Influence of age and vaccination interval on canine parvovirus, distemper virus, and adenovirus serum antibody titers.

Canine core vaccine titer screenings are becoming increasingly popular in veterinary practice as a tool to guide vaccination decisions, despite a lack of supportive, peer-reviewed evidence-based literature. Additionally, it has been suggested that the canine core vaccine duration of host protective immunity can persist past the currently recommended vaccination interval. Thus, this study evaluated serum antibody titers against three core antigens in dogs with known vaccination histories and lifestyles, analyzing the effect of life stage, exposure risk, and time since last vaccination (TSLV). Clinically healthy dogs (n = 188) presenting to the primary care services of three colleges of veterinary medicine were selected to represent a variety of ages, breeds, and vaccination history. Serum antibody titers for canine parvovirus (CPV), canine distemper virus (CDV), and canine adenovirus-2 (CAV2) were measured via virus neutralization and hemagglutination inhibition. CAV2 and CPV titers decreased, while CDV titers had a decreasing trend with increasing time since last vaccination or vaccination interval. When assessing circulating antibody levels historially associated with protective immunity across various vaccination intervals, 62% (95%CI 36-82%; 8/13) of dogs had positive titers for CDV 5 years post last vaccination, while 92% (95%CI 67-99%; 12/13) of dogs were positive for CAV2 and CPV. Both advanced age and life stage were associated with lower titers and thus, identify a canine population cohort likely at higher disease risk. The results of this study revealed that patient duration of core vaccine-mediated immunity changes with a number of variables, with animal aging and time since vaccination influencing host humoral immunity. This provides further support for the performance of canine core antibody titers to assess whether a vaccine booster and/or specific type of booster is warranted.

Bortezomib inhibits the proteasome, leading to cell death via apoptosis in feline injection site sarcoma cells in vitro.

OBJECTIVE: To determine the in vitro effects of the proteasome inhibitor bortezomib in feline injection site sarcoma (FISS) cell lines. SAMPLE: In vitro cultures of the FISS cell lines Ela-1, Hamilton, and Kaiser. PROCEDURES: Cells were treated with increasing doses of bortezomib or vehicle alone (dimethyl sulfoxide) and evaluated for cell viability via an adenosine triphosphate concentration assay, proteasome activity via a commercially available proteasome assay, accumulation of ubiquitinated proteins via Western blot, and apoptosis via flow cytometry. RESULTS: All 3 cell lines were sensitive to bortezomib with a 50% inhibitory concentration after 48 hours of treatment at 17.46 nM (95% CI, 15.47 to 19.72 nM) for Ela-1, 19.48 nM (95% CI, 16.52 to 23.00 nM) for Hamilton, and 21.38 nM (95% CI, 19.24 to 23.78 nM) for Kaiser. In the Ela-1 cell line, 20 nM bortezomib inhibited 20S proteasome activity by 90.9% compared with the vehicle-only control. In the Kaiser cell line, 20 nM bortezomib decreased 20S proteasome activity by 70%, compared with the untreated vehicle-only control. Last, treatment with bortezomib (25 and 40 nM) resulted in statistically significant decreases in viable cells accompanied by a statistically significant increase in apoptotic cells. CLINICAL RELEVANCE: Treatment options for FISS, especially nonresectable FISS, are currently very limited. These results support further investigation of bortezomib either alone or in combination with other treatments in such cases.

Variability in peripheral blood enrichment techniques can alter equine leukocyte cellularity, viability and function.

Peripheral blood is commonly sampled to assess the health status of human and veterinary patients. Venous blood collection is a minimally invasive procedure, and in the horse, the common collection site is the jugular vein. Post blood collection, sample processing for leukocyte enrichment can vary by research laboratory with the potential to yield different effects on the enriched cells and their function. The focus of the present study was to compare a common blood dilution-leukocyte enrichment technique using a Histopaque gradient medium (His) to a modified leukocyte buffy coat syringe-lymphocyte separation medium technique (Syr- LSM) with peripheral blood from 12 healthy horses. The endpoints examined included cell recovery/mL of blood, cell viability, leukocyte enrichment purity, leukocyte cell marker subset phenotype, leukocyte spontaneous and mitogen-induced proliferation and secretory TNFα concentrations. Leukocyte cell recovery/mL of whole blood and cell viability was significantly increased in enriched leukocytes from the Syr-LSM technique. Interestingly, the percentage of CD8+ and CD21+ were significantly increased with the His technique as was Con A-induced proliferation. Still, leukocyte cell purity and TNFα concentrations from the 72 h cell culture supernatants were comparable across the two enrichment techniques. To summarize, the type of whole blood leukocyte enrichment technique employed can affect the results of immunologic assay endpoints possibly altering data interpretation.

