Taurine Partially Improves Abnormal Anxiety in Taurine-Deficient Mice.

Taurine is abundant in various tissues including the brain, muscle, heart, spleen, liver and kidney with various physiological functions. Since taurine is produced by cysteine sulfinic acid decarboxylase (CSAD) in the liver and kidney, taurine-deficient mice without CSAD have been investigated for abnormal physiological functions such as retinal development, immune, pancreatic and liver function. In this study, the behavioral effects and abnormal brain development caused by low taurine in the developing brain were examined. In neonatal brains of homozygous CSAD knockout mice (HO), taurine was reduced by 85%, compared to wild-type mice (WT). Taurine was reduced by 35% in the brains of 2 month-old HO, compared to WT. Anxiety, motor coordination and autistic-like behaviors were evaluated at 2 months of age using five behavioral tests: elevated plus maze, open field, social approach, marble burying and accelerating rotarod. Mice were tested from 3 groups including WT, HO and HO with oral treatment of 0.2% taurine in the drinking water (HOT). HOT were born from HO dams treated with taurine from before pregnancy and were continuously treated with taurine in the drinking water after weaning. The taurine levels in the brain and plasma of HOT were restored to WT at 2 months of age. Taurine-deficiency did not lead to changes in autistic-like behaviors as the HO were not significantly different from WT in marble burying and social approach. However, taurine-deficiency increased anxiety-like behavior in HO in the elevated plus maze and open field, compared to WT. Taurine treatment significantly restored the HOT to WT levels of anxiety-like behavior in the elevated plus maze. However, changes in exploratory activity in the open field were not improved with taurine treatment. There was a slight difference in motor ability as the WT mice stayed on the accelerating rotarod longer that the HO and HOT, but the difference was significant in the HOT during the first trial only, compared to WT.These data support hypothesis that taurine is essential for the emotional development of the brain. First, taurine is remarkably low in the neonatal brain of HO, compared to the adult brain of HO. Second, taurine treatment in HO partially improves anxiety-like behavior to WT.

Is Taurine a Biomarker in Autistic Spectrum Disorder?

Taurine is a sulfur-containing amino acid which is not incorporated into protein. However, taurine has various critical physiological functions including development of the eye and brain, reproduction, osmoregulation, and immune functions including anti-inflammatory as well as anti-oxidant activity. The causes of autistic spectrum disorder (ASD) are not clear but a high heritability implicates an important role for genetic factors. Reports also implicate oxidative stress and inflammation in the etiology of ASD. Thus, taurine, a well-known antioxidant and regulator of inflammation, was investigated here using the sera from both girls and boys with ASD as well as their siblings and parents. Previous reports regarding taurine serum concentrations in ASD from various laboratories have been controversial. To address the potential role of taurine in ASD, we collected sera from 66 children with ASD (males: 45; females: 21, age 1.5-11.5 years, average age 5.2 ± 1.6) as well as their unaffected siblings (brothers: 24; sisters: 32, age 1.5-17 years, average age 7.0 ± 2.0) as controls of the children with ASD along with parents (fathers: 49; mothers: 54, age 28-45 years). The sera from normal adult controls (males: 47; females: 51, age 28-48 years) were used as controls for the parents. Taurine concentrations in all sera samples were measured using high performance liquid chromatography (HPLC) using a phenylisothiocyanate labeling technique. Taurine concentrations from female and male children with ASD were 123.8 ± 15.2 and 145.8 ± 8.1 µM, respectively, and those from their unaffected brothers and sisters were 142.6 ± 10.4 and 150.8 ± 8.4 µM, respectively. There was no significant difference in taurine concentration between autistic children and their unaffected siblings. Taurine concentrations in children with ASD were also not significantly different from their parents (mothers: 139.6 ± 7.7 µM, fathers: 147.4 ± 7.5 µM). No significant difference was observed between adult controls and parents of ASD children (control females: 164.8 ± 4.8 µM, control males: 163.0 ± 7.0 µM). However, 21 out of 66 children with ASD had low taurine concentrations (<106 µM). Since taurine has anti-oxidant activity, children with ASD with low taurine concentrations will be examined for abnormal mitochondrial function. Our data imply that taurine may be a valid biomarker in a subgroup of ASD.

