Suppression of the immune response in C3H/HeJ mice by protein-free lipopolysaccharides.

The experiments described herein demonstrate the plaque-forming cell response of C3H/HeJ mice can be suppressed by a Boivin type lipopolysaccharide (LPS) and a deproteinized glycolipid. Suppression was observed both in vivo and in vitro, and could be transferred to normal cells in coculture experiments. This newly discovered effect of LPS in C3H/HeJ mice indicates that the adjuvant and inhibitory action of LPS may be distinct phenomena which are under different genetic regulation. Thus, the C3H/HeJ strain provides a convenient animal model for study of immunosuppression independent of the adjuvant effect.

Regulation of the immune system by synthetic polynucleotides. 3. Action on antigen-reactive cells of thymic origin.

Polyadenylic-polyuridylic acid complexes, a potent adjuvant to the immune response, were tested for action on thymic-influenced and bone marrow-derived lymphocytes in model systems deficient in one or the other of these cells. Adult mice, thymectomized at birth or mice treated with heterologous antithymocyte serum produced 90-95% fewer splenic rosette-forming cells than normal mice in response to an injection of sheep erythrocytes. Intravenous injection of complexes of homoribopolynucleotides, polyadenylic and polyuridylic acids, poly A:U with SRBC restored immunologic competence to NTx- or ATS-treated mice such that they produced normal or near normal levels of splenic RFC. In addition, injection of poly A:U enabled NTx mice to reject allogeneic skin grafts at the same rate as control mice with an intact thymus. Further reduction in residual thymocytes by combining neonatal thymectomy with ATS treatment reduced the number of anti-SRBC RFC induced by poly A:U. Lethally irradiated mice which received SRBC, excess bone marrow cells, and as few as 40,000 thymic lymphocytes were stimulated by poly A:U to produce RFC. No adjuvant effect was observed when irradiated mice received excess thymic lymphocytes and low doses of bone marrow cells with poly A:U. The results suggested that the adjuvant action of poly A:U was exerted on the thymic-influenced, antigen-reactive cell and that restoration of immunocompetence to NTx- or ATS-treated mice was caused by amplification of a small number of residual antigen-reactive cells which were influenced by the thymus in utero before thymectomy, or which survived treatment with ATS.

Regulation of the immune system by synthetic polynucleotides. II. Action on peritoneal exudate cells.

Incubation of antigen with normal mouse peritoneal exudate cells in vitro and subsequent reinjection of the washed cells into syngeneic mice resulted in increased antibody titers as compared to mice injected with antigen alone. Several of the variables influencing this system were studied with and without the stimulus of complex homoribopolynucleotides (poly A:U or poly I:C) as adjuvants to determine the cellular site of action of the latter. It was found that addition of poly A:U or poly I:C caused a further rise in circulating antibody levels which correlated with increased RNA synthesis, suggesting that the macrophage was one cell affected by this adjuvant. Actinomycin D was found to inhibit the rise in titer induced by PEC and this inhibition could be overcome by poly A:U. Injection of the polynucleotides 18 hr before antigen resulted in depression of circulating antibody levels, and poly A:U or poly I:C injected 18 hr before harvesting PEC and incubation with antigen also inhibited the capacity of the PEC to increase antibody levels. A 4S RNA-rich fraction was purified after treatment with phenol of PEC exposed to antigen in vitro, and under the stimulus of poly A:U this RNA was capable of inducing specific antibody titers and rosette-forming cells on injection into mice. Antigen contamination of Pronase-treated RNA, active biologically, was below 10(-11) g as determined isotopically.

Regulation of the immune system by synthetic polynucleotides. V. Effect on cell-associated immunoglobulin receptors and immunological memory.

