) was purchased from Amersham/Searle (Arlington Heights, 111.) and had a radiopurity greater than 96% by zonal scanning (9) using a thin-layer system for bile acid conjugates (10). [⁹⁹Tc]Albumin was prepared using a commercial kit (New England Nuclear Radiopharmaceutical Division, North Billerica, Mass.). A solution of [l-¹⁴C]cholylglycine was mixed with [⁹⁹Tcjalbumin to give a concentration of 5 μCi of ^I4C and 15 μCi of ⁹⁹Tc radioactivity per milliliter.

Animal preparation. Male Sprague-Daw-ley rats weighing 250-350 g (Mayo Clinic stock) and adult golden Syrian hamsters (Engel Laboratory, Farmersburg, Ind.) were used.

Several T cell mitogens, con-canavalin A (Con A), phytohemagglutinin P (PHA-P), and staphylococcal enterotoxin A (SEA), were compared for their ability to inhibit the in vitro antibody response and to stimulate the production of mitogen (antigen) -type interferon in mouse spleen cell cultures. It was found that the ability to inhibit the plaque-forming cell (PFC) response to sheep red blood cells was proportional to the ability of these mitogens to induce interferon in the cultures. SEA was the most effective inhibitor of the PFC response and the best inducer of mitogen-type interferon, followed by Con A, with PHA-P being the least effective. The data suggest that SEA would be the most suitable inducer of mitogen-type interferon in quantity as a prerequisite to purification and characterization of the molecule. The data are supportive of previous studies suggesting a role for mitogen (antigen) -type interferon in regulation of the immune response via suppressor T cells.

We wish to thank Ms. Phyllis Myers and Mr. Kley Hughes for their skillful technical assistance.

Acute (single dose) and chronic (multiple dose) ethanol intoxication has been produced in rats and mice so that the effects on survival and on ethanol metabolism could be determined in animals pretreated with ascrobic acid and/or zinc sulfate. Only 13 of 40 mice (32%) injected ip with a fixed amount of ethanol on 2 consecutive days survived. In contrast, the survival rates in matched animals pretreated with ascrobic acid (25 mg) or zinc (1 μM) were 100 and 90%, respectively (20 animals per group). Smaller amounts of zinc (0.2 and 0.4 μM) improved the survival after the first injection of ethanol (24 hr), but not after the second (48 hr). Similar observations were made in rats given repeated injections of ethanol over a 4-week period. Serial blood ethanol concentrations were determined in rats pretreated with ascrobic acid, zinc, a combination of ascorbic acid and zinc, pyrazole (an alcohol dehydrogen-ase inhibitor), or saline (control). Blood ethanol concentrations were significantly lower (P < 0.05) 1 hr after ip ethanol injections in animals pretreated with ascorbic acid and/or zinc when compared to saline control animals. Pyrazole, in contrast, maintained increased blood ethanol levels. These studies indicate that both ascorbic acid and zinc exert protective effects in ethanol-in-toxicated rodents.

[l-¹⁴C]Naphthalene and the [l-¹⁴C]naphthalene complexed with BSA at 8-12 ppb in flowing sea water produced 100% mortality in 24-36 hr in newly hatched Dungeness crab (Cancer magister) zoea and stage I and V spot shrimp (Pandalus platyceros). Maximum accumulation of [1-¹⁴C]naphthalene in the stage V spot shrimp exposed to [l-¹⁴C]naphthalene was nearly four times greater than in shrimp exposed to the [l-¹⁴C]naphthalene-BSA complex (820 and 220 ppb, respectively). These values represent a magnification of 25-100 times the exposure levels. Metabolic products of [l-¹⁴C]naphthalene in larval tissues exposed to the complexed and noncomplexed naphthalene reached a maximum of 21 and 9%, respectively, of the total radioactive compounds (based on the molecular weight of naphthol). [l-¹⁴C]Naphthalene was almost entirely depurated from the tissues in 24-36 hr, whereas metabolic products were strongly resistant to depuration.

This research was supported in part by NOAA's Environmental Research Laboratories Outer Continental Shelf Environmental' Assessment Programs under funding from the Bureau of Land Management.

Ontogeny of thymus cell function in humoral antibody response was studied in vitro using cells from different embryonic stages and neonates of BDF₁ mice. As early as the sixteenth day of embryonic development, the thymus contained cells that were readily activated in vitro by con A to function as helper cells in an antibody response against sheep erythrocytes. The helper function of embryonic thymocytes increased rapidly, reaching adult levels before birth. It has also been shown that con A-treated thymus cells are less susceptible to anti-Ø serum and correspondingly more susceptible to anti-H-2 serum; the implication of this finding in thymus cell maturation has been discussed.

