Inhibiting Microglia Expansion Prevents Diet-Induced Hypothalamic and Peripheral Inflammation.

Cell proliferation and neuroinflammation in the adult hypothalamus may contribute to the pathogenesis of obesity. We tested whether the intertwining of these two processes plays a role in the metabolic changes caused by 3 weeks of a high-saturated fat diet (HFD) consumption. Compared with chow-fed mice, HFD-fed mice had a rapid increase in body weight and fat mass and specifically showed an increased number of microglia in the arcuate nucleus (ARC) of the hypothalamus. Microglia expansion required the adequate presence of fats and carbohydrates in the diet because feeding mice a very high-fat, very low-carbohydrate diet did not affect cell proliferation. Blocking HFD-induced cell proliferation by central delivery of the antimitotic drug arabinofuranosyl cytidine (AraC) blunted food intake, body weight gain, and adiposity. AraC treatment completely prevented the increase in number of activated microglia in the ARC, the expression of the proinflammatory cytokine tumor necrosis factor-α in microglia, and the recruitment of the nuclear factor-κB pathway while restoring hypothalamic leptin sensitivity. Central blockade of cell proliferation also normalized circulating levels of the cytokines leptin and interleukin 1ß and decreased peritoneal proinflammatory CD86 immunoreactive macrophage number. These findings suggest that inhibition of diet-dependent microglia expansion hinders body weight gain while preventing central and peripheral inflammatory responses due to caloric overload.

Low dietary iron intake restrains the intestinal inflammatory response and pathology of enteric infection by food-borne bacterial pathogens.

Orally administrated iron is suspected to increase susceptibility to enteric infections among children in infection endemic regions. Here we investigated the effect of dietary iron on the pathology and local immune responses in intestinal infection models. Mice were held on iron-deficient, normal iron, or high iron diets and after 2 weeks they were orally challenged with the pathogen Citrobacter rodentium. Microbiome analysis by pyrosequencing revealed profound iron- and infection-induced shifts in microbiota composition. Fecal levels of the innate defensive molecules and markers of inflammation lipocalin-2 and calprotectin were not influenced by dietary iron intervention alone, but were markedly lower in mice on the iron-deficient diet after infection. Next, mice on the iron-deficient diet tended to gain more weight and to have a lower grade of colon pathology. Furthermore, survival of the nematode Caenorhabditis elegans infected with Salmonella enterica serovar Typhimurium was prolonged after iron deprivation. Together, these data show that iron limitation restricts disease pathology upon bacterial infection. However, our data also showed decreased intestinal inflammatory responses of mice fed on high iron diets. Thus additionally, our study indicates that the effects of iron on processes at the intestinal host-pathogen interface may highly depend on host iron status, immune status, and gut microbiota composition.

Positional identification of RT1-B (HLA-DQ) as susceptibility locus for autoimmune arthritis.

Rheumatoid arthritis (RA) is associated with amino acid variants in multiple MHC molecules. The association to MHC class II (MHC-II) has been studied in several animal models of RA. In most cases these models depend on T cells restricted to a single immunodominant peptide of the immunizing Ag, which does not resemble the autoreactive T cells in RA. An exception is pristane-induced arthritis (PIA) in the rat where polyclonal T cells induce chronic arthritis after being primed against endogenous Ags. In this study, we used a mixed genetic and functional approach to show that RT1-Ba and RT1-Bb (RT1-B locus), the rat orthologs of HLA-DQA and HLA-DQB, determine the onset and severity of PIA. We isolated a 0.2-Mb interval within the MHC-II locus of three MHC-congenic strains, of which two were protected from severe PIA. Comparison of sequence and expression variation, as well as in vivo blocking of RT1-B and RT1-D (HLA-DR), showed that arthritis in these strains is regulated by coding polymorphisms in the RT1-B genes. Motif prediction based on MHC-II eluted peptides and structural homology modeling suggested that variants in the RT1-B P1 pocket, which likely affect the editing capacity by RT1-DM, are important for the development of PIA.