Addition of polyadenylic-polyuridylic acid in complex form (poly A:U) without antigen to a suspension of spleen cells obtained from BALB/Aj mice primed 6 wk previously with human gamma-globulin (HGG) resulted in an immediate fourfold increase over background number of anti-HGG rosette-forming cells (RFC). Culture of similar cells in the presence of puromycin for 1-6 hr before poly A:U did not significantly reduce the response. Continued culture of primed spleen cells in the presence of poly A:U, resulted in a decrease of RFC to background levels within an hour followed by an increase again 6 hr later. This later increase in RFC was inhibited by addition of puromycin to the culture medium. The nonspecific stimulation by poly A:U of antibody production by primed spleen cells also was induced in vivo. Increases in splenic RFC were detectable 6 hr after intravenous injection of poly A:U alone, without antigen, into primed mice. The response peaked at 18 hr and had dissipated completely within 3 days. A second injection of poly A:U 24 hr or later after the first injection resulted in a second response, similar to the first with respect to kinetics and intensity. Rosette formation by poly A:U-stimulated cells could not be inhibited by mitotic poisons, but was inhibited by treatment of the cells with goat anti-mouse gamma-globulin serum, suggesting that the antibody involved was a 7S gamma-globulin. The decrease in RFC induced by culture of primed cells for 1 hr in poly A:U paralleled a decrease in secondary responsiveness of the cells to antigen. This poly A:U-induced inhibition of secondary responsiveness could be reversed by suspending the treated cells in supernatant fluids derived from poly A:U-stimulated cultures. The reversal was specific in that supernatant fluids removed from bovine serum albumin (BSA)-primed cells by poly A:U did not stimulate the response of HGG-primed cells to HGG. However supernatant fluids from BSA-primed cells caused the production of anti-HGG RFC if BSA rather than HGG was used as triggering antigen. The active factor in the supernatant fluids appeared to be a 7S gamma-globulin since activity was lost after 45 min incubation of the supernatant fluids in the presence of goat anti-mouse 7S gamma-globulin serum.

Characterization of the renin-antirenin system.

Rabbit antibody capable of neutralizing the pressor activity of the enzyme renin, derived from hog kidneys, was characterized with respect to type and capacity to react serologically. Three antirenin antibodies were detected, both a precipitating and nonprecipitating, neutralizing antibody as well as a neutralizing antibody capable of being absorbed by inactivated renin. The precipitating, neutralizing antibody was purified and used successfully in an assay for hog renin by means of a hemagglutination inhibition test. By immunofluorescent methods utilizing the purified antiserum, renin was found to be localized in the macula densa of the distal convoluted tubules and the juxtaglomerular cells of the afferent arteriole in both hog and dog kidneys.

Radioautographic and electron-microscopic evidence of rapid uptake of antigen by lymphocytes.

Iodine-125-labeled ferritin molecules were detected by radioautography in the sinuses of the rat popliteal lymph node shortly after injection into the foot pad; they appeared to be taken up by macrophages and phagocytic reticular cells. Electron microscopic examination of the same tissue also revealed ferritin molecules within small lymphocytes as early as 5 minutes after injection. The antigen appeared to be taken up by the process of pinocytosis and was distributed throughout the cytoplasm and nucleus. While the number of ferritin molecules observed in the lymphocyte was much less than that taken into the inacrophage, the observation is significant in understanding the role lymphocytes play during the early phase of antibody response.

A randomized, double-blind, placebo-controlled, parallel-group study to assess the efficacy and safety of dual ACE/NEP inhibitor GW660511X in mild-to-moderate hypertensive patients.