The author is grateful to Professor Robert Auerbach for his encouragement and criticisms during the course of this work. This study was supported in part by Research Grant CA 13548 from the National Institute of Health.

Malignant lymphoid cell lines are unusually sensitive to the toxic effects of BUDR. Incubation of mouse myeloma MOPC 315 with 1 and 0.1 μg of BUDR/ml results in an acute toxic effect by Days 3 and 5, respectively. Subcultivation of MOPC 315 in 0.01 and 0.001 μg of BUDR/ml typically results in on Days 12 and 18, respectively. Similar patterns of acute toxicity occur with mouse lymphoma S49.1, human leukemia CCRF/CEM, and mouse myeloma MPC 11. The dose of BUDR required to cause a 50% inhibition of proliferation (ID₅₀) ranges from 0.09 to 0.3 μg of BUDR/ml. In contrast, the ID₅₀ for a variety of cell lines of alternate origins range from 3.14 μg of BUDR/ml for human cervical adenocarcinoma HeLa to 6.23 μg of BUDR/ml for mouse fibroblast L929. Fibroblasts L929 and HeLa cells were grown for 20 and 25 days, respectively, in 2 μg of BUDR/ml with no toxicity.

The toxicity of 1 μg of BUDR/ml to MOPC 315 and MPC 11 is partially prevented by 1 μg of thymidine/ml and completely prevented by 10 μg of thymidine/ml. Addition of 1 μg of thy midine/ml completely protects lymphoma S49.1 from the effects of 1 μg of BUDR/ml.

We wish to thank Dr. Charlotte Friend and Dr. William Scher for their helpful discussions and critical evaluation of this manuscript.

Interstitial fluid pressure (P_if) of the dog was measured from capsules implanted in subcutaneous tissue of the upper and lower hindleg 4 to 8 weeks prior to use. (The capsules were a disk-shaped hollow (6 mm x 9 mm i.d.) porous polyethylene matrix (Bolab, Inc.) with an average pore size of 65 μm.) Total tissue pressure (P_tot) was measured either with small flaccid balloons inserted subcutane-ously or, more frequently, by the needle method for measuring total tissue pressure (see (3) for a discussion of these methods).

Pressure transmission was examined in two different preparations. With the first, the intact hindleg of the pentobarbital anesthetized dog was placed up to the hip in a water filled plethysmograph and P_if and P_tot measured while applying positive or negative pressure to the leg. With the second preparation, the hindlimb was totally isolated from the dog and artificially perfused with whole blood as described elsewhere (4). The isolated hindlimb was then placed entirely inside the plethysmograph with only the arterial, venous, and pressure measurement catheters extending out of the plethysmograph.

Using the intact hindlimb preparation, the response of interstitial fluid pressure to alterations in ambient pressures was determined as P_if was increased above its normal value in the intact hindleg. In order to increase P_lf, warmed Tyrode's solution was infused intravenously. When P_if approached or exceeded atmospheric pressure and the infusion was terminated, P_if at first decreased with time as if the tissues were undergoing stress-relaxation. Therefore, measurements were made about 0.5 hr after the infusion of Tyrode's solution when F_if appeared to have reached a steady value.

With changes in applied pressure, a small amount of fluid (less than 1 μ1) is displaced into or out of the pressure transducer.

Extracts of HSV-2-trans-formed hamster cells inhibit plaque formation of HSV-1 and HSV-2 in normal hamster cells. Extracts also inhibit replication of HSV. The inhibitor is a soluble component of the cells, and probably does not act directly on extracellular virus. The inhibitor is not a classicial interferon since the inhibition is temperature-sensitive and the replication of VSV is not impaired.

Dr. Ronald Duff supplied helpful suggestions for this study. This investigation was supported by Contract NO1 CP 53516 within the Virus Cancer Program of the National Cancer Institute, NIH, PHS.

Sustained static (isometric) contraction of striated muscle to fatigue was carried out in normal, untrained, young (22 to 29 years) male subjects in order to compare the hemodynamic response of large (ankle plantar flexor) and small (little finger adductor) muscle contraction. There were pronounced increases in systolic and dia-stolic pressures and heart rates in all cases. The increases in systolic pressure were similar during static contraction of the two different muscle groups; however, diastolic pressure increases were greater and heart rate increases less with finger contraction as compared to leg contraction.