A Novel Cysteine Sulfinic Acid Decarboxylase Knock-Out Mouse: Taurine Distribution in Various Tissues With and Without Taurine Supplementation.

Taurine, a sulfur containing amino acid, has various physiological functions including development of the eye and brain, immune function, reproduction, osmo-regulatory function as well as anti-oxidant and anti-inflammatory activities. In order to understand the physiological role, we developed taurine deficient mice deleting a rate-liming enzyme, cysteine sulfinic acid decarboxylase (CSAD) for biosynthesis of taurine. Taurine was measured in various tissues including the liver, brain, lung, spleen, thymus, pancreas, heart, muscle and kidney as well as plasma from CSAD knock-out mice (CSAD KO) with and without treatment of taurine in the drinking water at the age of 2 months (2 M). Taurine was determined using HPLC as a phenylisothiocyanate derivative of taurine at 254 nm. Taurine concentrations in the liver and kidney from homozygotes of CSAD KO (HO), in which CSAD level is high, were 90% and 70% lower than WT, respectively. Taurine concentrations in the brain, spleen and lung, where CSAD level is low, were 21%, 20% and 28% lower than WT, respectively. At 2 M, 1% taurine treatment of HO restored taurine concentrations in all tissues compared to that of WT. To select an appropriate taurine treatment, HO were treated with various concentrations (0.05, 0.2, 1%) of taurine for 4 months (4 M). Restoration of taurine in all tissues except the liver, kidney and lung requires 0.05% taurine to be restored to that of WT. The liver and kidney restore taurine back to WT with 0.2% taurine. To examine which enzymes influence taurine concentrations in various tissues from WT and HO at 2 M, expression of five taurine-related enzymes, two antioxidant enzymes as well as lactoferrin (Lft) and prolactin receptor (Prlr) was determined using RT2 qPCR. The expression of taurine transporter in the liver, brain, muscle and kidney from HO was increased except in the lung. Our data showed expression of glutamate decarboxylase-like 1(Gadl-1) was increased in the brain and muscle in HO, compared to WT, indicating taurine in the brain and muscle from HO was replenished through taurine transporter and increased biosynthesis of taurine by up-regulated Gadl-1. The expression of glutathione peroxidase 3 was increased in the brain and peroxireductase 2 was increased in the liver and lung, suggesting taurine has anti-oxidant activity. In contrast to newborn and 1 month CSAD KO, Ltf and Prlr in the liver from CSAD KO at 2 M were increased more than two times and 52%, respectively, indicating these two proteins may be required for pregnancy of CSAD KO. Ltf in HOT1.0 was restored to WT, while Prlr in HOT1.0 was increased more than HO, explaining improvement of neonatal survival with taurine supplementation.These data are essential for investigating the role of taurine in development of the brain and eye, immune function, reproduction and glucose tolerance.

A Novel Cysteine Sulfinic Acid Decarboxylase Knock-Out Mouse: Immune Function (II).