This multicentre, double-blind, placebo-controlled, parallel-group study determined the efficacy and safety of GW660511 200 mg, a dual inhibitor of angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP), in mild-to-moderate hypertensive patients (diastolic blood pressure (DBP), > or =90 and < or =109 mm Hg; systolic blood pressure (SBP), > or =150 and < or =180 mm Hg). After a single-blind 2- to 4-week placebo run-in period, 123 patients (aged 18-65 years) were randomized to either placebo (n=62) or to active treatment (n=61) consisting of two consecutive 3-day dose titration periods of GW660511X 50 mg once daily and 100 mg once daily followed by GW660511X 200 mg once daily for 14 days. GW660511X 200 mg significantly lowered (baseline and placebo-corrected) both trough mean cuff SBP (-8.00 mm Hg, P=0.002) and DBP (-5.38 mm Hg, P=0.003). GW660511X 200 mg significantly reduced placebo-corrected mean 24-h and daytime but not night-time ambulatory SBP and DBP. Over the 0-24 h time period following GW660511X 200 mg, there were significant (P<0.001) reductions in serum ACE activity and significant (P<0.001) increases in plasma ANP concentration compared with placebo in terms of both peak and trough effects. In addition, treatment with GW660511X 200 mg significantly (P=0.003) increased (placebo-corrected, 1.52-fold) urinary excretion of cGMP over the 0-24 h interval. Treatment-related adverse events were experienced by 43% of the patients administered GW660511X 200 mg and 44% of those dosed with placebo with headache the most commonly reported. In conclusion, GW660511X 200 mg is an effective antihypertensive in mild-to-moderate hypertensive patients with potent effects on biological markers of ACE and NEP inhibition.

Macrophage activation by adjuvants in aging mice.

Peritoneal macrophages from young (3-8 mo) and aging (12-29 mo) mice of the C58, BALB/c, C3H/He, C57BI/6J, and B6D2F1 stains were compared for their capacity to become activated by various adjuvants in four assays. In chemiluminescence, activation by phorbol myristic acetate or zymosan of macrophages from aging mice of the C58, BALB/c, and C3H/He strains was increased approximately twofold greater than that of cells from young mice. A reversal of this was seen in the same three strains when measuring activation of phagocytosis by lipopolysaccharide, polyadenylate:polyuridylate (polyA:poly U), or muramyl dipeptide in that increased activity was induced readily in macrophages from young but not aging mice. Similarly, tumoricidal activity of macrophages from young but not aging mice was stimulated 6.0- and 4.4-fold by lipopolysaccharide and poly A:poly U, respectively, in the C58 strain (the only strain studied). Activation by lipopolysaccharide and poly A:poly U of the hexose monophosphate shunt in macrophages from the C58 and C3H/He strains also was significant in young but not aging mice, whereas it occurred in both age groups of the BALB/c and C57B1/6J mice. A reversal of response patterns was observed between aging female virgin and breeder C58 mice in the chemiluminescence and hexose monophosphate shunt assays in that the breeding mice mimicked the young virgin mice.

Glycolipid induced proliferation of lipopolysaccharide hyporesponsive C3H/HeJ splenocytes.

Although the C3H/HeJ mouse is hyporesponsive to lipopolysaccharides (LPS), certain forms of the lipid A fraction have been shown to stimulate cells from this mouse strain. To determine the role of the oligosaccharide chain length on the lipid A-induced proliferation of C3H/HeJ splenocytes, a panel of glycolipids from R-chemotypes (Re, Rc, and Rd) and a nontoxic monophosphoryl lipid A (MPL) were tested. The MPL cells isolated from the MPL of Salmonella minnesota, Salmonella typhimurium, and the Reglycolipids isolated from Escherichia coli were found to be effective at stimulating the LPS-hyporesponsive spleen cells. A Re-glycolipid isolated from a different strain of E. coli cells was inactive, as were the S. minnesota Rc and Rd chemotypes. Proliferation induced by MPL and the active Re preparations was dose dependent and was inhibited by polymyxin B. Thus, if contamination of the Re-LPS or MPL with lipid A-associated protein occurred, it was below functional levels. The data suggest that the C3H/HeJ spleen cells are capable of responding to certain glycolipids, but they may lack the ability to convert native LPS into a stimulatory signal. In addition, a monosaccharide precursor of lipid A (lipid X), and a monoacyl glucosamine phospholipid derivative of lipid X (MaGP), were capable of inhibiting the proliferation induced by the MPL and Re-glycolipids. These data are compatible with the existence of a spleen cell receptor for lipid A.

Activation of macrophages from aging mice by detoxified lipid A.