Upon three weekly repetitions, the cardiovascular responses were not significantly altered although the force output was increased with repetition in the case of both muscle groups. The results indicate that (i) although the size of the muscle mass influences the diastolic pressure and heart rate response, the relative muscle tension is the major factor determining the hemodynamic result; (ii) the divergence of blood pressure and heart rate responses suggest a dissociation of the mechanisms governing these two variables in static exercise; and (iii) the relative consistency of the hemodynamic changes during these tests indicates that isometric exercise is a simple and reliable method to study cardiovascular response to stress.

Lewis rats injected ip with 100 mg of rat cord homogenate developed a mild degree of experimental allergic en-cephalomyelitis (EAE). One iv injection of 0.6 mg of the polyribonucleotide complex, polyinosinic acid:polycytidylic acid (I:C), administered at the time of encephalitogenic challenge, increased the incidence and severity of EAE whereas polyadenylic acid: polyuridylic acid (A:U) and the single-stranded polyinosinic acid (I) or polycyti-dylic acid (C) had no effect. Injection of I:C at the time of onset of clinical signs of EAE did not influence the course of the illness.

The mechanism of enhancement of EAE by I:C was investigated with the aid of the passive transfer system. Unexpectedly, I:C inhibited the development of EAE lesions when injected into recipients along with the fully immunized cells from donors with EAE. The inhibitory effect of I:C on EAE cells was transient and nondestructive. Inhibition by I:C was not mediated by the adrenal gland or by induction of interferon. The apparently opposite effects of I:C on active and passive production of EAE may be explained by its biphasic effects on lymphocytes and reticuloendothelial function.

Dithiothreitol (DTT) increases the generation of angiotensin when bovine renin acts on bovine substrate or hog substrate. Kinetic analysis indicates that the V_max increases without a change in K_m. Pretreatment of bovine renin with DTT followed by dilution of the DTT to a low concentration does not augment renin activity, while pretreatment of the substrate and a similar dilution reveals marked potentiation. N-Ethylmaleimide (NEM) does not inhibit renin activity but does prevent the augmenting effect of DTT present during the incubation. It is concluded that DTT augments renin activity by an action on renin substrate and that renin does not fall in the category of sulfhydryl-dependent enzymes.

We acknowledge the valuable technical assistance of Mr. Paul Arnold and Mrs. Roselle Poisner.

It is shown that appropriate xenogeneic rabbit and horse antiserum, as well as allogeneic rat serum, can serve as sources from which ¹²⁵I-labeled antibody to a strong histocompatibility antigen expressed on Fischer-344 strain rat cells can be prepared. After iv administration all three types of antibody would localize preferentially in F-344 skin grafts made 4 days earlier on Buffalo strain rats. After 24 hr, 125I localization ranged from 10 to 20% of the injected dose on F-344 skin grafts averaging 0.3 g weight on 150-190-g Buffalo rats. The horse serum used as an antibody source was a commercially available anti-rat-lymphocyte serum. Prior ip administration of two or three doses of 0.5 ml of this serum largely blocked uptake by F-344 skin grafts of both the ¹²⁵I-labeled alloantibody and the ¹²⁵I-labeled xenogeneic histocompatibility antibody prepared from this horse antiserum.

This work was supported by USPHS Research Grant CA-16749 from the National Cancer Institute and under contract with the U.S. Energy Research and Development Administration with the University of Rochester and has been assigned Report No. UR-3492-913

CC1₄ depressed hepatic excretory function in adult and developing rats. In this regard, plasma ouabain concentrations were significantly higher and biliary (intestinal) ouabain was significantly lower in the treated animals. Of particular interest was the observation that hepatic ouabain was significantly lower than controls in CCI₄ treated young rats (decreased uptake into liver), but significantly higher than controls in treated older rats (decreased secretion from liver into bile). These results suggest that the “site” of altered biliary function following CCI₄-induced liver damage is age dependent.

This investigation was supported in part by National Institute of Health Grant ES00560. S.Z.C. was supported by a predoctoral fellowship from the Eli Lilly Co. The authors acknowledge the technical assistance of Ms. Harriett Sherman.