Resistin concentrations in serum and stifle synovial fluid from normal and cruciate deficient dogs with osteoarthritis.

OBJECTIVE: To compare synovial fluid (SF) resistin concentrations in healthy dogs to dogs with osteoarthritis (OA) secondary to cranial cruciate ligament (CrCL) injury and to correlate resistin concentrations with body condition score (BCS) and evaluate resistin release from peripheral blood mononuclear cells (PBMC) and adipocytes. STUDY DESIGN: Controlled, prospective, clinical study ANIMALS: Thirty-nine client-owned dogs, 13 healthy and 26 with secondary OA, were enrolled. Blood was collected from six healthy purpose-bred dogs for PBMC culture. An additional six mixed-breed dogs were used for adipocyte collection and culture. METHODS: Resistin concentrations were measured with a canine-specific enzyme-linked immunoabsorbent assay. Resistin was compared between healthy SF and OA SF with Student's t test. Correlation of resistin concentrations to BCS was performed. Peripheral blood mononuclear cells and adipocytes were cultured under three conditions: negative control, lipopolysaccharide, and concanavalin A (Con A). A linear mixed model was used to determine differences in resistin concentrations among treatments. RESULTS: Resistin concentrations in OA SF were comparable to healthy SF. Neither serum nor SF resistin was correlated with BCS. Cultured PBMC stimulated with Con A released resistin, while adipocytes did not. CONCLUSION: Neither serum nor SF resistin were altered in dogs with OA secondary to CrCL insufficiency. In addition, resistin was not correlated with canine body fat and did not appear to function as adipocytokine in the dog. CLINICAL SIGNIFICANCE: Resistin may not be involved in the pathogenesis of OA. However, resistin may be important in inflammation because it is released from inflammatory cells.

Elevated Synovial Fluid Concentration of Monocyte Chemoattractant Protein-1 and Interleukin-8 in Dogs with Osteoarthritis of the Stifle.

Chemokines such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) have been shown to cause monocyte and natural killer cell chemotaxis and polymorphonuclear cell chemotaxis, respectively. Additionally, MCP-1 signalling has been implicated in modulating pain. Elevated synovial fluid concentrations of MCP-1 and IL-8 have been demonstrated in humans with osteoarthritis, but currently there are no studies evaluating synovial MCP-1 or IL-8 concentrations in dogs. Additionally, there are no canine studies evaluating the correlation between these chemokines and caregiver perceived pain and mobility, as measured by the clinical metrology instrument, Liverpool Osteoarthritis in Dogs. This study documented elevated synovial fluid concentrations of IL-8 and MCP-1 in the stifle of dogs with secondary osteoarthritis compared with normal stifles. However, this study found no correlation between MCP-1 or IL-8 and Liverpool Osteoarthritis in Dogs or radiographic severity of osteoarthritis.

Correlation of serum and synovial leptin concentrations with body condition scores in healthy and osteoarthritic dogs.

OBJECTIVE: To evaluate the relationship between serum and synovial fluid (SF) leptin concentrations and body condition score (BCS) in healthy and osteoarthritic dogs. STUDY DESIGN: Controlled, prospective, clinical study. ANIMALS: Nineteen healthy dogs and 29 dogs with osteoarthritis (OA) secondary to cranial cruciate ligament injury. METHODS: Synovial fluid was obtained from the femorotibial joint under sedation (healthy dogs) or during surgery (OA dogs). Serum and SF leptin and interleukin (IL)-1ß concentrations were measured via enzyme-linked immunosorbent assay. Dogs were classified as optimal weight (BCS 4-5/9) or overweight (BCS >5/9). Radiographs were scored for OA severity by a radiologist. Owners completed the Liverpool Osteoarthritis in Dogs (LOAD) questionnaire. RESULTS: Mean (± SD) SF leptin (4.09 ± 4 ng/mL) was lower than serum leptin (6.88 ± 5.52 ng/mL, P < .0001). Synovial fluid leptin was higher in overweight (5.28 ± 4.21) than in optimal body weight dogs (1.54 ± 1.72 ng/mL, P < .0001). Serum (P < .001) and SF leptin (P = .004) concentrations were associated with BCS. Concentration of SF leptin did not differ between healthy (2.4 ± 2.04 ng/mL) and OA (4.9 ± 4.3 ng/mL, P = .25) dogs. Synovial fluid leptin and LOAD scores were weakly associated (P = .03). No association was detected between SF leptin and radiographic score or IL-1ß (P = .73). CONCLUSION: Serum and SF leptin correlated with BCS in this population. Synovial fluid leptin was weakly associated with LOAD scores but not with radiographic severity of OA or IL-1ß. CLINICAL SIGNIFICANCE: Serum and SF leptin concentrations do not predict radiographic severity of canine OA but contribute to joint pain and dysfunction.

