Experimental Biology and Medicine

An inhibitor of lectin-induced splenocyte proliferation from serum of normal chickens has been characterized. This suppressive factor, found in both serum and plasma and at concentrations as low as 3%, causes a 50% inhibition in proliferative responses to T-cell lectins of autologous and heterologous lymphoid cells. The inhibitor in serum also dramatically suppresses murine IL-2 synthesis, proliferation of murine spleen cells stimulated with PHA, and synthesis of DNA in xenogeneic-transformed mammalian lymphoblastoid cell lines. Serum does not block binding of the lectin to lymphoid cells and the suppressive activity cannot be overcome by any dose of lectin. The inhibitor of DNA synthesis is destroyed by pepsin. NH₄₍₂₎SO₄ (50%) and TCA (15%) treatments both precipitate the suppressor factor, which further indicates that the suppressive factor is a protein. A 330-fold purification of the inhibitory protein from serum was obtained when boiled serum was passed over a Sepharose 6B and then a DEAE-Sephacel column which was washed at pH 5.0 and eluted with 0.2 M NaCl. SDS-PAGE with silver staining revealed a nonreduced protein with an apparent molecular weight of 61 kDa. Less than 2 μg of the protein thus obtained caused a 50% inhibition in the proliferation of chicken lymphoid cells to Con A. The inhibitor of DNA synthesis is therefore not cytotoxic, does not bind to Con A or to mannose or glucose residues on lymphocytes, is acid and heat stable, and is associated with a protein that has a molecular weight of 61 kDa. Since such low concentrations of this naturally occurring, protein-aceous, immunosuppressive factor cause substantial inhibition of IL-2 synthesis and proliferative activity of T cells, this protein may be a very important immunomodulator.

[³H]Spiperone was administered (20 μCi/kg, 0.0003 mg/kg, sc) to mice. In agreement with other published reports, 2 hr later the accumulation of tritium was three to four times greater in the corpus striatum than in the cerebellum. Ascorbic acid (100, 1000, 2000 mg/kg, ip, 30 min) reduced the 2-hr accumulation in the corpus striatum 16, 42, and 63%, respectively, with only the highest dose producing any significant (18%) reduction in the cerebellum. The effect was still evident in striatum 18 hr after a single dose of 1000 mg/kg. Striatal minces taken from mice treated 1 or 2 hr earlier with ascorbic acid (2000 mg/kg, ip) showed no reduction in [³H]spiperone binding. However, preincubation of striatal minces for 2 hr with ascorbic acid (10⁻³ M) produced an 82% reduction in specific binding while not having any effect on nonspecific binding. While it cannot be certain that the reduction of striatal [³H]spiperone concentrations after ascorbic acid in vivo was not a result of some nonspecific alteration in the pharmacokinetics of [³H]spiperone, the in vitro observation strongly suggests that it resulted from an alteration of binding characteristics at the receptor level.

The suppressive effects of Δ⁹-tetrahydrocannabinol (THC) on the proliferation of lymphocytes from the spleen, lymph node, and thymus of weanling animals vs adult animals to the T-cell mitogen PHA were examined. THC had a suppressive effect on thymus cells from animals of both younger and older mice. THC suppressed spleen and lymph node cells responses to phytohemagglutinin (PHA) more readily when the cells were obtained from young mice rather than older animals. Suppression by THC in the adult mice was greater in an organ containing fewer mature T lymphocytes such as the thymus in comparison to lymphocytes in secondary organs such as the spleen and lymph nodes which contain more mature lymphocytes.