Three peritoneal lavages at intervals of approximately 8-10 hr were performed in each of four splenectomized dogs. Induction of peritoneal inflammation and removal of neutrophilic cells from the peritoneal cavity was associated with a rapid mobilization of the neutrophilic band and segmented forms from the marrow into the blood and increased granulocytopoiesis in the marrow. The fluids removed from the peritoneal cavity contained both neutrophilic leukocytosis-inducing factor (LIF) and colony-stimulating factor (CSF) activities. The intensity of blood neutrophilia noted in dogs following the iv injection of fluids was considered to reflect LIF activity. CSF activity was assayed by the capacity of the lavage fluids to stimulate dogs'marrow cells to form granulocytic-macrophage colonies in vitro in soft agar medium. The increased LIF activity level was observed in lavages I, II, and III to approximately the same extent. This observation indicated that the production of LIF, having the function of mobilizing mature neutrophils from the marrow into the blood, continued as long as the peripheral demand for the cells existed. The CSF activity was maximum in lavage I and was precipitously reduced in lavages II and III. Following the increase of CSF level in lavages, the marrow M:E ratio, the percentage of myeloblasts + promyelocytes + myelocytes, and the granulocytic cellularity increased, which suggested that the CSF was influencing marrow granulocytopoiesis.

The pattern of fluctuation of the CSF activity in the lavages was different from that of the LIF activity, which indicated that CSF and LIF are two different entities.

We acknowledge the technical help of Ms. Louise Honikel and Mr. James Lehmann.

Cyclic somatostatin, at a concentration which inhibited both phases of glucose-induced insulin release, completely suppressed monophasic insulin secretion elicited by ionophore A23187. The ability of A23187 to initiate insulin secretion is believed to be highly dependent upon the drug's capacity to influence calcium transport. Thus, the present findings suggest that the negative influence of somatostatin on the insulin secretory process may be exerted on some calcium-related event critical to the release mechanism.

We are grateful for the technical assistance of Mr. Ken Milburn and the secretarial aid of Mrs. Ruth Garrett and Miss Mitzi Spence.

Extraction of CEF plasma membranes with LIS solubilizes membrane components which interact directly with ATV in vitro, antagonizing ATV binding to CEF plasma membranes and reducing the transforming capacity of these viruses. This activity is specific to CEF; approximately 200 human erythrocytes must be extracted with LIS to produce the activity equivalent to that extractable from a single CEF.

The effects of several cortico-steroids on the sudden neutropenia which occurs in animals exposed to activated complement components were studied. Solu-Medrol and Solu-Cortef in high concentrations inhibited such neutropenia but only if present before or during the period of complement activation; Decadron was much less efficient. The observed dose and temporal relationships are strikingly similar to those noted in previous studies of corticosteroid protection from endotoxin shock, suggesting that complement-mediated alterations in neutrophils may be critical in this entity.

This work was supported in part by grants from the National Institutes of Health, AM 15730, CA 15627, and HL 19725.

Rats fasted for periods of 24, 48, or 72 hr showed no changes in plasma glucagon concentration despite lowered blood glucose concentration. Islets isolated from both fed and fasted animals elicited biphasic glucagon and insulin secretion when stimulated with arginine and glucose, respectively. There were no differences in glucagon secretion rates between islets prepared from fed and fasted animals. Significant reductions in both the first and second phases of insulin secretion were observed in islets prepared from fasting animals.

The authors wish to thank Ms. Carol Grimme and Ms. D. Barr for typing the manuscript, and Dr. A. Pagliara for anti-glucagon serum. This work was made possible by Public Health Service Grant AM-165 34.

Lymphotoxin (LT) is a cytotoxin released by antigen- or mitogen-stimulated lymphocytes in vitro (1). The inhibition of lymphocyte-mediated cytolysis by anti-LT serum (2), as well as other lines of evidence (1), suggest that this lymphokine has a role in lymphocyte-mediated cytotoxicity in tissue culture. Recently, Henney (3) and Ferluga and Allison (4) reported the protective effect of dextrans on lymphocyte-mediated target-cell destruction.

In this study, we examined the effect of high molecular-weight dextran (83,000) on lymphotoxin-induced cytolysis in tissue culture and compared our findings with those observed with dextran in cell-mediated cytolysis in vitro.

Materials and assays, target cells. Mono-layers of mouse L fibroblasts, clone 929, grown for 24 hr in round-bottomed tubes, were used as targets. The tissue culture medium was RPMI 1640 with 5% fetal calf serum (FCS).