Taurine deficient mice lacking cysteine sulfinic acid decarboxylase (CSAD KO) were developed for investigating the various physiological roles of taurine including the development of the brain and eye as well as immune function. Due to severe abnormalities of immune function in a taurine deficient cat, the immune function including adoptive and innate immunity in taurine-deficient mice have been studied. Previously we demonstrated that B cell function in CSAD KO was reduced in both females and males. However, T cell function was significantly reduced only in females. In this study, we have examined innate immunity using macrophage activation with LPS or/and IFN-γ and polymorphonuclear leukocytes (PMN) activation with phorbol myristate acetate (PMA). Pro- and anti-inflammatory cytokines including IL-6, TNF-α and IL-10 as well as nitric oxide (NO) were determined using ELISA and Griess reagent, respectively. Peritoneal macrophages were activated with 1 µg/mL of lipopolysaccharide (LPS) and/or 50 U/mL of IFN-γ. In addition, superoxide anion was measured using peritoneal PMN activated with PMA in the presence and absence of superoxide dismutase. Superoxide anion production in activated PMN from CSAD KO homozygotes (HO) was not significantly different from wild-type (WT) with and without 25 mM taurine. IL-10 and TNF-α production in both female and male CSAD KO were not significantly different. IL-6 and NO were significantly lower only in females as previously observed in Con A-activated cellular proliferation of splenocytes. Cytokine production with 10 mM of taurine was not different, indicating the reduction of NO and IL-6 in females may be due to the absence of the CSAD gene, not due to low taurine concentrations.These data indicate that some measures of innate immunity were altered in female CSAD mice.

Development of a novel cysteine sulfinic Acid decarboxylase knockout mouse: dietary taurine reduces neonatal mortality.

We engineered a CSAD KO mouse to investigate the physiological roles of taurine. The disruption of the CSAD gene was verified by Southern, Northern, and Western blotting. HPLC indicated an 83% decrease of taurine concentration in the plasma of CSAD(-/-). Although CSAD(-/-) generation (G)1 and G2 survived, offspring from G2 CSAD(-/-) had low brain and liver taurine concentrations and most died within 24 hrs of birth. Taurine concentrations in G3 CSAD(-/-) born from G2 CSAD(-/-) treated with taurine in the drinking water were restored and survival rates of G3 CSAD(-/-) increased from 15% to 92%. The mRNA expression of CDO, ADO, and TauT was not different in CSAD(-/-) compared to WT and CSAD mRNA was not expressed in CSAD(-/-). Expression of Gpx 1 and 3 was increased significantly in CSAD(-/-) and restored to normal levels with taurine supplementation. Lactoferrin and the prolactin receptor were significantly decreased in CSAD(-/-). The prolactin receptor was restored with taurine supplementation. These data indicated that CSAD KO is a good model for studying the effects of taurine deficiency and its treatment with taurine supplementation.

Treatment of recalcitrant warts with occlusive warming patches.

Prior studies have identified local heat therapy as a treatment for recalcitrant warts. We have employed a thermal pad that raises local temperature to 42-43ºC for at least 2 hours in a proof of concept study of three patients with recalcitrant warts. The recalcitrant warts cleared in all three patients beginning in the fourth and fifth weeks after daily treatment with the pads. There were no adverse events. We conclude that the timing of clearance following use of these thermal pads is likely via direct viral killing and immunologic mechanisms. Further controlled trials are underway.

Altered lymphocyte proliferation and innate immune function in scrapie 139A- and ME7-infected mice.

Lymphoid organs play an important role in prion disease development and progression. While the role of lymphoid organs and changes in immune-related genes have been extensively investigated in scrapie-infected animals, innate immunity has not. Previous studies examined lymphocyte function in scrapie-infected C3H/HeJ mice, which exhibit defects in lipopolysaccharide (LPS) response now known to result from a mutation in Toll-like receptor (TLR) 4. We examined immune function in scrapie-infected CD1 mice, which are LPS responders. Lymphocyte proliferation from CD1 mice infected with either 139A or ME7 scrapie was measured in response to concanavalin (Con) A or LPS at 1 and 3 months after infection. Following LPS exposure, mice infected 3 months with ME7, but not 139A, demonstrated significantly decreased lymphocyte proliferation compared to controls. After Con A exposure, lymphocyte proliferation in scrapie-infected mice did not differ from controls. Gender-specific comparison of lymphocyte proliferation showed significant decreases in mitogenic responses in females infected 3 months with either 139A or ME7, compared to controls. Males infected for 3 months with ME7, but not 139A, showed significantly decreased proliferation after lymphocyte exposure to LPS, but not Con A. Neither gender showed changes in lymphocyte proliferation after 1 month of scrapie infection. Innate immune activation of peritoneal macrophages was determined via production of nitric oxide (NO), IL-6, and TNF-α after exposure to TLR ligands. TNF-α and IL-6 production were reduced in macrophages from females infected with either scrapie strain for 3 months, while NO production after TLR agonist plus IFN-γ exposure was decreased in both females and males infected for 3 months with 139A, compared to ME7. These data demonstrated altered innate immunity, suggesting hormonal and/or other gender-specific regulation may contribute to gender differences in some immune functions. Our data demonstrate lymphocyte proliferation and innate immune functioning in scrapie-infected mice deteriorate with disease progression.