Immunomodulatory in vitro effects of oclacitinib on canine T-cell proliferation and cytokine production.

BACKGROUND: Oclacitinib is a Janus kinase inhibitor used to control pruritus and skin lesions in canine allergic skin disease; its effect on canine T cells is not well-characterized. HYPOTHESIS/OBJECTIVES: To evaluate the impact of oclacitinib on cultured T cells using peripheral blood mononuclear cells from dogs. ANIMALS: Six bluetick coonhounds. METHODS AND MATERIALS: Lymphocyte-enriched cells were incubated with or without the T-cell mitogen concanavalin A (Con A), oclacitinib (0.5, 1 or 10 µM), ciclosporin (200 ng/mL), Con A + oclacitinib 1 µM and Con A + ciclosporin. We assessed both T-cell proliferation and the secretion of cytokines. RESULTS: Ciclosporin and oclacitinib both inhibited the spontaneous proliferation of T cells; this effect was significant only after incubation with oclacitinib at 10 µM. At this concentration, oclacitinib significantly reduced the spontaneous secretion of clonal activator cytokines [interleukin (IL)-2, IL-15], pro-inflammatory cytokines (interferon-gamma (IFN-γ), IL-18) and the regulatory cytokine IL-10; tumour necrosis factor alpha (TNF-α) and IL-6 cytokine production was mildly inhibited. After Con A stimulation, only T cells co-treated with ciclosporin achieved a significant proliferation inhibition and reduction of IL-2, IL-10, IL-15, IL-18, IFN-γ and TNF-α. Surprisingly, oclacitinib at 1 µM (337 ng/mL, corresponding to the oral dosage of 0.4-0.6 mg/kg) did not significantly affect Con A-stimulated T-cell proliferation nor cytokine production (IL-2, IL-10, IL-15, IL-18, IFN-γ and TNF-α). CONCLUSIONS: Although a limited number of dogs were investigated, these preliminary results suggest that oclacitinib appears to have immunosuppressive properties, but only at dosages above those used to treat allergic pruritus in dogs.

Acute Lameness in a Roller Pigeon ( Columba livia ) with Multicentric Lymphosarcoma.

A 3-yr-old adult female roller pigeon ( Columba livia ) used as part of a breeding pair for an ongoing research study presented with acute left limb lameness. Palpation of the left leg and region revealed a large lump near the coxofemoral joint. The bird was able to ambulate in the cage, but would not brood her hatchling. The bird was humanely euthanized and necropsy was performed. Grossly, multiple large white to pale tan nodules were noted in the pancreas, lung, rib cage, intestines, and unilaterally in the left kidney. Microscopic examination of the various organs revealed neoplastic proliferation of round cells consistent with lymphoblasts. Immunohistochemistry was performed with the use of antibodies to CD3, CD79a, CD20, and CD21 to phenotype the cells. The results indicated that the neoplastic infiltrating cells were predominantly of T-cell origin.

Effects of Chicken Interferon Gamma on Newcastle Disease Virus Vaccine Immunogenicity.

