Transcription of the murine iNOS gene is inhibited by docosahexaenoic acid, a major constituent of fetal and neonatal sera as well as fish oils.

Macrophage activation is deficient in the fetus and neonate when the serum concentrations of docosahexaenoic acid (DHA) are 150 microM, or 10-50-fold higher than in the adult. We now show that DHA inhibits production of nitric oxide (NO) by macrophages stimulated in vitro by IFNgamma plus LPS, or by IFNgamma plus TNFalpha. The half-maximal inhibitory activity of DHA was approximately 25 microM. There were strict biochemical requirements of the fatty acid for inhibition. Polyenoic fatty acids with 22 carbons were more inhibitory than those with 20 carbons. Among 22-carbon fatty acids, those with a greater number of double bonds and a double bond in the n-3 position were more inhibitory. DHA was the most inhibitory of the polyenoic acids we tested. Inducible nitric oxide synthase (iNOS) is the enzyme responsible for the production of NO by macrophages. NO production is initiated after new iNOS enzyme is synthesized following transcription of the iNOS gene. In macrophages stimulated by IFNgamma plus LPS, DHA inhibited accumulation of iNOS mRNA, as measured by Northern blotting, and iNOS transcription, as measured by nuclear run-on assays. We transfected RAW 264.7 macrophages with a construct containing the iNOS promoter fused to the chloramphenicol acetyl transferase gene. DHA inhibited activation of this promoter by IFN gamma plus LPS. By inhibiting iNOS transcription in the fetus and neonate, DHA may contribute to their increased susceptibility to infection.

Docosahexaenoic acid, a constituent of fetal and neonatal serum, inhibits nitric oxide production by murine macrophages stimulated by IFN gamma plus LPS, or by IFN gamma plus Listeria monocytogenes.

Murine macrophage activation is deficient in the fetus and the neonate, and in areas of the placenta perfused by the fetal circulation. Fetal and neonatal serum concentrations of docosahexaenoic acid (DHA) are 150 microM, or approximately 50-fold higher than in the adult. We previously showed that DHA inhibits activation of the gene for inducible nitric oxide synthase (iNOS) in murine macrophages stimulated in vitro with interferon gamma (IFN gamma) plus lipopolysaccharide (LPS). We have now pursued these observations in greater depth. An assay system was developed which separated the stimulation of macrophages by IFN gamma plus LPS, and the actual production of nitric oxide (NO). It was found that macrophages do not produce NO until they have been stimulated by IFN gamma plus LPS for a period of 10 h. NO is produced during the subsequent 10 h, even though IFN gamma plus LPS are not longer present. DHA, if present, inhibited only during the initial 10 h stimulation; DHA did not inhibit the production of NO by macrophages which had previously been stimulated by IFN gamma plus LPS, and were already producing NO. It was also found that DHA was less inhibitory if given prior to the IFN gamma plus LPS stimulation. In a dose-responsive manner, DHA inhibited the increased abundance of iNOS mRNA by macrophages stimulated by IFN gamma plus LPS. NO contributes to the host defense against Listeria monocytogenes and other intracellular pathogens. We therefore investigated the ability of DHA to inhibit NO production by macrophages stimulated by IFN gamma plus Listeria monocytogenes in vitro; DHA inhibited transcription of the iNOS gene and also the listeriocidal activity of activated macrophages. Inhibition of NO production by DHA may contribute to the increased susceptibility of the fetoplacental unit and neonate to intracellular infections.

Immunosuppressive effect of 2'-deoxycoformycin (Pentostatin) for rat islet allotransplantation.