Continuous infusion of a gram-negative bacterial endotoxin in relatively small doses into rats by means of an implanted osmotic pump was studied. The model system was designed to examine the effects of endotoxin on the blastogenic response of spleen cells to the endotoxin itself and to a nonspecific T-cell mitogen, concanavalin A (Con A). Rats were implanted with an osmotic pump which delivered saline for the first 42 hr to provide postsurgical recovery before the onset of endotoxin infusion. Previous studies had shown that during the first 1–4 days after administration of endotoxin marked alterations of metabolism and some changes in physiologic parameters such as blood pressure and in vitro myocardial performance occurred. In the present study the blastogenic responsiveness of spleen cells to endotoxin itself as well as to the nonspecific T-cell mitogen Con A was markedly decreased after several days of continuous administration of endotoxin. Control animals receiving only saline for the same period of time showed a similar depression of blastogenic responsiveness to the lipopolysaccharide (LPS), as well as to Con A, however, with a delay of 2–4 days before comparable levels of suppression became evident. These results indicate that marked alterations of immune competence as measured by blastogenesis of spleen cells to Escherichia coli LPS and to a mitogen such as Con A may occur after implantation of an osmotic pump, with or without continuous infusion of endotoxin. Further studies seem warranted to determine the role of the foreign body reaction to the osmotic pump as well as to the endotoxin administered by the pump.

Effects of neural blockages on pancreatic enzyme secretion in response to infusing fatty acid into the lumen were investigated using anesthetized rats, equipped with bile-pancreatic and duodenal cannulae, to evaluate the relative contribution of the neural and the hormonal mediations in the pancreatic response. Oleate (0.2 ml) was injected as a bolus into the rat duodenum, and the trypsin output in bile-pancreatic juice was monitored to determine the pancreatic enzyme secretion response with continuous return of bile-pancreatic juice to the intestine. When anticholinergic agents such as atropine sulfate and scopolamine were administrated, although basal level in pancreatic enzyme secretion fell, the increase of pancreatic enzyme secretion from basal level after stimulation by oleate was not significantly different from that of the control (no blockage). However, the ganglion blocker, hexamethonium, inhibited the pancreatic enzyme secretion in response to oleate by 94%. An adrenergic blocker, guanethidine, also led to as much of a decrease as the ganglion blocker-induced decrease. Adrenergic α- and β-blockers partially, but not completely, inhibited the enzyme secretion. Adrenergic blockage also suppressed the basal level in pancreatic enzyme secretion. On the other hand, a specific CCK antagonist, proglumide, significantly inhibited pancreatic enzyme secretion induced by oleate in the presence of scopolamine. These observations suggest that pancreatic enzyme secretion in response to oleate is primarily mediated by CCK and that adrenergic modulation may play an important role in the CCK-mediated pancreatic enzyme secretion in response to oleate, although interpretation of these results may have some restriction related to anesthesia.

Weanling male guinea pigs (Cavia porcellus), 2–3 weeks of age, with initial body weights of 207–271 g were exposed for 2–16 weeks to constant cold (6°C) and hypoxia (P _{o 2} = 85 Torr) equivalent to 4800 m above sea level. Their growth rates and body weights did not differ from those of control animals of the same age maintained under normoxic conditions (22°C, P _o2 = 133 Torr). After 2, 3, 4, 6, 10, or 16 weeks exposure the animals were sacrificed, the hearts were removed, the ventricles were separated and weighed, and myoglobin concentrations were determined. Total heart weight as well as both right and left ventricular weights increased linearly with age. By the second week of exposure of the guinea pigs to cold plus hypoxia the total heart and right ventricular weights were 25 and 50% greater than those of the normoxic control animals. Both weights increased at greater rates than those of the controls until Week 6 and then remained at 30 and 80% throughout the 16th week. The weights of the left ventricles in these animals were only slightly greater than those of the controls. In spite of the severe right ventricular hypertrophy these animals showed no clinical signs of right heart failure. Myoglobin concentrations were significantly greater in both ventricles for the cold-plus-hypoxic animals than for the controls.