Inflammatory mediators are inhibited by a taurine metabolite in CpG oligodeoxynucleotide and IFN-r activated macrophage cell line.

Taurine plays an important role in brain and retinal development, and has an antiinflammatory and antioxidant function. Taurine chloramine (Tau-Cl) is produced in polymorphonuclear leukocytes via the myeloperoxidase/halide system. We previously demonstrated that Tau-Cl inhibits the production of nitric oxide (NO) and TNF-α in human and murine macrophages activated with IFN-γ in combination with individual Toll-like receptor (TLR) ligands including those for TLR2 and/or TLR4. In the current study, we further explored the effects of Tau-Cl in RAW 264.7 cells stimulated with the TLR9 ligand CpG oligodeoxynucleotide (ODN). Specifically, we examined the effect of CpG ODN plus IFN-γ on the production of NO and TNF-α, and the effect of Tau-Cl on this process. Our findings show that CpG ODN plus IFN-γ-activated RAW 264.7 cells secrete high levels of NO and TNF-α, and that Tau-Cl (0.8 mM) inhibits this effect in a dose-dependent manner, more potently inhibiting the production of NO (99% inhibition) than that of TNF-α (48% inhibition). Nitric oxide synthase (iNOS) protein was also induced by CpG ODN plus IFN-γ, and was also inhibited by Tau-Cl. Furthermore, while CpG ODN plus IFN-γ induced TNF-α and iNOS mRNAs, Tau-Cl transiently suppressed this effect. Taurine itself had no effects on any of these processes. Our findings in a macrophage cell line demonstrate that Tau-Cl inhibits proinflammatory mediators resulting from TLR9 activation, and have implications for the utility of Tau-Cl in scenarios where such activation is deleterious such as in autoimmune conditions or infections in which overwhelming inflammation may occur. CpG ODNs and Tau-Cl both have potential for topical treatment of autoimmune conditions, including psoriasis, vitiligo, and alopecia areata. As CpG ODNs may, under some conditions, up-regulate Tregs, addition of Tau-Cl to CpG ODN topical formulations has potential for improving cancer immunotherapy.

Taurine chloramine inhibits NO and TNF-α production in zymosan plus interferon-γ activated RAW 264.7 cells.