Lymphotoxin. Human lymphotoxin was purified from supernatants of phytohemag-glutinin (PHA)-stimulated human adenoidal lymphocytes as previously described (5). The activity of LT is expressed in cytotoxic units (CU) as formerly defined (5). Lymphotoxin was dissolved in a buffer consisting of 0.1 M glycine-0.15 M NaCl, pH 9.5.

Spleen cells. Sensitized spleen cells were obtained from BALB/c mice which had received intraperitoneal and, 3-4 weeks afterward, intrasplenic injections of living L cells, derived from a C3H mouse as previously described (6). Spleens were harvested 3-6 days later; RBCs were lysed in hypo-tonic solution, and the spleen cells were resuspended in tissue culture medium. Control spleen cells were taken from nonin-jected BALB/c mice.

Dextran solutions. Dextran, average mol wt 83,000 (Sigma Chemical Co., St. Louis, Mo.), was dissolved in RPMI 1640 and di-alyzed against this medium. After dialysis, the concentration of dextran was adjusted to the desired molarity by adding RPMI 1640. Then, the material was filtered through Mil-lipore membranes (pore size, 0.22 μm), and FCS was added to yield a final concentration of 5% serum.

Cerebrospinal fluid (CSF) was collected repetitively from the third ventricle of three ovariectomized sheep. Immuno-reactive LHRH could not be detected (<100 pg/ml) in any of 70 samples of CSF collected from untreated, estradiol-treated, and LHRH-treated ewes. The LHRH im-munoassay accurately measured the hormone in extracts of ovine stalk-median eminence tissue. Furthermore, no immunoreactive LHRH was lost when synthetic LHRH was added to freshly collected CSF and stored in the manner used for the attempted measurement of endogenous LHRH. Intravenous injection of LHRH increased plasma concentrations of the hormone to 420 pg/ml but LHRH could not be detected (<100 pg/ ml) in CSF collected concurrently.

Leukocytic pyrogen was subjected to a five-step purification scheme, and quantitative assays for both leukocytic pyrogen and leukocytic endogenous mediator (LEM) were made. At each step of the purification scheme, the active fractions, in addition to producing fever, lowered plasma iron and zinc concentrations, elevated fi-brinogen concentrations, and caused a release of neutrophils from bone marrow. It appears, therefore, that LEM and leukocytic pyrogen are the same molecule.

Three nitrogen-containing can-nabinoids caused a dose-dependent fall of intraocular pressure when administered either topically or orally to rabbits. Concentrations of 0.001 to 1% applied topically and 0.3 mg/kg orally were effective. Topical concentrations of 0.5% caused a substantial decrease of intraocular pressure in rhesus monkeys. These compounds may prove therapeutically useful in the treatment of elevated intraocular pressure.

This work was supported in part by PHS Research Grants EY 01413, EY 01590 and DA 01213. We thank Dr. Harry Pars of SISA, Inc., Cambridge, Mass., for the generous supplies of the benzopyranopyridines, Ms. D. Hancock and Mrs. I. Prior for secretarial assistance, Ms. K. Bowman and Ms. D. Johnson for technical assistance, and Drs. M. Kling, R. Shimp and Mr. R. Morrison for their help with the monkey experiments.

Conscious sheep with chronically implanted vascular catheters and a renal artery flow probe were used to study the effects of papaverine alone or papaver-ine plus furosemide on renin secretion. Papaverine infused into the renal artery at 7 mg/min produced a significant renal vasodi-lation which was not further enhanced by increasing the papaverine infusion to 10 mg/ min. GFR decreased significantly as did the filtered load of Na. The increased Na excretion reported in anesthetized dogs was not observed in conscious sheep. Renin secretion was not altered despite these changes in renal function. When 35 mg of furosemide was administered intravenously in addition to the renal artery papaverine infusion, no further change in RPF or GFR was observed. Na excretion was significantly elevated at each clearance period after furosemide administration and these increases were closely paralleled in time by large increases in renin secretion. The increased renin secretion observed from the papaverine blocked kidney after furosemide administration appears to be mediated by changes in ion transport by the renal tubules and not by activation of the intrarenal baroreceptor.

The author expresses his gratitude to Hoechst-Rous-sel Pharmaceutical Corporation for the furosemide used in this study and to Miss Maureen Garver for her expert technical assistance

Cell proliferation is inhibited in rat glioma (strain C6) monolayer cultures treated with cortisol (>1. μM) or dexameth-asone (>0.001 μM). Transient growth-inhibitory responses occur at 3 μM cortisol and 0.01-0.03 μM dexamethasone. At higher glucocorticoid concentrations, non-transient inhibitory responses persist for 5 days. Dose-response curves show that dexamethasone is 250 to 300 times more potent than cortisol in suppressing C6 growth.