More effective vaccines are needed to control avian diseases. The use of chicken interferon gamma (chIFNγ) during vaccination is a potentially important but controversial approach that may improve the immune response to antigens. In the present study, three different systems to co-deliver chIFNγ with Newcastle disease virus (NDV) antigens were evaluated for their ability to enhance the avian immune response and their protective capacity upon challenge with virulent NDV. These systems consisted of: 1) a DNA vaccine expressing the Newcastle disease virus fusion (F) protein co-administered with a vector expressing the chIFNγ gene for in ovo and booster vaccination, 2) a recombinant Newcastle disease virus expressing the chIFNγ gene (rZJ1*L/IFNγ) used as a live vaccine delivered in ovo and into juvenile chickens, and 3) the same rZJ1*L/IFNγ virus used as an inactivated vaccine for juvenile chickens. Co-administration of chIFNγ with a DNA vaccine expressing the F protein resulted in higher levels of morbidity and mortality, and higher amounts of virulent virus shed after challenge when compared to the group that did not receive chIFNγ. The live vaccine system co-delivering chIFNγ did not enhanced post-vaccination antibody response, nor improved survival after hatch, when administered in ovo, and did not affect survival after challenge when administered to juvenile chickens. The low dose of the inactivated vaccine co-delivering active chIFNγ induced lower antibody titers than the groups that did not receive the cytokine. The high dose of this vaccine did not increase the antibody titers or antigen-specific memory response, and did not reduce the amount of challenge virus shed or mortality after challenge. In summary, regardless of the delivery system, chIFNγ, when administered simultaneously with the vaccine antigen, did not enhance Newcastle disease virus vaccine immunogenicity.

Serine protease inhibition attenuates rIL-12-induced GZMA activity and proinflammatory events by modulating the Th2 profile from estrogen-treated mice.

Estrogen has potent immunomodulatory effects on proinflammatory responses, which can be mediated by serine proteases. We now demonstrate that estrogen increased the extracellular expression and IL-12-induced activity of a critical member of serine protease family Granzyme A, which has been shown to possess a novel inflammatory persona. The inhibition of serine protease activity with inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride significantly diminished enhanced production of proinflammatory interferon-γ, IL-1ß, IL-1α, and Granzyme A activity even in the presence of a Th1-inducing cytokine, IL-12 from splenocytes from in vivo estrogen-treated mice. Inhibition of serine protease activity selectively promoted secretion of Th2-specific IL-4, nuclear phosphorylated STAT6A, signal transducer and activator of transcription (STAT)6A translocation, and STAT6A DNA binding in IL-12-stimulated splenocytes from estrogen-treated mice. Inhibition with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride reversed the down-regulation of Th2 transcription factors, GATA3 and c-Maf in splenocytes from estrogen-exposed mice. Although serine protease inactivation enhanced the expression of Th2-polarizing factors, it did not reverse estrogen-modulated decrease of phosphorylated STAT5, a key factor in Th2 development. Collectively, data suggest that serine protease inactivity augments the skew toward a Th2-like profile while down-regulating IL-12-induced proinflammatory Th1 biomolecules upon in vivo estrogen exposure, which implies serine proteases as potential regulators of inflammation. Thus, these studies may provide a potential mechanism underlying the immunomodulatory effect of estrogen and insight into new therapeutic strategies for proinflammatory and female-predominant autoimmune diseases.

Endocrine-disrupting chemical exposure and the American alligator: a review of the potential role of environmental estrogens on the immune system of a top trophic carnivore.

Endocrine-disrupting chemicals (EDCs) alter cellular and organ system homeostasis by interfering with the body's normal physiologic processes. Numerous studies have identified environmental estrogens as modulators of EDC-related processes in crocodilians, notably in sex determination. Other broader studies have shown that environmental estrogens dysregulate normal immune function in mammals, birds, turtles, lizards, fish, and invertebrates; however, the effects of such estrogenic exposures on alligator immune function have not been elucidated. Alligators occupy a top trophic status, which may give them untapped utility as indicators of environmental quality. Environmental estrogens are also prevalent in the waters they occupy. Understanding the effects of these EDCs on alligator immunity is critical for managing and assessing changes in their health and is thus the focus of this review.

Parabens: potential impact of low-affinity estrogen receptor binding chemicals on human health.

Parabens, alkyl esters of p-hydroxybenzoic acid, are widely used in cosmetics, pharmaceuticals, personal care products and as food additives to inhibit microbial growth and extend product shelf life. Consumers of these compounds are frequently exposed via the skin, lips, eyes, oral mucosa, nails, and hair. Parabens are estrogenic molecules but exert weaker activity than natural estrogens, which would imply a low risk. Consistent with this idea, a number of recent commission reports from different countries suggested that parabens pose a negligible endocrine-disrupting risk at the recommended doses. However, individuals are not routinely exposed to a single paraben, and most of the available paraben toxicity data, reviewed in these reports, are from single-exposure studies. Further, assessing the additive and cumulative risk of multiple paraben exposure from daily use of multiple cosmetic and/or personal care products is presently not possible based on current studies. In this review, current and recent studies of paraben exposure and public health policies as well as critical gaps in the knowledge are discussed and new research directions regarding multiple exposures and novel target cohorts are recommended.