Taurine is present abundantly in various tissues, especially in leukocytes embattled to foreign invaders such as microorganisms or oxidants. Taurine-chloramine (Tau-Cl) is produced from taurine at the site of inflammation via the myeloperoxidase-halide pathway in leukocytes induced by oxidants and/or infectious materials. Previously, our data demonstrated that Tau-Cl inhibited nitric oxide (NO) production and TNF-α secretion induced by lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR-4) ligand or lipoarabinomannan (LAM), a TLR-2 ligand plus interferon-γ (IFN-γ) in peritoneal macrophages or RAW 264.7 cells. Zymosan, a ß-glucan of yeast cell wall, is a ligand for TLR-2 and dectin-1 and stimulates macrophages to produce proinflammatory mediators such as NO and TNF-α. Based on our previous data, we examined the effect of zymosan and IFN-γ induced production of NO and TNF-α in the absence or presence of Tau-Cl or taurine using RAW 264.7 cells. Production of NO and secretion of TNF-α is increased when zymosan is combined with IFN-γ. Tau-Cl inhibited production of NO and secretion of TNF-α in zymosan plus IFN-γ activated RAW 264.7 cells in a dose-dependent manner (99% vs. 48% using 0.8mM Tau-Cl). Taurine was without effect. Nitric oxide synthase protein (iNOS), induced by zymosan plus IFN-γ, was inhibited by Tau-Cl (0.8mM) as measured using western blot analysis. NOS mRNA was inhibited by Tau-Cl at four, eight and 16 hours post activation, but not at 24 hours. TNF-α mRNA was inhibited at four hours and eight hours, but not at 16 and 24 hours. These data suggest that expression of both iNOS and TNF-α mRNAs are inhibited by treatment with Tau-Cl within four and eight hours, but not at later time points. Transient suppression of activation of RAW 264.7 cells induced by zymosan may play a critical physiological role for taurine in protecting against tissue injury from initial overt inflammation. This study indicates that tropical treatment of taurine may ameliorate inflammatory dermatoses caused by an environmental yeast or abnormal immune function.

Effects of parathyroid hormone on immune function.

Parathyroid hormone (PTH) function as immunologic mediator has become interesting with the recent usage of PTH analogue (teriparatide) in the management of osteoporosis. Since the early 1980s, PTH receptors were found on most immunologic cells (neutrophils, B and T cells). The in vitro evaluations for a possible role of PTH as immunomodulator have shown inconsistent results mainly due to methodological heterogeneity of these studies: it used different PTH formulations (rat, bovine, and human), at different dosages and different incubating periods. In some of these studies, the lymphocytes were collected from uremic patients or animals, which renders the interpretation of the results problematic due to the effect of uremic toxins. Parathyroidectomy has been found to reverse the immunologic defect in patients with high PTH levels. Nonetheless, the clinical significance of these findings is unclear. Further studies are needed to define if PTH does have immunomodulatory effects.

Effect of thalidomide on nitric oxide production in lipopolysaccharide-activated RAW 264.7 cells.

Thalidomide is anti-inflammatory under some conditions, yet has been reported to up-regulate Th1 (T helper 1) immunity measured by increased IL-2 (Interleukin-2) and gamma interferon. The authors have assessed the effect of thalidomide and analogues, di- and tri-thiothalidomide, on a lipopolysaccharide (LPS) activated macrophage cell line (RAW 246.7 cells). The authors' findings showed that nitric oxide (NO) was significantly inhibited by thalidomide (15%) and its analogues (di-thiothalidomide; 15%, tri-thiothalidomide; 32%). The proinflammatory molecules TNF-alpha (tumor necrosis factor-alpha) and IL-6 were not significantly inhibited. Pretreatment with thalidomide and analogues before activation was not different from simultaneous treatment. Inhibition of inducible nitric oxide synthase (iNOS) may prove to be an important target for the anti-inflammatory and anti-cancer effects of thalidomide and related immunomodulatory drugs (IMiDs).

Protection of bleomycin-induced fibrosis and inflammation by taurine.

Taurine has been shown to protect against lung injury induced by various oxidants including ozone, nitrogen dioxide, amiodarone, and paraquat and to protect against bleomycin-induced lung injury in combination with niacin. In this study, Spraque-Dawley rats were treated with 5% taurine in the drinking water for 10 days prior to bleomycin instillation. Fibrosis in the rats pretreated with taurine (BT) was absent, along with fewer inflammatory infiltrates compared to the untreated rats (BW). A significant decrease in the number of PMNs and a decrease in hydroxyproline levels were found in the bronchoalveolar lavage fluid in the BT group compared to the BW group. By immunohistochemical staining, inducible nitric oxide synthase was evident in the lungs of bleomycin-treated rats, and minimal when rats were treated with taurine. Tumor necrosis factor-alpha (TNF-alpha) as measured by immunohistochemical staining, was present in lungs of both taurine-treated and untreated rats, but was more abundant in the BW group compared to the BT group. In addition, decreased ICAM presentation was detected by EM immunogold staining in the BT group compared to the BW group. These data demonstrate that rats pretreated with 5% taurine in their drinking water prior to bleomycin instillation are protected from fibrosis, inflammatory infiltrates, as well as nitric oxide and TNF-alpha production, which are hallmarks of bleomycin lung injury.