Responsiveness to synthetic LHRH, as measured by increments in plasma LH following its sc injection, was evaluated in castrated rats with median eminence lesions and compared to that in sham-operated controls and controls with intact brains. Median eminence lesions in castrates were followed by a decline in plasma LH to approximately 30% of control values at 1, 7, and 14 days postlesions. This was associated with an increase in responsiveness to LHRH at 1 day, followed by a return to normal responsiveness at 7 and 14 days postlesions. Even though plasma LH was lowered in the animals with lesions, pituitary LH was little changed, which indicates that these lesions, although effective to inhibit LH release, were probably incomplete.

It is concluded that LH release is inhibited by lesions in the median eminence in the face of normal or even increased responsiveness to LHRH. The cause of the increased responsiveness to LHRH which appeared at 1 day after lesions remains to be elucidated.

The passive hemagglutination test was used to determine the presence of antibodies against the lipoprotein (LP) component of the cell wall of members of the family Enterobacteriaceae in the serum of healthy subjects and of patients with varied enterobacterial infections. LP antibodies were not demonstrated in the serum of healthy children and in only 4% of healthy adults. In contrast, 18% of patients with peritonitis complicating appendicitis, 24% of children with enterobacterial infection of the respiratory tract complicating cystic fi-brosis, and 42% of patients with enterobacterial bacteremia complicating malignancy had these antibodies. LP antibodies were not detected in the serum of patients with Salmonella or Shigella enteritis or enterobacterial infection of the urinary tract. The specificity of the antibody was documented by the observation that with 10 serum specimens containing antibodies essentially identical titers were obtained with either the murein-lipoprotein or the lipoprotein preparation cleaved from murein as indicator antigens. The reason for these striking differences in the immune response to this common antigen remains to be elucidated.

The authors express appreciation to Dr. Volkmar Braun, Universitat Tubingen, Tubingen, Germany, for his generous supply of lipoprotein.

A new technique for preparing intestinal fistulae in rats is presented. Perfusion of the fistula is facilitated by inserting small-diameter tubing into the closed orad end of the fistula. The distal end protrudes a 0.5 cm through the belly wall for quantitative. Collection of perfusate or secretion. Continuity of the gut is reestablished by a side-to-side anastomosis. Analysis of the mucosa from the fistula and from the intact intestine demonstrated that the mucosae are comparable during the week the animals are used for perfusion experiments. The results also demonstrate that duodenal infusion of raw egg white stimulates the output of protein, DNA, peptidase and alkaline phos-phatase activity in the jejunal fistula perfusate.

In both intact adult male and female rats anesthetized with tribromoe-thanol, the iv injection of thyrotropin-re-leasing hormone (TRH) evoked GH release within 2 min following the injection. The GH response to TRH was greater in males than in females and this response was significantly diminished by short-term (6 days) orchidectomy. In females, the GH response to TRH was maximal in estrus and minimal in diestrus day 2, showing intermediate values at proestrus and diestrus day 1. Long-term ovariectomy decreased the response. Pituitary GH content and concentration were higher in males than in females. Orchidectomy was followed by a significant decrease in pituitary GH values, whereas ovariectomy resulted in increased GH content and concentration. The results indicate that the gonads can modify pituitary GH response to TRH and, therefore, play a physiological role in modulating the stimulatory effect that the hypothalamus exerts on GH release through the secretion of substances with GH-releasing activity.

We appreciate the technical assistance of Miss Helen Jameson. We also thank Miss Cheri Reimann for secretarial assistance.

Peripheral blood leukocytes from 14 healthy, nonimmune, human donors were capable of destroying CMV-in-fected human fibroblasts, but non unin-fected fibroblasts. Antibody did not appear to mediate the response. Cytotoxicity in a similar range was noted when three transformed human cell lines were used as target cells. Unlike adult leukocytes, leukocytes separated from three samples of cord blood were ineffective in destroying CMV-in-fected target cells.

We thank Dr. Roberto Kretschmer for providing cord blood and Ricki Peitchel for technical assistance.

Indirect immunofluorescence revealed that 80 ± 4% of isolated mouse thymocytes were found to have membrane uptake of poly (A:U). Mice previously injected with hydrocortisone demonstrated a reduction to 20-30% in numbers of fluorescent thymocytes. Preincubation of thymocytes with DEAE-dextran did not increase the percentage of fluorescent cells.