A detoxified derivative of endotoxic lipopolysaccharides (LPS), monophosphoryl lipid A (MPL), which is capable of inducing nonspecific resistance against several infectious organisms, was tested for its capacity to activate peritoneal macrophages (M phi) from young and immunodeficient aging BALB/c and C3H/HeN mice. Superoxide generation and hydrogen peroxide release by M phi from aging mice were elevated following intraperitoneal injection with 25 micrograms of LPS or MPL, although they did not reach the peak levels achieved in LPS or MPL-treated young mice. Nitroblue tetrazolium reduction (NBT) by peritoneal M phi from aging C3H/HeN mice treated with MPL was higher than that in control aging mice, equalling that from MPL-treated young mice. LPS, its toxic counterpart, however, failed to increase NBT reduction in either group. MPL enhanced lysozyme activity in M phi from both aging and young C3H/HeN mice above initial control levels. On the other hand, LPS suppressed lysozyme activity in M phi from young, but not aging mice. Phagocytosis of Candida albicans by M phi from BALB/c mice was increased in both groups when stimulated by MPL, but not LPS. Similarly, MPL enhanced the ability to kill Candida in both aging and young BALB/c mice. This effect was not seen with LPS. Thus, a detoxified derivative of LPS was found capable of activating the respiratory burst, NBT reduction, elevating lysozyme activity, as well as phagocytosis and killing of Candida in murine peritoneal M phi from both young and aging mice.

A comparison of the immunomodulating properties of two forms of monophosphoryl lipid A analogues.

This investigation compared the immunomodulating activities of two forms monophosphoryl lipid A, which are analogues of bacterial lipopolysaccharides with little or no toxicity. Tested were a synthetic compound designated 504 and a purified compound, isolated from bacterial cell walls designated MPL. Both of these clinical adjuvant candidates were effective in mice in exerting strong immunomodulating activity in the following areas: (a) enhancing antibody production in young and aging mice; (b) suppressing antibody formation under different experimental conditions; (c) activating macrophages to secrete interleukin 1, hydrogen peroxide, and superoxide anion; and (d) stimulating proliferation of spleen cells from C3H/HeN mice. Both exhibited considerably reduced toxicity in LD50 assays when compared to native lipopolysaccharides (LPS). The LD50 for MPL was 225 times and that of compound 504, 40 times that of native LPS in the exquisitely sensitive, galactosamine-loaded C57BL/6 murine strain.

Polyadenylic: polyuridylic acid-induced determinants of host resistance to cytomegalovirus and their potentiation by hyperthermia.

The ability of spleen cells from poly A:poly U-treated mice to inhibit murine cytomegalovirus (MCMV) replication in confluent monolayer cells of secondary mouse embryo fibroblasts (MEFs) cultured at 37 and 40 degrees C was investigated. When spleen cells from BALB/c mice injected 48 h earlier with poly A:poly U were added to MEFs infected 2 h previously with MCMV, 37% less plaques were observed than in cultures containing control cells. Of interest, the poly A:poly U-induced antiviral activity at the elevated temperature (40 degrees C) resulted in a further drop to 61% in MCMV-induced plaques compared to those of the normothermic (37 degrees C) cultures. The antiviral function of spleen cells induced by poly A:poly U was evident in the supernatant fluid when cultured for 48 h at 37 degrees C. MCMV-induced plaques were reduced to 52 and 5% of controls in the plaque assays performed at 37 and 40 degrees C, respectively. Supernatant fluids generated at 40 degrees C, however, inhibited MCMV replication only when incubated at 40 degrees C. No direct inhibitory effect of the supernatant fluids on MCMV was evident; rather, inhibition was effected directly on the MEFs. The NK cell fraction of spleen cells from poly A:poly U-treated mice alone showed only a slight inhibitory effect at 40 degrees C. However, in the presence of the supernatant fluid from poly A:poly U-exposed spleen cells, the antiviral activity of NK cells was significantly increased both at 37 and 40 degrees C. The cellular source of the culture fluid showing poly A:poly U-induced antiviral activity appeared to be in the T-cell population. It was completely neutralized by monoclonal anti-IFN gamma antibody but not by anti-IFN beta, anti-IL4, anti-transforming growth factor, or anti-prostaglandin E2. In conclusion, these data document the ability of spleen cells from poly A:poly U-treated mice to inhibit MCMV replication and this activity is potentiated by hyperthermic conditions. The antiviral function of poly A:poly U-treated spleen cells appeared to be due mainly to the action of IFN gamma produced by T cells. The enhanced antiviral activity by hyperthermia appeared to be related to the action of IFN gamma rather than its production.