Accelerated prion disease pathogenesis in Toll-like receptor 4 signaling-mutant mice.

Prion diseases such as scrapie involve the accumulation of disease-specific prion protein, PrP(Sc), in the brain. Toll-like receptors (TLRs) are a family of proteins that recognize microbial constituents and are central players in host innate immune responses. The TLR9 agonist unmethylated CpG DNA was shown to prolong the scrapie incubation period in mice, suggesting that innate immune activation interferes with prion disease progression. Thus, it was predicted that ablation of TLR signaling would result in accelerated pathogenesis. C3H/HeJ (Tlr4(Lps-d)) mice, which possess a mutation in the TLR4 intracellular domain preventing TLR4 signaling, and strain-matched wild-type control (C3H/HeOuJ) mice were infected intracerebrally or intraperitoneally with various doses of scrapie inoculum. Incubation periods were significantly shortened in C3H/HeJ compared with C3H/HeOuJ mice, regardless of the route of infection or dose administered. At the clinical phase of disease, brain PrP(Sc) levels in the two strains of mice showed no significant differences by Western blotting. In addition, compared with macrophages from C3H/HeOuJ mice, those from C3H/HeJ mice were unresponsive to fibrillogenic PrP peptides (PrP residues 106 to 126 [PrP(106-126)] and PrP(118-135)) and the TLR4 agonist lipopolysaccharide but not to the TLR2 agonist zymosan, as measured by cytokine production. These data confirm that innate immune activation via TLR signaling interferes with scrapie infection. Furthermore, the results also suggest that the scrapie pathogen, or a component(s) thereof, is capable of stimulating an innate immune response that is active in the central nervous system, since C3H/HeJ mice, which lack the response, exhibit shortened incubation periods following both intraperitoneal and intracerebral infections.

CpG oligodeoxynucleotide-enhanced humoral immune response and production of antibodies to prion protein PrPSc in mice immunized with 139A scrapie-associated fibrils.

Prion diseases are characterized by conversion of the cellular prion protein (PrP(C)) to a protease-resistant conformer, the srapie form of PrP (PrP(Sc)). Humoral immune responses to nondenatured forms of PrP(Sc) have never been fully characterized. We investigated whether production of antibodies to PrP(Sc) could occur in PrP null (Prnp(-/-)) mice and further, whether innate immune stimulation with the TLR9 agonist CpG oligodeoxynucleotide (ODN) 1826 could enhance this process. Whether such stimulation could raise anti-PrP(Sc) antibody levels in wild-type (Prnp(+/+)) mice was also investigated. Prnp(-/-) and Prnp(+/+) mice were immunized with nondenatured 139A scrapie-associated fibrils (SAF), with or without ODN 1826, and were tested for titers of PrP-specific antibodies. In Prnp(-/-) mice, inclusion of ODN 1826 in the immunization regime increased anti-PrP titers more than 13-fold after two immunizations and induced, among others, antibodies to an N-terminal epitope, which were only present in the immune repertoire of mice receiving ODN 1826. mAb 6D11, derived from such a mouse, reacts with the N-terminal epitope QWNK in native and denatured forms of PrP(Sc) and recombinant PrP and exhibits a K(d) in the 10(-)(11) M range. In Prnp(+/+) mice, ODN 1826 increased anti-PrP levels as much as 84% after a single immunization. Thus, ODN 1826 potentiates adaptive immune responses to PrP(Sc) in 139A SAF-immunized mice. These results represent the first characterization of humoral immune responses to nondenatured, infectious PrP(Sc) and suggest methods for optimizing the generation of mAbs to PrP(Sc), many of which could be used for diagnosis and treatment of prion diseases.

