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In the setting of radiation-induced trauma, exposure to high levels of radiation can cause an acute radiation syndrome (ARS) causing bone marrow (BM) failure, leading to life-threatening infections, anemia, and thrombocytopenia. We have previously shown that human macrophages educated with human mesenchymal stem cells (MSCs) by coculture can significantly enhance survival of mice exposed to lethal irradiation. In this study, we investigated whether exosomes isolated from MSCs could replace direct coculture with MSCs to generate exosome educated macrophages (EEMs). Functionally unique phenotypes were observed by educating macrophages with exosomes from MSCs (EEMs) primed with bacterial lipopolysaccharide (LPS) at different concentrations (LPS-low EEMs or LPS-high EEMs). LPS-high EEMs were significantly more effective than uneducated macrophages, MSCs, EEMs, or LPS-low EEMs in extending survival after lethal ARS in vivo. Moreover, LPS-high EEMs significantly reduced clinical signs of radiation injury and restored hematopoietic tissue in the BM and spleen as determined by complete blood counts and histology. LPS-high EEMs showed significant increases in gene expression of STAT3, secretion of cytokines like IL-10 and IL-15, and production of growth factors like FLT-3L. LPS-EEMs also showed increased phagocytic activity, which may aid with tissue remodeling. LPS-high EEMs have the potential to be an effective cellular therapy for the management of ARS.

Despite being closely related, bonobos and chimpanzees exhibit several behavioral differences. For instance, studies indicate that chimpanzees are more aggressive, territorial, and risk-taking, while bonobos exhibit greater social tolerance and higher rates of socio-sexual interactions. To elucidate the potential neuroanatomical variation that accompanies these differences, we examined the microstructure of selected brain areas by quantifying the neuropil fraction, a measure of the relative tissue area occupied by structural elements of connectivity (e.g., dendrites, axons, and synapses) versus cell bodies. In bonobos and chimpanzees, we compared neuropil fractions in the nucleus accumbens (NAc; core and shell), amygdala (whole, accessory basal, basal, central and lateral nuclei), anterior cingulate cortex (ACC; dorsal and subgenual), anterior insular cortex (AIC), and primary motor cortex (M1). In the dorsal ACC and frontoinsular cortex (FI) we also quantified numbers of von Economo neurons (VENs), a unique subset of neurons thought to be involved in rapid information processing during social interactions. We predicted that the neuropil fraction and number of VENs in brain regions associated with socio-emotional processing would be higher in bonobos. In support of this hypothesis, we found that bonobos had significantly greater neuropil in the central and accessory basal nuclei of the amygdala, as well as layers V–VI of the subgenual ACC. However, we did not find a difference in the numbers of VENs between the two species. These findings support the conclusion that bonobo and chimpanzee brains differ in the anatomical organization of socio-emotional systems that may reflect species-specific variation in behavior.

Gyroviruses are small, single stranded DNA viruses in the family Anelloviridae. In chickens, the type virus (chicken anemia virus; CAV) causes epidemic disease in poultry flocks worldwide. In 2007 and 2008, young crested screamers (Chauna torquata) at a zoo in Wisconsin, USA, died of neurologic disease with clinical and pathological features resembling CAV infection. Conventional diagnostics were negative, but molecular analyses revealed coinfection of an affected bird with three variants of a novel Gyrovirus lineage, GyV10. Analysis of ten additional screamers from this and another zoo revealed infection in all but one bird, with co-infections and persistent infections common. The association between GyV10 ("screamer anemia virus," provisionally) and the disease remains unproven, but certain immunological and neurologic features of the syndrome would expand the known pathologic consequences of Gyrovirus infection. To control the virus, autogenous vaccines, environmental decontamination, and management strategies to limit vertical and horizontal transmission might prove effective.