The kinetics of mycophenolic acid and its glucuronide metabolite in adult kidney transplant recipients.

BACKGROUND: Mycophenolic acid kinetics have been reported to vary after renal transplantation, and mycophenolic acid area under the concentration-time curve (AUC) is the best predictor of suppression of graft rejection. METHODS: To determine whether mycophenolic acid kinetics vary after renal transplantation and to examine the potential role of enterohepatic recirculation, we investigated the kinetics of mycophenolic acid and mycophenolic acid glucuronide on days 2, 5, and 28 after transplantation in 10 kidney transplant recipients (male/female ratio, 1.5; mean age, 41.7 +/- 5.0 years) given 1 g mycophenolate mofetil twice a day. To facilitate therapeutic drug monitoring, we examined a limited sampling strategy for estimating 12-hour mycophenolic acid [AUC(0-12)]. RESULTS: The mean +/- SE AUC(0-12) for mycophenolic acid on day 28 was 38.5 +/- 1.6 mg x h/L, with a secondary peak 4 to 8 hours after dosing that was attributable to enterohepatic recirculation. Marked variability was shown in the kinetic profile of mycophenolic acid among patients across the three sampling days. Mycophenolic acid AUC(0-12) was positively predicted by both serum creatinine (P = .01) and serum albumin (P = .03) but not by time after transplantation, body weight, or trough concentration. Limited sampling (at 0, 1, 3, and 6 hours) accounted for 84.1% of the variability in the mycophenolic acid AUC(0-12) data and predicted the AUC(0-12) closely (r2 = 0.954) when evaluated in 10 different kidney transplant recipients. CONCLUSIONS: Mycophenolic acid AUC(0-12) is predicted by serum albumin and creatinine after kidney transplantation, and the AUC(0-12) may be determined during the early posttransplant period while the patient remains hospitalized with use of a limited sampling strategy to facilitate therapeutic drug monitoring.

Investigation by immunofluorescence of arterial lesions in rabbits on two different lipid supplements and treated with pyridinol carbamate.

Rabbits maintained on a pellet diet supplemented with cholesterol, or on a semi-synthetic diet containing beef fat but no added cholesterol, have been studied in relation to their development of hyperlipidaemia and of lipid-filled arterial lesions. The influence of pyridinol carbamate on animals on both diets was also examined but found to produce no significant effect. Animals on both diets developed a hyperlipoproteinaemia. In cholesterol-fed animals this developed quickly, became gross, and was characterized by the presence of an anomalous lipoprotein of very low density, large molecular size and abnormally high cholesterol content. Beef fat fed animals showed a more moderate hyperlipidaemia which developed more slowly and the lipoproteins qualitatively resembled those in normal rabbits. Differences in the rate and severity of development of aortic lesions between the two different dietary supplements were found to reflect differences in the duration and intensity of hyperlipoproteinaemia between the groups. Arterial lesions in cholesterol-fed animals were more extensive and contained larger numbers of fat-filled cells than those in beef fat-fed animals. Comparisons were made (in many cases on the identical section) between lesions treated with a fluorescein labelled antiserum to total rabbit serum low density lipoproteins (TLDL) and with a conventional lipid stain. Precise agreement was found between the distribution of lipid reacting with Oil red 0 and specific fluorescence for TLDL in endothelial cells, in extracellular deposits in the intimal ground-substance and in medial smooth muscle cells. But fat-filled cells in the intima and in reticulo-endothelial tissue showed variable immunofluorescent reactivity. The reason for this discrepancy is discussed. Agreement between the distribution of conventional lipid staining and specific immunofluorescence for TLDL was also found in extracellularly distributed material in arterioles and smaller vessels at certain sites. It is suggested that these results establish that rabbit TLDL serve as the vehicles transporting lipid into the experimental lesions, just as the homologous human lipoproteins do in human atherosclerosis.