Is Taurine A Biomarker?

Taurine, a sulfur-containing amino acid present in high concentrations in mammals, plays an important role in several essential biological processes. Taurine is not incorporated into protein and is the most abundant free amino acid in the heart, retina, skeletal muscle, brain, and leukocytes. The ideal biomarker or biological measure should be reliable, reproducible, noninvasive, simple to perform, and inexpensive. Samples for biological measures should be easily obtained from physiological fluids such as blood or urine. Taurine levels in physiologic fluids have been useful for both diagnosing pathology and establishing a disease modifying therapy. In the specific case of taurine, it is important that patient information include nutritional supplementation as well as information on disease status and medications. Taurine has been measured in biological fluids due to the importance of this simple amino acid and its relative ease of determination. Taurine has been measured in animal models of disease as well as a variety of human conditions. However, it remains unclear how taurine should be used as a biomarker and in which situations this measurement would be a good prognostic or diagnostic indicator.

Anti-inflammatory activity of herbal medicines: inhibition of nitric oxide production and tumor necrosis factor-alpha secretion in an activated macrophage-like cell line.

Houttuynia cordata Thunb. (HC), Glycyrrhiza uralensis Fischer (GU), Forsythia suspense (Thunb.) Vahl (FS), and Lonicera japonica Thunb. (LJ) are Chinese herbs known to possess anti-inflammatory properties. The effects of aqueous extracts of these herbs on the production of the pro-inflammatory mediators, nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) were examined in an activated macrophage-like cell line, RAW 264.7 cells. Aqueous extracts from FS at 0.0625-2.0 mg/ml inhibited in vitro production of NO and secretion of TNF-alpha in a dose-dependent manner. FS at 1.0-2.0 mg/ml and 0.125-2.0 mg/ml significantly inhibited NO production and TNF-alpha, respectively. An extract of LJ demonstrated potent inhibition of both NO production and TNF-alpha secretion in a dose-dependent manner. An aqueous extract from HC inhibited NO production in a dose-dependent manner, but minimally (approximately 30%) inhibited TNF-a secretion at 0.0625 and 0.125 mg/ml. In contrast, an aqueous extract of GU had a minimal effect on both the production of NO and the secretion of TNF-alpha. Viability of cells at all concentrations studied was unaffected as determined by MTT cytotoxicity assay and trypan blue dye exclusion. These results suggest that aqueous extracts from FS, LJ and HC have anti-inflammatory actions as measured by inhibition of NO production and/or TNF-alpha secretion.

Exposure to alcohol on E9 raises poststress corticosterone in mature but not old mice.

Prenatal exposure to alcohol alters postnatal function of the hypothalamic-pituitary-adrenal axis. Hyperresponsiveness to stress, or increased secretion of corticosterone, is a commonly studied effect in offspring of rats exposed to alcohol during a substantial period of gestation. No studies have reported on stress hormone secretion following alcohol exposure on a single day during embryonic development even though exposure at this time may damage the hypothalamus and pituitary. To explore the effect of an acute exposure, we used the offspring of C57BL/6J mice exposed to alcohol or saline on embryonic day (E) 9 (2.9 g/kg administered twice, 4h apart). At 7.5 or 22 months of age these mice were subjected to a 12-h restraint stress, or merely kept in the same environment without restraint. After the 12-h period, a blood sample was obtained from the retro-orbital plexus, and analyzed for the amount of corticosterone. The 7.5-month old group of alcohol-treated offspring were indeed hyperresponsive to restraint stress, but the 22-month old mice were not. Whether the normal-appearing corticosterone response of the old alcohol-exposed mice indicated adaptation to restraint, an aging-associated ceiling effect in corticosterone secretion, or an expression of pathology, cannot be decided on the basis of present data.