Influence of different cell storage/culture conditions on spontaneous proliferation and level of tyrosine kinase receptor inhibition in two feline injection-site sarcoma cell lines.

Optimizing cell culture conditions is important when studying cell proliferation and viability, particularly in response to cytotoxic compounds. Altered cell storage conditions can adversely impact proliferation and viability in mortal cell lines. However, little is known regarding the effects on immortal feline cell lines. In the present study, two feline injection-site sarcoma (ISS) cell lines were evaluated under standard culture conditions and three alternative storage/culture conditions for spontaneous proliferation rate and sensitivity to masitinib, a highly selective tyrosine kinase inhibitor with activity against primary and metastatic ISS cell lines. Cell viability was assessed by 7-aminoactinomycin D and cytology. Spontaneous proliferation did not significantly differ across the FBS concentrations (10% vs. 1%) for one cell line, however, with the other cell line spontaneous proliferation was significantly decreased in the 1% FBS 1-step technique, and the cold step technique at both 1% and 10% FBS. When normalized to untreated control cells, the IC50 values for masitinib were comparable across all culture techniques. Furthermore, apoptosis appeared to be the primary mechanism of this proliferation inhibition. Our preliminary findings suggest that select feline sarcoma cell lines cultured in 10% FBS yield comparable cytotoxicity data even when subjected to varying storage/culture conditions.

Multiple environmental stressors elicit complex interactive effects in the western fence lizard (Sceloporus occidentalis).

Evaluation of multiple-stressor effects stemming from habitat degradation, climate change, and exposure to chemical contaminants is crucial for addressing challenges to ecological and environmental health. To assess the effects of multiple stressors in an understudied taxon, the western fence lizard (Sceloporus occidentalis) was used to characterize the individual and combined effects of food limitation, exposure to the munitions constituent 2,4,6-trinitrotoluene (TNT), and Plasmodium mexicanum (lizard malaria) infection. Three experimental assays were conducted including: Experiment I--TNT × Food Limitation, Experiment II--Food Limitation × Malaria Infection, and Experiment III--TNT × Malaria Infection. All experiments had a 30 day duration, the malaria treatment included infected and non infected control lizards, food limitation treatments included an ad libitum control and at least one reduced food ration and TNT exposures consisting of daily oral doses of corn oil control or a corn oil-TNT suspension at 5, 10, 20, 40 mg/kg/day. The individual stressors caused a variety of effects including: reduced feeding, reduced testes mass, anemia, increased white blood cell (WBC) concentrations and increased mass of liver, kidney and spleen in TNT exposures; reduced cholesterol, WBC concentrations and whole body, testes and inguinal fat weights given food limitation; and increased WBC concentrations and spleen weights as well as decreased cholesterol and testes mass in malaria infected lizards. Additive and interactive effects were found among certain stressor combinations including elimination of TNT-induced hormesis for growth under food limitation. Ultimately, our study indicates the potential for effects modulation when environmental stressors are combined.

Serine protease inhibitor, 4-(2-aminoethyl)-benzene sulfonyl fluoride, impairs IL-12-induced activation of pSTAT4ß, NFκB, and select pro-inflammatory mediators from estrogen-treated mice.

Estrogen, a natural immunomodulator, is believed to be involved in the regulation of not only normal immune responses, but also pathological conditions such as inflammatory and autoimmune diseases. We have previously reported that estrogen exposure induces several pro-inflammatory molecules including nitric oxide, cytokines and chemokines (IFNγ, IL-12, MCP-1, etc.) and modifies transcription factors (preferential expression of STAT4ß, increased NFκB p50/p50 DNA binding, and enhanced T-bet and Bcl-3) from activated splenocytes. Given that estrogen promotes diverse range of pro-inflammatory molecules, and modifies transcription factors, it is plausible that estrogen upregulates a common set of molecular event(s) that favors inflammation. Serine proteases are thought to play an important role in inflammation. Therefore in this study, we investigated the consequence of exposure of splenocytes stimulated with a key Th1/IFNγ-inducing cytokine IL-12 or ConA from estrogen-treated mice to a serine protease inhibitor, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), on inflammatory cytokines (IFNγ, IL-12) and related transcription factors (STAT4α/ß, T-bet, NFκB). Exposure of splenocytes to AEBSF for 3h noticeably inhibited the induction of IFNγ, IL-12, and IL-12-induced STAT4ß, mRNA expression of T-bet and IL-12Rß2. The AEBSF-mediated inhibition of cytokines was accompanied by the expression of a normal-sized NFκB, downregulation of p50/p50 DNA binding but did not alter Bcl3. These findings provide a new understanding of inflammation and inhibition of serine proteases has important implications for designing novel therapeutic strategies for a broad range of inflammatory diseases.

Developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters postnatal T cell phenotypes and T cell function and exacerbates autoimmune lupus in 24-week-old SNF1 mice.

BACKGROUND: Untreated, more than 95% of female SWR x NZB: F(1) (SNF(1)) mice spontaneously develop a fatal lupus-like glomerulonephritis by 8 months-of-age, while disease onset in males is much slower. METHODS: : Timed-pregnant SNF(1) mice (10 per treatment) were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on gestational day (GD) 12 by oral maternal gavage with 0, 40, or 80 microg/kg TCDD. RESULTS: Offspring of the TCDD-exposed dams showed numerous alterations in T lineage cells at 24 weeks-of-age. Females but not males showed decreased CD4(+)8(+) and increased CD4(-)8(-) thymocytes. Females also showed increased autoreactive CD4(+)Vbeta17(a+) axillary and inguinal lymph node T cells. Concanavalin A-stimulated splenocytes from prenatal TCDD-treated mice produced decreased interleukin 17 (IL-17) in the females while males showed increased IL-2 and IFN-gamma, and diminished IL-4. Mitogen-stimulated pan-lymphoproliferative responses were significantly increased across sex by TCDD. Anti-IgG and anti-C3 immune complex deposition in kidneys was present in the males after TCDD, and visibly worsened in females. CONCLUSIONS: Developmental TCDD exposure can permanently alter T lymphopoiesis in autoimmune-prone SNF1 mice. The alteration profile is beyond the classic immune suppression response, to also include exacerbation and induction of a lupuslike autoimmune disease.

Signal transducer and activation of transcription (STAT) 4beta, a shorter isoform of interleukin-12-induced STAT4, is preferentially activated by estrogen.

Estrogen, a natural immunomodulatory compound, has been shown to promote the induction of a prototype T helper 1 cytokine, interferon (IFN)-gamma, as well as to up-regulate IFNgamma-mediated proinflammatory molecules (nitric oxide, cyclooxygenase 2, monocyte chemoattractant protein 1). Because IL-12 is a major IFNgamma-inducing cytokine, in this study we investigated whether estrogen treatment of wild-type C57BL/6 mice alters IL-12-mediated signaling pathways. A recent study has shown that IL-12 activates two isoforms of signal transducer and activation of transcription (STAT) 4, a normal-sized (full-length STAT4alpha) and a truncated form (STAT4beta). Interestingly, we found that estrogen treatment preferentially up-regulates the phosphorylation of STAT4beta in splenic lymphoid cells. Time kinetic data showed the differential activation of STAT4beta in splenic lymphoid cells from estrogen-treated mice, but not in cells from placebo controls. The activation of STAT4beta was mediated by IL-12 and not IFNgamma because deliberate addition or neutralization of IL-12, but not IFNgamma, affected the activation of STAT4beta. In contrast to IL-12-induced activation of STAT4beta in cells from estrogen-treated mice, STAT4alpha was not increased, rather it tended to be decreased. In this context, STAT4alpha-induced p27(kip1) protein was decreased in concanavalin A + IL-12-activated lymphocytes from estrogen-treated mice only. By using the in vitro DNA binding assay, we confirmed the ability of pSTAT4beta to bind to the IFNgamma-activated sites (IFNgamma activation sequences)/STAT4-binding sites in estrogen-treated mice. Our data are the first to show that estrogen apparently has selective effects on IL-12-mediated signaling by preferentially activating STAT4beta. These novel findings are likely to provide new knowledge with regard to estrogen regulation of inflammation.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or diethylstilbestrol (DES) cause similar hematopoietic hypocellularity and hepatocellular changes in murine fetal liver, but differentially affect gene expression.