Evaluation of the potential interaction between NaCl and prostaglandin inhibition in elderly individuals with isolated systolic hypertension.

OBJECTIVE: To evaluate whether prostaglandin inhibition with the non-steroidal anti-inflammatory drug (NSAID), indomethacin (I) interacts synergistically with different doses of salt (NaCl) in elevating systolic blood pressure (SBP). DESIGN AND METHODS: This randomized, placebo-controlled, double-blind, crossover study examined the interaction between NaCl and the prostaglandin inhibitor, I in 31 healthy elderly individuals with a mean age (+/- SD) of 68.7+/-5.7 years (range 61-85 years). Participants aged more than 60 years on a 140 mmol/day NaCl dose for 6 weeks were chosen with normal blood pressure [24-h SBP <148 mm Hg, diastolic blood pressure (DBP) <85 mm Hg on the Takeda Ambulatory Blood Pressure Monitor (TABPM); n = 15] and isolated systolic hypertension (ISH), [24-h SBP >148 mm Hg, 24-h DBP <85 mm Hg on TABPM; n = 16]. Exclusion criteria included uncontrolled hypertension (SBP >220 mm Hg and/or DBP >110 mm Hg), cardiac disease, creatinine clearance <60 ml/min, dementia and recent cerebrovascular accident or secondary hypertension. A 2x2 Latin square design was structured using four treatment groups [low salt (NaCl = 90 mmol/day) + I placebo, high salt (NaCl = 240 mmol/day) + I placebo, low salt + I (25 mg three times daily) and high salt + I] for 2 weeks each, balanced and interspersed with 2 week washout periods to minimize carryover effects. Twenty-four hour SBP, DBP and heart rate were measured and summarized using a moving interval averaging technique. The mean change in 24-h SBP, DBP, heart rate, urinary Na+, K+, protein and creatinine, creatinine clearance and serum electrolytes were compared across treatments in the total cohort and in ISH and control groups separately using ANCOVA (SAS). RESULTS: In the total cohort, compared with low NaCl, chronic high NaCl increased mean SBP (5.76 mm Hg; P = 0.0002) and DBP (3.36 mm Hg; P = 0.002). Indomethacin significantly increased mean SBP (2.66 mm Hg, P = 0.015) but not DBP (0.31 mm Hg, P = 0.419). High salt and I were additive (SBPT, DBPT) but there was no interaction (P = 0.795 and P = 0.739, respectively). Additionally, chronic high NaCl increased serum Na (P = 0.0001) and 24-h urinary Na (P = 0.0001) as expected. Indomethacin significantly decreased mean heart rate (P = 0.018). The effects of NaCl and I on SBP, DBP and heart rate were not modified by age, alcohol intake, serum K+, body mass index or treatment order. In the ISH group, NaCl dose significantly elevated SBP (9.87 mm Hg; P = 0.0001) and DBP (5.26 mm Hg, P = 0.006) but did not significantly alter blood pressure in the normotensive group. Indomethacin significantly elevated SBP (P = 0.03) in normotensive individuals but had no effect on blood pressure in the ISH group. CONCLUSIONS: Chronic high salt diet elevated blood pressure more than I in the total cohort of elderly individuals. No interaction was demonstrated and their effects were additive. In the ISH group, chronic high salt diet significantly increased SBP and DBP while I failed to alter blood pressure. In the normotensive group, I, but not salt, elevated SBP. Patients with ISH are sensitive to the pressor effect of NaCl but resistant to the pressor effect of prostaglandin inhibition in contrast to elderly normotensive control individuals where the reverse was found.

Blood pressure is linked to salt intake and modulated by the angiotensinogen gene in normotensive and hypertensive elderly subjects.