The effect of the smooth-muscle relaxing agents, papaverine, sodium nitroprusside, and verapamil, on the replication of human cytomegalovirus (CMV) was investigated. At a concentration of 100 μM, infectious yields of CMV were reduced by 1.23 to 5.72 log₁₀ by these drugs (papaverine, 5.72 log₁₀; nitroprusside, 1.85 log₁₀; verapamil, 1.23 log₁₀). The ED₅₀ for papaverine was found to be somewhat less than 1 μM, a concentration which appears to be within the range achieved clinically. Papaverine did not irreversibly modify treated cells to a virus-resistant state since treatment of cells with papaverine from 24 hr before until immediately prior to CMV infection did not significantly reduce CMV yields. Replication of CMV was most sensitive to inhibition when papaverine was added at or before 6 hr after CMV infection. Addition of papaverine at later times resulted in a substantial reduction of the inhibitory effect on virus yields, suggesting that the phase of CMV replication sensitive to papaverine inhibition occurred early in the replication cycle. These results, particularly in light of the potency of papaverine, indicate that some smooth-muscle relaxing agents have significant antiviral activity toward the replication of CMV.

The pathogenesis of anemia in patients with chronic renal failure was studied by analyzing the effect of uremic sera on the in vitro colony growth of erythroid (CFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells. Uremic sera from 20 of 30 patients inhibited erythroid colony growth below 70% of control even when cultured with normal human bone marrow of the same blood type. On the other hand, only one of the sera inhibited colony growth of CFU-GM as compared with normal sera. On Sephadex G-15 gel filtration, the CFU-E-inhibiting activity appeared in two different fractions: the void volume peak and the delayed eluant before the second peak. The inhibiting activity in the former fraction was noted only in uremic sera. The results of this study suggest the existence of a serum inhibitor(s) of erythropoiesis with a relative molecular mass of more than 1500 Da which are virtually impossible to dialyze by conventional membranes.

C57BL/10 mice exhibit major histocompatibility complex linked nonresponsiveness to hen egg white lysozyme (HEL). When these animals are primed with HEL in Freund's complete adjuvant (FCA), their secondary splenic plaque forming cell responses to aqueous HEL challenge are minimal to nonexistent. This notwithstanding, we show here that concomitant priming with both HEL and keyhole limpet hemocyanin (KLH) leads to an enhanced response to the HEL component following secondary challenge with an HEL-KLH conjugate. This enhancing effect can be transferred by nylon wool nonadherent spleen cells from HEL/FCA primed animals. Adoptive transfer studies with fractionated spleen cell populations suggest also that B cells are primed in these animals. Thus, animals which are incapable of mounting a secondary response to this antigen nevertheless appear to be primed at both the T-cell and B-cell levels following exposure to the antigen in FCA. The implications of this finding are discussed.

Various rat mammary tumors were analyzed for the presence of a milk-specific Ca²⁺-stimulated RNase (Ca²⁺-RNase). When crude extracts of some differentiated tumors—adenocarcinomas of MT/W9, MT/W9a, R3230AC, DMBA-1, DMBA-8, and DMBA-14 and 3MN squamous cell carcinoma—were assayed for RNase activity under various ionic conditions, it was always highest in the presence of Ca²⁺/EDTA than under any other ionic condition. The opposite was true in invasive MT/W449a and 13762 adenocarcinomas, poorly differentiated SMT/2A carcinomas, MAMF₂/TC fibrosarcoma, and MT/A fibroadenoma. Sephacryl S-200 chromatography separation of tumor extracts confirmed the presence of Ca²⁺-RNase in those differentiated tumors and absence of the enzyme from other tumors. Expressing the activity as a ratio of Ca²⁺/EDTA to either Mg²⁺/EDTA or EDTA alone to more clearly represent the relative level of Ca²⁺-RNase activity further illustrates the distinct differences between tumor classes. Thus Ca²⁺-RNase is a sensitive marker for use in the characterization of rat tumors with respect to differentiated mammary functions.