These findings suggest that medullary thymocytes bind poly (A:U) to a lesser degree than cortical thymocytes. Several mechanisms of thymocyte uptake of poly (A:U) are suggested including adsorption to the membrane and/or rapid penetration through the membrane.

Rabbit lung lavage cells (LLC) were incubated with 0, 200, 300, and 400 ppm (0, 1.53, 2.29, and 3.05 mM) of 3-methylindole (skatole, 3MI) or 0, 400, 600, and 800 ppm (0, 3.42, 5.13, and 6.84 mM) of indole for up to 4 hr at 37°. The release of βgalactosidase and βglucuronidase activities was compared to the total enzyme activity solubilized by sonication. Significant increases in soluble enzyme activity were obtained with these concentrations of 3MI, and the amount of enzyme activity solubilized depended upon the 3MI concentration and incubation time. After incubation with 400 ppm of 3MI for 4 hr, 58% of the total βgalactosidase and 46% of the βglucuron-idase activities was solubilized from LLC. This activity was greater than that released by freezing and thawing two times. At higher concentrations, indole caused a similar release of these lysosomal enzyme activities. Significantly more βgalactosidase activity was released when LLC were incubated with 3MI in combination with low concentrations of acetylsalicylic acid, reti-noic acid, Triton X-100, and phosphatidyl serine than by incubation with each agent alone. Incubation with DMSO decreased enzyme release by 3MI, and 3MI inhibited some of the membrane-labilizing effects of lysolecithin. The data indicate that 3MI and indole can cause the release of marker lysosomal enzymes from LLC and that the effects of 3MI can be enhanced or decreased by the presence of some other lipotrophic agents.

Using renal micropuncture techniques, the effect of luminal PGE₂ on the net efflux of simultaneously administered ²²Na was examined in rats in hyper-tonic saline diuresis. [³H]Inulin and ²²NaCl were microinjected into the lumen of late-proximal convoluted tubules with and without PGE₂, and the urinary recovery of ³H and ²²Na were followed. Sodium recovery was significantly higher in the presence of PGE₂ while renal function and the method and site of injection were similar.

I wish to thank Dr. E. E. Elko and Dr. F. D. Sticht for the critical reading of this manuscript.

A new derivative of arsonic acid, arsanil isothiocyanate was used to couple arsonic acid via an is othiocyanate group to amino groups. Immunization of guinea pigs with this determinant attached to e-amino carproic acid in complete Freund's adjuvant led to the production of delayed hypersensitivity toward this haptenic determinant, as indicated by positive skin tests to compounds to which the determinant was coupled to egg albumin and bovine serum albumin. In addition, lymphocytes from these immunized animals responded to these antigens in vitro by undergoing blast transformation as measured by incorporation of tritiated thymidine.

The dominance of the arsonic acid group in the determinant was seen in the form of positive skin tests as well as in in vitro stimulation with the various conjugates in animals immunized with either Ars-NCS-EACA or ABA-tyr.

The response by the homologous compounds were, however, generally better than those of the heterologous compounds. These results suggest an important contribution to the reactivity of the immunodomi-nant arsonic acid group by the neighboring site of attachment.

Bond-phase enzyme lmmu-noassay (EIA) and radioimmunoprecipitation tests were developed for detection of antibodies to Rochalimaea (Rickettsia) quintana in trench fever. Both were equally sensitive, and both detected more positive sera than either passive hemagglutination or immunofluorescence tests. The advantages of EIA are the stability of label and ease and safety of use.

Butylated hydroxyanisole (BHA), an anti-oxidant food additive, inhibited the primary in vitro antibody (PFC) response of C57B1/6 spleen cells to both a thymus-dependent antigen (SRBC) and a thymus-independent antigen (E. coli 0127:B8); the PFC response of athymic nude spleen cells to E. coli 0127:B8 was inhibited to the same degree. BHA inhibited both B- and T-lymphocyte function. BHA needed to be present only for the first 4 hr of culture to exert its inhibitory effect on the PFC response. DNA synthesis in C57B1/6, athymic nude, and T-cell mito-gen-induced C57B1/6 spleen cell cultures was inhibited by BHA; however, short-term exposure of cultures to BHA at certain PFC inhibitory concentrations resulted in stimulation of DNA synthesis. The mechanism of the inhibitory effect of BHA on the in vitro PFC response is unknown, but may possibly involve activation of regulatory cell activity. Certainly, the data warrant further study of the effect of this ubiquitous food additive on the immune response.