TCDD and DES have immunotoxic effects, including selective diminution of T lymphocyte progenitors in the fetal liver. The histologic presentation of fetal liver after exposure to either chemical has not been described. Similarly, limited information exists regarding mechanisms by which TCDD or DES may alter fetal hematopoiesis. Treatment of pregnant C57BL/6 mice with either 10 micro g/kg/day TCDD or 48 micro g/kg/day DES on gestation days (gd) 14 and 16 led to increased fetal liver weight on gd 18. Moderate anisocytosis and anisokaryosis with increased cytoplasmic and nuclear sizes, and increased cytoplasmic basophilia were present within hepatocytes after TCDD or DES. Both chemicals also decreased the presence of hematopoietic cells, however megakaryocyte numbers were unaffected. In contrast to these similar outcomes, real time quantitative PCR using a preliminary panel of 4 genes suggested that the chemicals act through different gene targets. TCDD increased c-jun gene expression in fetal liver, and decreased p53 without alteration in bcl-2 expression, indicating possible pro-proliferative and antiapoptotic effects. DES decreased c-jun and bcl-2, without altering p53, suggesting a shift away from proliferation. Both agents decreased PKCalpha expression, which may suggest shared decreased phosphorylation of substrates required for normal cell cycle progression.

IFN-gamma-inducing transcription factor, T-bet is upregulated by estrogen in murine splenocytes: role of IL-27 but not IL-12.

Estrogen is believed to be involved in regulation of the differentiation, survival, or function of diverse immune cells as well as in many autoimmune and inflammatory diseases. However, the mechanisms behind the immunomodulatory effects of estrogen are poorly understood. Previously, we have shown that natural estrogen can upregulate IFN-gamma and IFN-gamma-mediated-inflammatory events (iNOS, nitric oxide, COX-2). Since IFN-gamma is regulated by T-bet, in this study, we investigated whether estrogen induces T-bet expression in primary murine splenocytes. We found that in vivo estrogen treatment primes splenocytes for early upregulation of T-bet upon activation by T cell stimulants, Concanavalin-A (Con-A) or anti-CD3 antibodies. The expression of T-bet protein was not altered by IL-12 while IFN-gamma had partial effects on T-bet in splenocytes from estrogen-treated mice. Notably, T-bet expression increased in Con-A-activated splenocytes from estrogen-treated mice in the presence of IL-27. Together, our studies show that in vivo estrogen exposure primes lymphocytes towards Th1 type development by promoting/upregulating T-bet expression, which is upregulated in part by IFN-gamma and IL-27. Given that T-bet is a potent inducer of IFN-gamma, these studies may lead to new lines of investigation in relation to many female-predominant autoimmune diseases and inflammatory disorders.

Influence of oral 2,4-dinitrotoluene exposure to the northern bobwhite (Colinus virginianus).

Military activities associated with training, munitions manufacturing, and demilitarization has resulted in soil residues of munition compounds and their breakdown products. Two isomers of dinitrotoluene (2,4- and 2,6-) are often found in soil associated with those activities at considerable concentrations. Consequently, issues regarding the effects of exposure to birds that visit these habitats require evaluation. To provide data useful to a risk assessment approach, we followed a controlled dosing regime (gavage) using 2,4-dinitrotoluene (2,4-DNT) in the Northern Bobwhite (Colinus virginianus) for 60 days following a 14-day range-finding study and the determination of a LD50 using the up/down method. The LD50 was determined to be 55 mg/kg using corn oil as a vehicle. Individuals dosed exceeding this level were moribund or died within 60 h of exposure. Morbidity and death occurred during the 14-day range-finding study at dosing regimens of 35 and 55, but not at 15, 5, and 0.5 mg/kg-day. Compound-related morbidity/mortality occurred in the 60-day study during the first week of exposure at 25 and 15, but not at 5, 1, and 0 mg/kg-day. Overt signs of toxicity occurred with both sexes at the onset of exposure. Signs included weight loss, diarrhea, and lethargy. Dose-related changes in egg production, ovary, kidney, and brain mass, and body weight, but not feed consumption, were found. Changes in kidney mass and histological observations suggest accumulation of nitrogenous waste may be the cause of morbidity. These data suggest that oral 2,4-DNT exposures are more acutely toxic and has a different etiology than 2,4,6-trinitrotoluene in birds.