OBJECTIVES: To evaluate salt sensitivity in elderly subjects with different forms of hypertension and controls and to investigate any modulation by genotype DESIGN: Randomized, double-blinded, placebo-controlled latin-square SETTING: Tertiary referral hospital PARTICIPANTS: Community subjects (n = 46) aged > or = 60 years classified as isolated systolic hypertension [ISH; systolic blood pressure (SBP) > or = 160, diastolic blood pressure (DBP) < 90 mmHg, n = 19], diastolic +/- systolic hypertension (SDH; DBP > or = 90 mmHg, n = 10) and normotension (SBP < 160, DBP < 90 mmHg, n = 17). INTERVENTION: Four 14 day treatments, 50, 100, 200 and 300 mmol/day of sodium chloride supplementation interspersed with 14 day washout periods on a salt-restricted diet. MAIN OUTCOME MEASURES: The 24 h blood pressure, heart rate, weight, urinary sodium and creatinine clearance measured during baseline, treatment and washout periods and angiotensinogen (AGT) and angiotensin converting enzyme (ACE) genotypes. RESULTS: For the entire cohort, the mean +/- standard error (SE) of change from baseline in SBP for 50, 100, 200 and 300 mmol/day salt was 7.7+/-2.4, 12.1+/-2.4, 16.6+/-3.0, 18.5+/-2.6 mmHg, respectively. For DBP, the respective changes were: -0.1+/-1.5, 2.4+/-1.6, 3.0+/-1.5, 5.8+/-1.7 mmHg. The increase in SBP among ISH subjects was significantly higher than among subjects in the SDH and normotensive groups (P < 0.05). AGT genotype influenced the effect of salt dose on the change in DBP (P = 0.006) but not SBP (P = 0.7). CONCLUSIONS: In healthy, older subjects, a linear increase in BP occurred with increasing salt dose, it appeared most pronounced in ISH subjects and could be modulated by AGT genotype.

M235-->T polymorphism of the angiotensinogen gene predicts hypertension in the elderly.

OBJECTIVE: To determine whether the M235-->T polymorphism (exon 2) of the angiotensinogen gene is associated with hypertension in elderly patients with isolated systolic hypertension [ISH: systolic blood pressure (SBP) > or = 160 mmHg, diastolic blood pressure (DBP) < 90 mmHg) or systolic-diastolic hypertension (SDH: DBP > or = 90 mmHg, SBP > or = 160 mmHg) compared with normotensive controls (SBP < 160 mmHg, DBP < 90 mmHg). DESIGN: A case-control study in 769 non-institutionalized, elderly (aged > or = 60 years; female:male ratio 0.85) residents of Dubbo, New South Wales. METHODS: Individuals were classified as having ISH (n = 171), having SDH (n = 218) and being normotensive controls (n = 366) with age and sex matching. MM, TT and MT genotypes were determined by a nested polymerase chain reaction strategy using DNA extracted from serum. The prediction of ISH or SDH by genotype or allele was examined in a multiple-logistic regression model that controlled for various confounders. RESULTS: SBP (mean +/- SD, mmHg)/DBP (mean +/- SD, mmHg) was 176 +/- 16/79 +/- 8 in the ISH group, 167 +/- 23/97 +/- 7 in the SDH group and 134 +/- 14/74 +/- 9 in the normotensive control group. The frequencies of M and T alleles in the normal population (0.69 and 0.31, respectively) were altered significantly in the ISH group (0.61 and 0.39, respectively; chi 2 = 6.0, P < 0.02) and the SDH group (0.62 and 0.38, respectively; chi 2 = 6.0, P < 0.02). The presence of the TT genotype predicted both ISH (odds ratio 1.9, 95% confidence interval 1.1-3.3) and SDH (1.7, 1.0-3.0) as did that of the T allele (ISH: 1.3, 1.0-1.7; SDH: 1.3, 1.0-1.7). CONCLUSIONS: The M235-->T polymorphism may be a marker for both forms of hypertension in the elderly. Whether the TT genotype represents a genetic risk factor for the development of hypertension in later life requires confirmation.

An improved method for estimating cholecystokinin in human serum.