The present study was undertaken to evaluate the effect of 24,25(OH)₂D₃ on serum calcium concentration in rats with reduced renal mass. Adult 5/6 nephrectomized male rats were divided into four groups: (i) control rats, (ii) rats treated with 1,25(OH)₂D₃, (iii) rats treated with 24,25(OH)₂D₃, and (iv) rats treated with 1,25(OH)₂D₃ and 24,25(OH)₂D₃. After 4 days, serum calcium in the 1,25(OH)₂D₃-treated group was 7.13 ± 0.32 meq/liter (P < 0.001 vs control). With the combination of 1,25(OH)₂D₃ and 24,25(OH)₂D₃ serum calcium was higher than that in control, 6.25 ± 0.5 meq/liter (P < 0.001 vs control), but lower than that in rats receiving 1,25(OH)₂D₃ alone (P < 0.05). No change in serum calcium was seen in animals treated with 24,25(OH)₂D₃ alone. On the eighth day serum calcium in the 1,25(OH)₂D₃-treated group, 6.52 ± 0.25, was higher than in the 1,25(OH)₂D₃ + 24,25(OH)₂D₃ group, 5.87 ±0.17 meq/liter, P < 0.05, P < 0.001 vs control. In both 1,25(OH)₂D₃- and 1,25(OH)₂D₃ + 24,25(OH)₂D₃-treated rats, hypercalciuria of similar magnitude occurred on the fourth and eighth day of treatment. No change in urinary calcium was seen in the control and 24,25(OH)₂D₃-treated rats. Thus, in 5/6 nephrectomized rats combined administration of 1,25(OH)₂D₃ and 24,25(OH)₂D₃ attenuates the calcemic response to 1,25(OH)₂D₃ without changes in urinary calcium excretion. These observations suggest that the effect of 24,25(OH)₂D₃ on serum calcium is different in 5/6 nephrectomized rats as compared to normal rats, in which an augmentation of serum calcium was observed following administration of both vitamin D metabolites. The effect of 24,25(OH)₂D₃ on serum calcium in rats with reduced renal mass may result from a direct effect of 24,25(OH)₂D₃ on the bone.

The temporal activation of the human interferon system by infection with virus was studied by serial measurements of both interferon in serum and activity of 2′,5′-oligo adenylate synthetase in peripheral mononuclear leukocytes. A frequency distribution of baseline values of synthetase was established for normal individuals. Following subcutaneous inoculation of rubella vaccine virus, serum interferon rose briefly with a peak on Day 14. The peak concentration of synthetase also occurred on Day 14 but remained elevated for greater than 1 week. After measles virus, serum interferon did not rise above baseline, but synthetase peaked on Day 14 and remained elevated. Subcutaneous inoculation of mumps vaccine virus was associated with a brief period of elevation of the synthetase and no interferon in the serum. Thus, the determination of synthetase levels in tissue may be useful in some situations to reflect a small or transient elevation of endogenous interferon.

Disease caused by the coccidian Toxoplasma gondii can be confined to the central nervous system, although the parasite is capable of infecting all organ systems. To determine whether neural cells are differentially susceptible to infection and destruction by T. gondii, infection of neonatal mouse brain monolayers was compared to infection of human fibroblast monolayers under the same conditions with equal inocula of two parasite strains. In preliminary experiments there was no difference in total parasite yield or in plaques per monolayer between rodent and human cells. A standardized inoculum of T. gondii RH strain caused 35.6 ± 6.4 (SD) plaques per well in neural explant monolayers compared to 39.3 ± 12.5 plaques per well in fibroblasts. T. gondii P strain produced 35.6 ± 8.9 infected foci per well in neural cells compared to 32.6 ± 9.3 foci in fibroblasts. Intrinsic properties of neural cells do not appear to cause a higher rate of infection than that in nonneural cells.