Dietary zinc is a key environmental modifier in the progression of IgA nephropathy.

IgA nephropathy (IgAN) shows diverse epidemiological characteristics, resulting from both genetic and acquired (e.g., environmental) causes. Environmental factors, such as diet or exposure to exogenous antigens, may prescribe the progression or prognosis of IgAN. It remains unclear as to how diet and infection influence susceptibility to IgAN. A relationship, such as Toll-like receptors (TLRs), especially TLR9 and TLR4, was demonstrated between IgAN and pathogen-recognition molecules. Recently, zinc (Zn) was discovered to be involved in various immune-related diseases, affecting B, T, and dendritic cells (DCs). This study investigates the relationship between dietary Zn and IgAN development in IgAN-prone mice. Seven-week-old IgAN-prone mice were divided into low, normal, and high Zn diet groups. To assess exogenous pathogen-mediated immune responses, lipopolysaccharide (LPS) was nasally administered. The activity of IgAN was biochemically and pathologically evaluated during the disease course. We also examined in vitro IgA production in spleen cells or in combinations of cocultured B, T, and DCs under various Zn conditions with or without LPS. Dietary conditioning with Zn affected serum immunoglobulins and urinary albumin levels, and mesangial deposition of IgA and IgG. Zn deficiency is associated with IgAN progression through the activation of the TLR4/TIR-domain-containing adapter-inducing interferon-ß (TRIF), but not the TLR9, in DCs. Zn supplementation prevented disease aggravation. Our findings indicate that immune conditioning with dietary Zn alters nephritogenic IgA production after mucosal infection.

Supplemental arginine administered in ovo or in the feed reduces the susceptibility of broilers to pulmonary hypertension syndrome.

1. Two experiments were conducted to determine if in ovo and in-feed arginine (ARG) supplementation is effective in the prevention of pulmonary hypertension syndrome (PHS) in broiler chickens reared at high altitude. 2. In Experiment I, a total of 300 fertile eggs were divided into two equal groups. On d 5 of incubation, one group was injected with 0.5 ml of ARG (20 mg/ml) and the other remained untreated and served as controls. After hatching, male chicks (64 chickens per treatment) were selected and given a commercial maize-soyabean meal diet up to 48 d of age. 3. In Experiment II, a total of 128 male broiler chickens (Ross 308) were randomly assigned to two treatments, a control group that were fed on a basal diet that met ARG requirements and the second was fed on the basal diet supplemented with 1.5 g ARG per kg of diet. 4. Cumulative mortality from ascites was recorded in both experiments. Results from Experiment I indicated that in ovo injection of ARG significantly decreased ascites mortality of broilers (18.8 vs. 43.8%). Results from Experiment II showed a similar effect so that ascites mortality in the group that were given Arg supplement was significantly lower than the control (28.1 vs. 43.8%).

The effect of dietary supplementation with the natural carotenoids curcumin and lutein on broiler pigmentation and immunity.