Adenosine deaminase (ADA) is an important enzyme for proper function of lymphocytes and congenital absence of ADA results in a form of severe combined immunodeficiency syndrome. 2'-Deoxycoformycin (Pentostatin, DCF) irreversibly inhibits ADA and therefore has been suggested as an immunosuppressive drug. The present study evaluated the immunosuppressive effect of DCF for islet allotransplantation in rats. Isolated islets (1,500 islets) from Lewis rats were transplanted into the kidney subcapsular space of streptozotocin-induced diabetic Wistar-Furth rats. DCF was administered IP either as a single injection at 1 mg/kg/wk, 1 mg/kg twice weekly, 5 mg/kg/twice weekly or 1 mg/kg/day, or as a continuous infusion at 0.8 or 1 mg/kg/day. Daily administration of DCF at 0.8 mg/kg in both methods, single daily injection or continuous infusion, resulted in a lymphopenia and a decrease in concanavalin A stimulation of splenic lymphocytes. However, DCF (in all doses) was not effective in preventing islet allograft rejection as evaluated by measuring the duration of normoglycemia following islet transplantation and by microscopic examination of the islet grafts. In fact, the duration of normoglycemia following islet transplantation was 7.5 +/- 0.9 and 9.0 +/- 1.0 days in rats receiving DCF in single daily injection or continuous infusion, respectively. This was not significantly different from control nontreated transplanted rats (8.5 +/- 0.7 days). Increasing the dose of DCF to 1 mg/kg, administered by continuous infusion, resulted in 100% mortality. For comparison, cyclosporine A (20 mg/kg, IP daily injection for 14 days) prolonged islet allograft survival to 27.3 +/- 1.5 days (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of macrophage nitric-oxide production and Ia-expression by docosahexaenoic acid, a constituent of fetal and neonatal serum.

PROBLEM: We previously demonstrated profound inhibition of macrophage activation in the murine placenta in vivo. Given the importance of macrophages both in initiating cellular immunity by presenting antigen in the context of Ia to CD4+ T cells, and in killing cellular targets by producing nitric oxide (NO), inhibition of macrophage functions in the placenta may account for the increased susceptibility of the placenta to infection. We have also showed that docosahexaenoic acid (DHA), at concentrations present in the fetal circulation, has a major role in inhibiting macrophage Ia-expression and NO production in the placenta. The concentration of DHA in fetal serum perfusing the placenta is 50x higher than in the adult. DHA has previously been reported to profoundly affect prostanoid production, to be metabolized by lipoxygenases, and to affect lipoxygenases. We now determine if these activities of DHA account for its inhibition of macrophage NO production and Ia-expression. METHODS: Murine macrophages were cultured in vitro, exposed to IFN gamma endotoxin, DHA, and various eicosanoids, and their ability to produce NO or express Ia determined. RESULTS: Although the cyclooxygenase inhibitor, indomethacin, did inhibit NO production, DHA inhibited by a different mechanism. DHA further inhibited NO production by macrophages exposed to doses of indomethacin known to maximally inhibit prostanoid production. Stable, biologically active prostanoids did not reverse the inhibitory effect of DHA. Although DHA is metabolized by lipoxygenases, the lipoxygenase inhibitor NDGA did not reverse the inhibition of either NO production nor Ia expression. This indicates that lipoxygenase products of DHA did not mediate inhibition. NDGA itself inhibited NO production and Ia expression. However, DHA did not inhibit by inhibiting lipoxygenase activity because DHA further inhibited macrophages exposed to doses of DHA known to maximally inhibit lipoxygenases. Furthermore, stable biologically active analogs of lipoxygenase products did not reverse DHA inhibition. DHA also did not inhibit by preventing PAF production because PAF did not reverse inhibition of NO production. CONCLUSION: DHA did not inhibit Ia-expression or NO production via its known effects on eicosanoid or PAF metabolism, nor by being metabolized by lipoxygenases.

Docosahexaenoic acid, a major constituent of fetal serum and fish oil diets, inhibits IFN gamma-induced Ia-expression by murine macrophages in vitro.