Six structural analogs of bradykinin were tested to determine whether they antagonize the vasodilator response to bradykinin. The dog hindlimb preparation was used as a bioassay. Mongrel dogs were anesthetized and the femoral arteries were isolated and fitted with a noncan-nulating electromagnetic flow probe. An indwelling catheter was also placed for administration of saline, bradykinin, or the various analogs. The vasodilatory responses of the hindlimb circulation to bolus doses of bradykinin from 1 of 20 ng were tested during vehicle or analog administration at 1 and 10 μg/min. Bradykinin analogs which were characterized by amino acid replacement by β-(2-thienyl)-L-alanine (Thi) at positions 5 and 8, D-phenylalanine (D-Phe) at position 7, and an additional replacement of hydroxyproline at position 2 or 3 were effective antagonists of bradykinin. The decapeptide bradykinin analog (BKA06) D-Arg-(Hyp³-Thi⁵-D-Phe⁷-Thi)-BK was the most potent analog tested, producing a full log dose shift in the dose-response curve to bradykinin at the 10 μg/min (4 nmole/min) infusion rate. None of the analogs we tested produced vasodilation or had any effect upon systemic blood pressure at the concentrations tested. Our results suggest that these structural analogs of bradykinin may be effective pharmacologic tools to study the role of endogenous kinins in the control of vascular resistance and circulatory homeostasis.

Plasma lipids (cholesterol, triglycerides, and phospholipids; mg/dl) and the fecal excretion (mg/day) of neutral steroids and bile acids were studied in layers (L), hereditary nonlayer hens (NL), and roosters (R) fed a basal cholesterol-free grain diet ad libitum. Each group had significantly (P < 0.05) different levels of plasma cholesterol, triglycerides, and phospholipids when compared to the other groups. The highest lipid values were found in the NL group (cholesterol, 798 ± 89; triglycerides, 8914 ± 679; phopholipids, 2458 ± 112). There was no difference in the fecal excretion of neutral steroids between L and NL; however, fecal bile acid excretion by these two groups was significantly different (P < 0.05) (L, 13.1 ± 1.7 vs NL, 26.9 ± 3.4). Fecal neutral steroid excretion by R was significantly greater (P < 0.05) than that by either L or NL (L, 6.4 ± 1.3; NL, 6.0 ± 1.4; R, 14.4 ± 1.2). While fecal excretion of bile acids by R (36.1 ± 4.0) was also greater than that by either L or NL, only the difference between R and L was statistically significant (P < 0.05). Since, in the steady state, fecal bile acid excretion is equal to its synthesis, these results suggest that bile acid metabolism in these animals can be affected by both sex and egg-laying status.

Phosphoribosylpyrophosphate (PRPP) levels increase several-fold in HL-60 cells adapted to folate deficiency either by continuous passage in folate-deficient medium or by short-term incubation with 10⁻⁸ M methotrexate (MTX). The addition of folic acid (PteGlu) or 5-formyltetrahydrofolic acid (5-CHO-H₄PteGlu) in the form of Leucovorin normalizes this effect. The reactions for measuring PRPP levels are time and temperature dependent and are influenced by PRPP-reacting substances in undialyzed serum. Inorganic phosphate (PO₄), when added to the assay, markedly stimulates PRPP levels in HL-60 cells and can be used to stress folate-dependent PRPP utilization for purine synthesis. The integrity of the folate-dependent pathways of purine-synthesizing cells can be sensitively assessed by measurement of PRPP levels during a 2-hr assay in the presence of PO₄ in medium free of folate but containing dialyzed serum. In HL-60 cells that are folate deficient or in the presence of MTX (as low as 2 × 10⁻⁹ M), PO₄-stimulated PRPP levels remain elevated due to ineffective utilization unless folate is added to the incubation mixture. The sensitivity of this PRPP assay to metabolically assess the integrity of folate-dependent reactions in purine synthesis is comparable to that of the deoxyuridine suppression assay. Inorganic phosphate can also be used to stimulate the incorporation of purine analogs, such as 6-mercaptopurine, into intact red blood cells which may have therapeutic implications for targeting drug delivery.