The objective of this study was to compare the effects of supplementation with 2 carotenoids, curcumin and lutein, on pigmentation and immunity in lipopolysaccharide (LPS)-stimulated broiler chicks. Two hundred forty 1-d-old Arbor Acres broilers were randomly distributed into 3 dietary treatment groups: a basal diet without carotenoid supplementation (control), a basal diet supplemented with 200 mg/kg of curcumin (CRM), or a basal diet supplemented with 200 mg/kg of lutein (LTN) for 42 d. The birds were vaccinated against Newcastle disease (ND) and avian influenza on d 10. At 16, 18, and 20 d of age, half of the chicks in each group were injected in the abdominal region with either LPS (250 mg/kg of BW) or an equal volume of 0.9% NaCl. The intensity of the shank skin color (Roche color fan score) and the b* (yellow) values of the breasts and thighs were highest in lutein-supplemented broilers, followed by curcumin-supplemented and control broilers, whereas the a* (red) value of the thigh muscle was highest in curcumin-supplemented LPS-induced birds. At 42 d, the relative weight of the abdominal fat was lowest in the CRM-supplemented group, followed by the LTN-supplemented and control groups; the spleen weight was lower in the non-LPS-induced LTN-supplemented group than the LPS-induced control group. The ND and avian influenza titers were significantly higher in the CRM-supplemented group than in the other groups at 20 d; at 30 d, the ND titer was significantly higher in the LPS-induced LTN group. Supplementation with curcumin significantly promoted B and T lymphocyte proliferation in both LPS- and non-LPS-induced birds at 21 d. Curcumin also promoted B lymphocyte proliferation in non-LPS-induced birds at 42 d. Curcumin significantly reduced alanine aminotransferase and aspartate aminotransferase activities at 42 d in non-LPS-treated birds, whereas lutein significantly increased the activities of these enzymes in LPS-induced birds. Both carotenoids significantly lowered lipid oxidation in the liver of supplemented birds. Thus, in broiler chickens, lutein-supplemented birds exhibited better pigmentation efficiency, whereas curcumin-supplemented birds exhibited improved immune responses.

Grape antioxidant dietary fiber inhibits intestinal polyposis in ApcMin/+ mice: relation to cell cycle and immune response.

Epidemiological and experimental studies suggest that fiber and phenolic compounds might have a protective effect on the development of colon cancer in humans. Accordingly, we assessed the chemopreventive efficacy and associated mechanisms of action of a lyophilized red grape pomace containing proanthocyanidin (PA)-rich dietary fiber [grape antioxidant dietary fiber (GADF)] on spontaneous intestinal tumorigenesis in the Apc(Min/+) mouse model. Mice were fed a standard diet (control group) or a 1% (w/w) GADF-supplemented diet (GADF group) for 6 weeks. GADF supplementation greatly reduced intestinal tumorigenesis, significantly decreasing the total number of polyps by 76%. Moreover, size distribution analysis showed a considerable reduction in all polyp size categories [diameter <1mm (65%), 1-2mm (67%) and >2mm (87%)]. In terms of polyp formation in the proximal, middle and distal portions of the small intestine, a decrease of 76, 81 and 73% was observed, respectively. Putative molecular mechanisms underlying the inhibition of intestinal tumorigenesis were investigated by comparison of microarray expression profiles of GADF-treated and non-treated mice. We observed that the effects of GADF are mainly associated with the induction of a G1 cell cycle arrest and the downregulation of genes related to the immune response and inflammation. Our findings show for the first time the efficacy and associated mechanisms of action of GADF against intestinal tumorigenesis in Apc(Min/+) mice, suggesting its potential for the prevention of colorectal cancer.

Multiparametric immunotoxicity screening in mice during early drug development.

Evaluation of potential adverse effects on the immune system should be incorporated into drug development prior to phase III clinical trials. In addition to standard toxicity results, T-dependent antibody response (TDAR) assays are widely used to evidence impaired immune function. The present study was aimed at validating a multiparametric screening approach in mice to investigate exaggerated pharmacologic or unintended immunosuppressive effects in early drug development. Male CD1 mice injected with a single IV dose of 2mg KLH displayed a robust anti-KLH IgM response that peaked on day +5. Anti-KLH IgM response, standard haematology parameters, and thymus/spleen weight and histology were examined in mice treated once daily for 4 days with cyclophosphamide (CY; 5-20mg/kg/day), cyclosporine (CS; 10-90mg/kg/day), dexamethasone (DX; 5-20mg/kg/day), prednisolone (PR; 3-30mg/kg/day) or chlorpromazine (CZ; 10-30mg/kg/day). CY and CS decreased anti-KLH IgM response at all dose levels. CY induced a marked decrease in WBC count and thymus/spleen weight with histological changes in both lymphoid organs. CS mainly decreased thymus weight (highest dose), which was associated with lymphoid depletion, without relevant effects on haematology parameters. Neither DX nor PR nor CZ induced significant changes in anti-KLH IgM response. DX and PR decreased lymphocyte counts and thymus/spleen weight, and induced histological changes in both lymphoid organs. CZ (higher doses) decreased lymphocyte count and thymus weight, and induced consistent histological changes in the thymus. This multiparametric study was able to detect 5 human drugs with variable immunosuppressive potency and thus may prove to be a useful early screening tool for predicting drug immunotoxicity.