Because of difficulties encountered in setting up radioimmunoassays for cholecystokinin (CCK) a sensitive and reliable biological method for estimating this hormone is still needed. The principles of such a biological technique and an improvement to it have already been described, but the serum levels of CCK reported were high and the technique required further refinement and validation. The strips of rabbit gall-bladder used to estimate the concentration of CCK increased in sensitivity to standard solutions of CCK over a 6--8 period before stabilizing, but a single sample of serum increased the sensitivity of the strips of gall-bladder to their maximum immediately. These two problems were eliminated by 'priming' the strips of gall-bladder by exposure to two serum samples before exposure to the standard solutions used for production of a dose--response curve. Thirdly, it was discovered that some non-peptide substances in serum possessed CCK-like activity; by extracting all the small peptides from serum with dextran-coated charcoal the residual activity could be measured and subtracted from the total CCK activity. Finally, the activity of CCK in the serum increased during processing before freezing. This increase was eliminated by taking the blood samples into aprotinin which has been shown to cause dramatic reduction in CCK activity in some experiments. When all these factors were taken into account and the technique suitably modified, the mean level of CCK in the serum of ten normal fasting subjects was found to be 28 milli Ivy Dog units/ml (2.4 pmol/ml), which is only one third of that reported previously.

Arginine vasopressin and osmolality in the elderly.

OBJECTIVES: To evaluate the influence of age on plasma arginine vasopressin (AVP) concentrations and the relationship between plasma AVP and serum osmolality in younger and older subjects, and in the elderly, to assess the effect of gender on plasma AVP concentration and to determine the impact of prostaglandin blockade on renal responsiveness to AVP. DESIGN: Cross-sectional study; randomized, double-blind, crossover, placebo-controlled study. SETTING: The Renal Laboratory, Royal North Shore Hospital (younger adults) and Clinical Room, St Vincents Hospital (elderly subjects). PARTICIPANTS: 45 younger adults (35 +/- 9 years), and 41 elderly subjects (29 males, 12 females; 78 +/- 3 years). All subjects were healthy and non-institutionalized. The elderly subjects were screened to exclude significant pathology (clinical assessment, multiple investigations). INTERVENTION: Blood samples were drawn from all younger and elderly subjects. The elderly subjects were randomly allocated indomethacin or placebo for 1 month. Following a 1 to 2-week washout, the alternative was administered for a further 1 month. MAIN OUTCOME MEASURES: Plasma AVP and serum osmolality and plasma AVP, serum, and urine osmolality at baseline were measured on indomethacin and placebo. RESULTS: In the elderly subjects, baseline plasma AVP concentration was significantly higher than in the younger subjects studied (4.7 +/- 0.7 vs 2.1 +/- 0.2 pg/mL respectively; P = 0.0003). Plasma AVP was strongly correlated with serum osmolality in the younger subjects (r = 0.76, P = 0.0001) but not in the elderly cohort (r = -0.18, P = 0.26). No difference was found between the sexes in plasma AVP (P = 0.89), and indomethacin treatment did not alter the plasma AVP/urine osmolality ratio (P = 0.85) in the elderly subjects. In addition, changes in plasma AVP with indomethacin therapy did not correlate with changes in serum osmolality (r = 0.16, P = 0.09). CONCLUSIONS: Aging is accompanied by an increase in plasma AVP concentrations. In healthy, elderly subjects, plasma AVP is not dependent on serum osmolality and is not influenced by gender. Indomethacin has no effect on the renal responsiveness to plasma AVP.

The mitogenic activity of polyadenylic-polyuridyclic acid complexes.

Polyadenylic-polyuridylic acid complexes (poly A:U) at the 1-5 mu g level, were mitogenic for spleen cells when given intravenously to normal Balb or cortisone-treated mice. Similarly, mitogenicity was evident when poly A:U was added to tissue culture fluids containing spleen cells from normal or cortisone-treated mice, or bone marrow cells from normal mice. Under these conditions, this adjuvant was not mitogenic for thymus cells or mesenteric lymph node cells, either in vivo or in vitro.