Many investigations have indicated a functional role for carbonic anhydrase in the mediation of hormone-stimulated bone resorption. These studies depend heavily on the use of heterocyclic sulfonamide inhibitors of carbonic anhydrase. These drugs have effects on many tissues other than bone, and some of these effects confound the interpretation of studies of the role of carbonic acid in bone metabolism. A novel, “bone-targeted” sulfonamide has been produced to obviate these extraosseous effects. This compound (designated WP-1) is the combination of tetracycline and acetazolamide, such that the acetazolamide is not an active inhibitor. Hydrolysis of WP-1 yields an active carbonic anhydrase inhibitor. WP-1 has a marked affinity for bone mineral, allowing deposition of the drug in bone. At a concentration of 10⁻⁵ M, WP-1 attenuates parathyroid hormone stimulated net release of calcium from neonatal rat calvaria in culture. WP-1 is the first member of a class of drugs which may prove useful as pharmacological probes in the study of bone metabolism.

Effects of ANF(8–33) and Auriculin A on renal variables were investigated in conscious water-diuretic dogs. The two substances were injected intravenously (1.08 μg/kg in 3 min) or ANF(8–33) was infused (0.2 μg/kg × min in 20 min). The effects were compared to those of an equinatriuretic dose of furosemide (1.0 μg/kg × min). Injections caused increases in sodium excretion, diuresis, and osmolar clearance. No significant change in exogenous creatinine clearance (C_CREA) occurred. Infusion of ANF(8–33) decreased blood pressure by 14% (P < 0.01) and increased sodium excretion by a factor of 10 (P < 0.01). The natriuresis was a function of increases in diuresis and urinary sodium concentration, the latter by a factor of 6 (P < 0.01). Diuresis and free-water clearance (C_H2O) increased by 60% (P < 0.01), but urine osmolality did not change significantly. After the infusion a significant decrease in PAH clearance (C_PAH) (P < 0.01) was observed. Filtration fraction (FF) did not change. The furosemide natriuresis appeared later than that of ANF without significant deviations in diuresis, C _H2O, C_CREA, C_PAH, and FF; urine osmolality increased by 35% (P < 0.01). The effects of ANF(8–33) differ from those of furosemide in several ways. First, the onset of natriuresis is faster; second, the natriuresis is associated by marked increases in diuresis and free-water clearance but not in urine osmolality; and third, natriuresis is followed by a reduction in renal blood flow. The rapid natriuresis of ANF can occur without changes in glomerular filtration rate.

Bovine milk was labeled intrinsically with enriched stable isotopic zinc for human bioavailability studies. Intrajugular administration of zinc isotopes temporarily increased the plasma zinc concentration of Ayrshire cows by as much as 76%, but milk zinc concentration and the distribution of zinc between casein and whey did not change appreciably. Milk zinc isotopic enrichment reached 105 and 613 atom % excess for ⁶⁷Zn and ⁷⁰Zn, respectively within 4-12 hr of zinc administration and decreased gradually over several days. This degree of isotopic enrichment is sufficient for testing bioavailability to infants of intrinsic zinc from milk-based formulas.

The effect of age on the density and the affinity of beta adrenergic receptors was determined in the hearts of Fischer 344 rats at three ages, 6, 12, and 24 months old. The binding of the beta adrenergic antagonist ¹²⁵I-iodohydroxybenzylpindolol (IHYP), was used to quantitate and characterize cardiac beta adrenergic receptors. The maximal number of binding sites (B_max = F moles/mg of protein) were 26.3±2.5, 25.4±0.99, and 24.5±2.4 and the dissociation constants (K_d = nM) were 0.166±0.014, 0.126±0.006, and 0.135±0.015 for 6, 12, and 24 months-old animals, respectively . There were no significant differences among the three ages. These results support the contention that neither beta adrenergic receptor density or affinity changes with age in the ventricles of the rat heart.