Effect of immune stress on body weight regulation is altered by ovariectomy in female rats.

It has been suggested that obesity and loss of ovarian function alter the inflammatory response to immune stress. Ovariectomized (OVX) rats, which are used as a model of human menopause, exhibit both hyperphagia-induced obesity and gonadal steroid deficiency. To evaluate the effects of ovariectomy on inflammatory responses, we compared the anorectic response to LPS in OVX rats and gonad intact female rats. As leptin and hypothalamic interleukin-1ß (IL1ß) play pivotal roles in the anorectic response to immune stress, these factors were also measured. It was found that the OVX rats exhibited an increased anorectic response to LPS compared with the sham-operated rats. The OVX rats showed higher serum leptin concentrations and a greater increase in hypothalamic IL1ß mRNA expression after LPS injection. In addition, in order to determine whether gonadal steroid deficiency contributes to the changes in the inflammatory responses of OVX rats, we compared responses between OVX rats treated with gonadal steroids and untreated OVX rats. There were no differences in appetite, the serum leptin level, and hypothalamic IL1ß mRNA expression between the two groups after LPS injection. These findings suggest that the loss of ovarian function increases the induction of leptin and hypothalamic IL1ß synthesis and consequently increases the anorectic response under immune stress conditions. It is possible that these alterations are caused by OVX-induced obesity rather than the direct effects of gonadal steroid deficiency.

Obesity-mediated inflammatory microenvironment stimulates osteoclastogenesis and bone loss in mice.

Clinical evidence indicates that fat is inversely proportional to bone mass in elderly obese women. However, it remains unclear whether obesity accelerates bone loss. In this report we present evidence that increased visceral fat leads to inflammation and subsequent bone loss in 12-month-old C57BL/6J mice that were fed 10% corn oil (CO)-based diet and a control lab chow (LC) for 6 months. As expected from our previous work, CO-fed mice demonstrated increased visceral fat and enhanced total body fat mass compared to LC. The adipocyte-specific PPARγ and bone marrow (BM) adiposity were increased in CO-fed mice. In correlation with those modifications, inflammatory cytokines (IL-1ß, IL-6, TNF-α) were significantly elevated in CO-fed mice compared to LC-fed mice. This inflammatory BM microenvironment resulted in increased superoxide production in osteoclasts and undifferentiated BM cells. In CO-fed mice, the increased number of osteoclasts per trabecular bone length and the increased osteoclastogenesis assessed ex-vivo suggest that CO diet induces bone resorption. Additionally, the up-regulation of osteoclast-specific cathepsin k and RANKL expression and down-regulation of osteoblast-specific RUNX2/Cbfa1 supports this bone resorption in CO-fed mice. Also, CO-fed mice exhibited lower trabecular bone volume in the distal femoral metaphysis and had reduced OPG expression. Collectively, our results suggest that increased bone resorption in mice fed a CO-enriched diet is possibly due to increased inflammation mediated by the accumulation of adipocytes in the BM microenvironment. This inflammation may consequently increase osteoclastogenesis, while reducing osteoblast development in CO-fed mice.

Effects of hydrolysed Saccharomyces cerevisiae yeast and yeast cell wall components on live performance, intestinal histo-morphology and humoral immune response of broilers.