Decreased Ia expression by macrophages may account for the increased susceptibility of fetuses and neonates to infection. We chose to investigate the role of docosahexaenoic acid (DHA), an omega-3 fatty acid, on Ia expression in vitro, because neonatal serum concentrations of DHA (100-150 microM) are approximately 50 times higher than in the adult. In addition, DHA is a major component of fish-oil diets that ameliorate some autoimmune diseases and prevent renal allograft rejection. DHA inhibited IFN-gamma-induced Ia expression with a half-maximal inhibitory concentration of 25 microM. The inhibition was not caused by nonspecific damage, because oxidative metabolism via the mitochondrial electron-transport chain was not inhibited. There were strict biochemical requirements for inhibition of Ia expression. Polyenoic fatty acids with 22 carbons were more inhibitory than those with 20 carbons. Among 22-carbon fatty acids, those with more double bonds, and, in particular, with a double bond in the omega-3 position, were more inhibitory. Although DHA is known to inhibit cyclooxygenase and thus the production of eicosanoids, indomethacin did not inhibit Ia expression. This indicated that inhibition of cyclooxygenase was not responsible for inhibition of Ia expression. We divided induction of Ia expression by IFN-gamma into four phases, with IFN-gamma being present only during the second phase. DHA was most inhibitory when given before or with the IFN-gamma. This indicated that DHA inhibited early steps in IFN-gamma-induced Ia expression. Consistent with this idea, we found that DHA inhibited the increase in mRNA transcripts for Ia beta b, as assayed by Northern blotting. In summary, we found that DHA, a major component of fetal and neonatal sera as well as fish-oil diets, inhibited IFN-gamma-induced macrophage Ia expression in vitro by preventing increases in Ia mRNA transcripts.

Effects of cooler temperature dialysate on hemodynamic stability in "problem" dialysis patients.

Symptomatic hypotension is a common and disabling complication of hemodialysis treatments. The incidence of symptomatic hypotensive episodes is particularly high in patients who have normal or low blood pressure at the initiation of dialysis and in patients who have large interdialytic weight gains. The aim of this study was to determine whether cooling the dialysate temperature from 37 degrees C to 35 degrees C improved tolerance to dialysis in a group of 12 of these "problem" patients. A double-blinded protocol was performed in six hypotension-prone and six large weight gainers who were subjected to two identical hemodialyses except for the dialysate temperature of 37 degrees C or 35 degrees C. Changes in biochemical parameters and weight were comparable during the two maneuvers. Recumbent blood pressure declined significantly (P < 0.01) during 37 degrees C dialysis but not 35 degrees C dialysis; blood pressure was significantly lower at 1, 2, and 3 hours of 37 degrees C dialysis compared to 35 degrees C dialysis (P < 0.05). Further, both supine and upright blood pressure was significantly lower following 37 degrees C dialysis (P < 0.02). This lower blood pressure was present in both subgroups of patients. All 18 episodes of symptomatic hypotension noted during the study occurred during 37 degrees C dialysis. A significantly greater increase in peripheral vascular resistance (calf blood flow was measured directly by venous occlusion plethysmography) occurred upon exposure to the 35 degrees C dialysate in both subgroups of patients (P < 0.01); supine and upright post-dialysis plasma norepinephrine values were also significantly greater (P < 0.001) after 35 degrees dialysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Xenotransplantation.

Transplantation of solid organs (heart, lung, liver, and kidney) from swine to humans would solve the current critical shortage of cadaver organs needed by patients with end-stage disease of these organs. In addition, transplantation between distant species (discordant xenografting) will require an understanding of a number of unique immunologic features. Discordant xenografts are rejected within minutes to hours after transplantation. This rejection is due to natural immunity by recipients never before exposed to the xenografts. In some species combinations, this fulminant rejection is due to naturally occurring pre-existing antibodies against the xenograft endothelium. In other species combinations, the xenograft activates the alternative pathway of complement. The swine to human species combination is the most clinically relevant. In this combination, natural human and private antibodies recognize alpha-galactosyl residues of glycoproteins and glycolipids. Potential future therapeutic measures to prevent natural immunity include the genetic engineering of human complement inhibitors into swine cell membranes or genetic "knock out" of the enzymes responsible for placing alpha-galactosyl residues on swine cell surfaces. There are also special considerations in acquired immunity against xenografts. Cytokines and adhesion molecules may not work across species lines. Xenograft antigens may have to be processed by host antigen-presenting cells in order to effectively stimulate the immune system.