1 he growth dynamics ot hemo-poietically active liver and spleen of neonatal mice have been used to study the association between hemopoietic stem cells (CFU_S) and a target cell for Friend virus, defined by its capacity, following transformation, for autonomous colonial growth in appropriate host mice (tumor colony forming units, tCFU). The kinetics of tCFU target cells and CFU_S are sufficiently dissimilar to allow the conclusion-that they do not constitute identical populations.

The technical assistance of A. Bulba, J. Jividen, B. Styles, and R. Reszka is gratefully acknowledged.

Daily determinations or fluid exchanges, urinary Na+ excretion, and plasma volume, Na+, K+, protein, and os-molality were made using 13 adult male rabbits during periods of ad libitum feeding, food deprivation for 4 days (water available), and 1 day of refeeding. Significant increases in urinary Na+ excretion and volume occurred after 24 hr of deprivation, and a significant increase in drinking took place after 2 days; maximal values were obtained by Day 3 for all three parameters. The plasma volume was significantly reduced from 33.4 to 29.3 ml/kg, coincident with the changes in urinary volume and Na+ excretion after 1 day of deprivation, and remained at this reduced level. Plasma osmo-lality was decreased after 1 day of deprivation and continued to decline. Plasma concentrations of Na+, K+, and protein did not change. In a second group of rabbits deprived of food and water, a marked natri-uresis was induced despite the expected reduction in urine volume. In a third group of rabbits, plasma glucose concentration was decreased after 24 hr of fasting but returned to the control level by Day 2 of fasting.

The data substantiate the assumption that the fasting-induced natriuresis and increased urine flow in the rabbit are accompanied by a reduction in plasma volume. The diuresis and natriuresis persist in fasting male rabbits with no further change in plasma volume after the first day of fasting. The natriuresis is clearly independent of the increased urine flow and fluid intake. The polydipsia is secondary to the reduction in plasma volume and enhances the increased urine flow to produce a marked polyuria.

The authors thank Mr. John Hegmann for his excellent technical assistance.

An instrument to determine the oxygen association-dissociation equilibrium of whole blood has been developed. Only one drop of blood sample is required to trace automatically the oxygen-red blood cell equilibrium curve in the direction of deoxygenation and reoxygenation within a total of less than 10 min. The whole blood is suspended in the form of a thin liquid film reenforced by a micromesh. The partial pressure of oxygen is changed continuously with a gas-mixing pump while simultaneously being recorded by polarographic oxygen electrodes. The spectral change of red blood cells is recorded by a dual-beam spec-trophotometer.

Using this instrument it is shown that the oxygen equilibrium curve of sickle cells obtained during deoxygenation is shifted to a higher oxygen affinity as compared to those obtained during reoxygenation. This hysteresis-like behavior of equilibrium curves is also reflected in the Hill constant, n, which is greater for deoxygenation than for reoxygenation. On the other hand, if deoxygenation is completed rapidly, the sigmoidness of the reoxygenation equilibrium curve and its oxygen affinity are greater than that obtained after slow deoxygenation. Hysteresis-like behavior is the postulated consequence of intracellular polymerization of sickle hemoglobin.

The authors wish to thank Dr. George Brewer from the University of Michigan for helpful comments, and to acknowledge the cooperation kindly extended to them by the Comprehensive Sickle Center of Wayne State University in supplying blood samples and the support of a WSU Faculty Research Award, and a grant from NHLI.

The process or bone mineralization in the tibial diaphysis, in response to treatment with a lathyrogen, βAminopropionitrile (BAPN), at a dose known to inhibit collagen crosslinking, was studied in growing rats. BAPN treatment impaired the initial rate of mineralization but not its onset. In addition, in the BAPN treated group, there was a 5% decrease in diaphyseal ash weight, a decrement estimated to be greater than expected from the decreased rate of mineralization. This suggests that the maximal level of mineral deposition was also decreased as a result of BAPN treatment.

Lumenal intracecal inoculation of viable malignant cells produces a protective effect against a secondary ip challenge with the same tumor cell. Cells inoculated in the lumen of the cecum showed immunizing properties as demonstrated by specific antibodies evidenced by a positive indirect im-munofluorescence test.

Furthermore, the protective effect can be transferred to an untreated animal by sensitized splenocytes.

This work was supported by the Danish Cancer Society.