1. The effects of enzymatically hydrolysed whole Saccharomyces cerevisiae yeast (HY) and the pellets of yeast cell wall (YCW) on production traits, the microbiology and histo-morphology of the small intestine, and humoral immune responses against Newcastle disease virus (NDV), of Ross 308 broilers were investigated. 2. The control group received a maize-soyabean meal based basal diet for 42 days. In the treated groups the basal diet was supplemented with 1 g/kg of HY and YCW. There were 8 replicate pens per group (n = 12 birds/pen). 3. HY and YCW supplementation improved live weight (P = 0·006) and FCR (P = 0·003) at 42-d as compared with the control group. 4. In the small intestine, Salmonella spp and Escherichia coli numbers were higher (P = 0·01) in the mucosa and lower (P = 0·01) in the digesta of the HY and the YCW fed groups at 25 d of age. Lactobacillus in the duodenal and jejunal digesta was higher (P < 0·05) in the HY and the YCW fed groups as compared with the control. 5. Following oral challenge with Salmonella pullorum, Escherichia coli and Lactobacillus increased (P < 0·05) in the mucosa and decreased in the digesta (P < 0·05) of the HY and YCW supplemented groups, relative to the control. 6. Supplementation of HY and YCW increased villus height in the jejunum (P = 0·02), width of villus in the ileum (P = 0·034) and number of goblet cells in villi of the jejunum (P = 0·006) and ileum (P = 0·01). 7. YCW increased antibody level against NDV at 21 and 42 d of age compared with the control and the HY supplemented diets (P < 0·05). 8. It was concluded that HY and YCW improved growth and feed efficiency in broilers, and considering the improvements in production traits and humoral immune responses, yeast cell wall may be a better dietary tool than the hydrolysed whole yeast cell as a performance enhancer for broilers.

Neonatal LPS injection alters the body weight regulation systems of rats under non-stress and immune stress conditions.

It has been reported that prenatal immune stress induced by lipopolysaccharides or cytokines increases food intake and leads to obesity and other features of metabolic syndrome in adulthood. Using Sprague-Dawley rats, we evaluated whether neonatal LPS injection altered their body weight regulation systems under non-stress and immune stress conditions. On Day 10 after birth, all pups were injected with LPS (100 microg/kg, i.p.) (PND(10)LPS) or saline (PND(10)Saline). After weaning, body weight was significantly elevated in PND(10)LPS compared with PND(10)Saline. Thereafter, the rats were injected with LPS (100 microg/kg, i.p.) or saline (used as a basal condition) from 7 to 8 weeks of age. Under basal conditions, cumulative food intake were significantly higher, serum leptin concentration was significantly increased, and hypothalamic NPY mRNA expression was significantly decreased in PND(10)LPS compared with PND(10)Saline. Under adult LPS injected conditions, body weight gain and cumulative food intake were suppressed in both the PND(10)LPS and PND(10)Saline groups compared with those observed under basal adult saline-injected conditions. The suppressive effects induced by adult LPS injection were less evident in the PND(10)LPS group than in the PND(10)Saline group. Adult LPS injection increased the serum leptin concentration in the PND(10)Saline rats, but not in the PND(10)LPS rats. In addition, adult LPS injection increased the mRNA expression of anorexinergic factors (IL-1beta, and TNF-alpha), and decreased that of the orexinergic factor NPY in both groups. However, the influence of adult LPS injection upon these factors was less evident in the PND(10)LPS group than in the PND(10)Saline group. These results suggest that neonatal LPS injection alters body weight regulation under both non-stress and immune stress conditions in male rats. Changes in the endocrine, neuropeptide, and cytokine regulation systems might be involved in these alterations.

Continuous orally administered coffee enhanced the antigen-specific Th1 response and reduced allergic development in a TCR-transgenic mice model.

Coffee is a globally consumed beverage. Although recent studies have suggested that coffee reduced the risk of lifestyle-related diseases, there are few studies regarding allergic response. This study investigates the effects of orally administered coffee (91 ml/kg/d) on allergic responses using a T cell receptor (TCR)-transgenic DO11.10 mouse allergic model. Splenocytes from coffee-administered naïve mice increased antigen (Ag)-specific interleukin (IL)-12p40 secretion. When Ag sensitization and coffee administration were concurrently performed, the splenocytes from coffee-administered mice showed a decrease of IL-2 and an increase of IL-12p40 secretion. The Ag-specific cutaneous response and serum IgE level were reduced in coffee-administered mice, although, after establishing the allergy, coffee administration did not suppress the allergic reaction. These results suggest that coffee could induce a Th1-type response of the immune system and prevent an allergy developing. Further studies on the optimum dose, cultivar differences, and roasted degree need to be undertaken.

Antibodies against the melanocortin-4 receptor act as inverse agonists in vitro and in vivo.

Functionally active antibodies (Abs) against central G-protein-coupled receptors have not yet been reported. We selected the hypothalamic melanocortin-4 receptor (MC4-R) as a target because of its crucial role in the regulation of energy homeostasis. A 15 amino acid sequence of the N-terminal (NT) domain was used as an antigen. This peptide showed functional activity in surface plasmon resonance experiments and in studies on HEK-293 cells overexpressing the human MC4-R (hMC4-R). Rats immunized against the NT peptide produced specific antibodies, which were purified and characterized in vitro. In HEK-293 cells, rat anti-NT Abs showed specific immunofluorescence labeling of hMC4-R. They reduced the production of cAMP under basal conditions and after stimulation with a synthetic MC4-R agonist. Rats immunized against the NT peptide developed a phenotype consistent with MC4-R blockade, that is, increased food intake and body weight, increased liver and fat pad weight, and elevated plasma triglycerides. In a separate experiment in rats, an increase in food intake could be produced after injection of purified Abs into the third ventricle. Similar results were obtained in rats injected with anti-NT Abs raised in rabbits. Our data show for the first time that active immunization of rats against the NT sequence of the MC4-R results in specific Abs, which appear to stimulate food intake by acting as inverse agonists in the hypothalamus.

Glutamine supplementation influences immune development in the newly weaned piglet.

A study was conducted to determine changes that occur in immune function during the early post-weaning period and the effect of supplementing glutamine (gln, 4% w/w) to the weaning diet of piglets. Dutch-Landrace piglets (n=10/group) were killed prior to weaning (21 d) or randomized to one of two nutritionally complete weaning diets with or without gln. With age there was an increased ability of peripheral blood mononuclear cells (PBMC) and mesenteric lymph nodes (MLN) cells to proliferate (rate of (3)H-thymidine uptake) to T cell mitogens (P<0.05). PBMC from older piglets produced less of a Th-1 type response after stimulation (P<0.05). Adding gln to the weaning diet significantly (P<0.05) modified immune cells in the MLN, in a potentially beneficial manner (with respect to mucosal infections) by preventing an increase in antigen naïve CD4+ cells, increasing the proliferative response to pokeweed mitogen and supporting a Th-1 type cytokine response after T cell (phytohemagglutinin) stimulation.

Evaluation of the Mtb72F polyprotein vaccine in a rabbit model of tuberculous meningitis.

Using a rabbit model of tuberculous meningitis, we evaluated the protective efficacy of vaccination with the recombinant polyprotein Mtb72F, which is formulated in two alternative adjuvants, AS02A and AS01B, and compared this to vaccination with Mycobacterium bovis bacillus Calmette-Guérin (BCG) alone or as a BCG prime/Mtb72F-boost regimen. Vaccination with Mtb72F formulated in AS02A (Mtb72F+AS02A) or Mtb72F formulated in AS01B (Mtb72F+AS01B) was protective against central nervous system (CNS) challenge with Mycobacterium tuberculosis H37Rv to an extent comparable to that of vaccination with BCG. Similar accelerated clearances of bacilli from the cerebrospinal fluid, reduced leukocytosis, and less pathology of the brain and lungs were noted. Weight loss of infected rabbits was less extensive for Mtb72F+AS02A-vaccinated rabbits. In addition, protection against M. tuberculosis H37Rv CNS infection afforded by BCG/Mtb72F in a prime-boost strategy was similar to that by BCG alone. Interestingly, Mtb72F+AS01B induced better protection against leukocytosis and weight loss, suggesting that the polyprotein in this adjuvant may boost immunity without exacerbating inflammation in previously BCG-vaccinated individuals.

t10,c12 Conjugated linoleic acid induces compensatory growth after immune challenge.

Previous work demonstrated that feeding commercial preparations of conjugated linoleic acid (CLA) [a 50:50 mixture of c9,t11 and t10,c12 CLA (cCLA)] partially overcame lipopolysaccharide (LPS)-induced growth depression. The objective of this study was to determine which CLA isomer was responsible for the reduction of LPS-induced growth depression. Dietary cCLA supplementation for 3 weeks protected mice from LPS-induced weight loss 24 h after injection compared to mice fed isocaloric and isonitrogenous control diets supplemented with either corn oil (CO) or a mixture of CO and olive oil. Dietary c9,t11 or t10,c12 CLA led to body weight loss intermediate to controls and cCLA. After LPS-induced weight loss, the t10,c12 CLA fed mice regained weight faster than the control or c9,t11 CLA fed mice. Dietary t10,c12 CLA and cCLA reduced plasma tumor necrosis factor 2 h after LPS stimulation. While neither c9,t11 nor t10,c12 CLA isomers alone protected from immune-induced weight loss, the t10,c12 CLA isomer induced compensatory gain.

Immunoglobulins in nasal secretions of dog puppies from birth to six weeks of age.

In order to investigate local immune defence mechanisms in the dog, the concentration of immunoglobulins (Ig) G, A and M in nasal secretions (NS) and serum of 42 healthy, neonatal Rottweiler puppies was determined. Ig were measured with a commercially available, dog-specific ELISA during the first six weeks of life. On average, IgG was the predominant Ig isotype during the first three days of life. The IgA:IgG ratio changed between weeks 1 and 3 due to markedly decreasing IgG concentrations. Between the fourth and sixth week, IgG predominated again. During the first week, only 21-39% of puppies had measurable amounts of IgM in NS, in week 2, this percentage increased to 69%. Marked differences between litters and between individual puppies within litters were found. No puppy diseased during the observation period and all developed normally.

Inhalation toxicity of brevetoxin 3 in rats exposed for 5 days.

Brevetoxins are potent neurotoxins produced by the marine dinoflagellate Karenia brevis. Exposure to brevetoxins may occur during a K. brevis red tide when the compounds become aerosolized by wind and surf. This study assesses possible adverse health effects associated with short-term inhalation exposure to brevetoxin 3. Male F344/Crl/Br rats were exposed to 500 microg brevetoxin 3/m3 by nose-only inhalation for 0.5 or 2 h/d for 5 consecutive days. Control rats were sham exposed for 2 h to vehicle. Calculated deposited brevetoxin doses were 8.3 and 33 microg/kg/d for the low- and high-dose groups, respectively. At the termination of exposures, only body weights of the high-dose group (Group B) were significantly below control values. By immunohistochemistry (IHC), small numbers of splenic and peribronchiolar lymphoid tissue macrophages stained positive for brevetoxin, while nasal mucosa, liver, and brain were IHC negative for brevetoxin. No gross or microscopic lesions were observed in any tissue examined. There was no biochemical evidence of cytotoxicity or inflammation in bronchoalveolar lavage fluid. Alveolar macrophages showed some evidence of activation following brevetoxin exposure. Humoral-mediated immunity was suppressed in brevetoxin-exposed rats as indicated by a >70% reduction in splenic plaque-forming cells in brevetoxin-exposed animals compared to controls. Results suggest that the immune system may be a target of toxicity following brevetoxin inhalation. Future studies will focus on identification of a no-effect level and mechanisms underlying brevetoxin-induced immune suppression.