Purpose To determine the feasibility of creating a clinically relevant hepatic ablation (ie, an ablation zone capable of treating a 2-cm liver tumor) by using robotically assisted sonic therapy (RAST), a noninvasive and nonthermal focused ultrasound therapy based on histotripsy. Materials and Methods This study was approved by the institutional animal use and care committee. Ten female pigs were treated with RAST in a single session with a prescribed 3-cm spherical treatment region and immediately underwent abdominal magnetic resonance (MR) imaging. Three pigs (acute group) were sacrificed immediately following MR imaging. Seven pigs (chronic group) were survived for approximately 4 weeks and were reimaged with MR imaging immediately before sacrifice. Animals underwent necropsy and harvesting of the liver for histologic evaluation of the ablation zone. RAST ablations were performed with a 700-kHz therapy transducer. Student t tests were performed to compare prescribed versus achieved ablation diameter, difference of sphericity from 1, and change in ablation zone volume from acute to chronic imaging. Results Ablation zones had a sphericity index of 0.99 ± 0.01 (standard deviation) (P < .001 vs sphericity index of 1). Anteroposterior and transverse dimensions were not significantly different from prescribed (3.4 ± 0.7; P = .08 and 3.2 ± 0.8; P = .29, respectively). The craniocaudal dimension was significantly larger than prescribed (3.8 ± 1.1; P = .04), likely because of respiratory motion. The central ablation zone demonstrated complete cell destruction and a zone of partial necrosis. A fibrous capsule surrounded the ablation zone by 4 weeks. On 4-week follow-up images, ablation zone volumes decreased by 64% (P < .001). Conclusion RAST is capable of producing clinically relevant ablation zones in a noninvasive manner in a porcine model. © RSNA, 2018.

Using a transgenic cross, we evaluated features of preeclampsia, renal injury and the sFlt1/VEGF changes. Transgenic hAGT and hREN, or wild-type (WT) C57Bl/6 mice were cross-bred: female hAGT × male hREN for preeclampsia (PRE) model and female WT × male WT for pregnant controls (WTP). Samples were collected for plasma VEGF, sFlt1, and urine albumin. Blood pressures (BP) were monitored by telemetry. Vascular reactivity was investigated by wire myography. Kidneys and placenta were immunostained for sFlt1 and VEGF. Eleven PRE and 9 WTP mice were compared. PRE more frequently demonstrated albuminuria, glomerular endotheliosis (80% vs. 11%; P = 0.02), and placental necrosis (60% vs. 0%; P < 0.01). PRE group demonstrated declining BPs with advancing gestation. Plasma sFlt1 increased across pregnancy in PRE; VEGF did not vary. IHC demonstrated the presence of sFlt1 in glomeruli, lymphatics, and collecting tubules of PRE kidneys, suggesting excretion. VEGF immunostaining was increased specifically in the glomeruli of PRE kidneys. Placenta in PRE showed marked immunostaining for sFlt1. We conclude that this transgenic model of preeclampsia recapitulates human preeclamptic state with high fidelity, and that, vascular adaptation to pregnancy is suggested by declining BPs and reduced vascular response to PE and increased response to acetylcholine. Placental damage with resultant increased release of sFlt1, proteinuria, deficient spiral artery remodeling, and glomerular endotheliosis were observed in this model of PRE. Increased VEGF binding to glomerular endothelial cells in this model of PRE is similar to human PRE and leads us to hypothesize that renal injury in preeclampsia may be mediated through local VEGF.

BACKGROUND The purpose of this study was to explore the role of perinatal vitamin-D intake on the development and characterization of hyperkyphosis in a porcine model. METHODS The spines of 16 pigs were assessed at 9, 13, and 17 weeks of age with radiography and at 17 weeks with computed tomography (CT), magnetic resonance imaging (MRI), histology, and bone-density testing. An additional 169 pigs exposed to 1 of 3 maternal dietary vitamin-D levels from conception through the entire lactation period were fed 1 of 4 nursery diets supplying different levels of vitamin D, calcium, and phosphorus. When the animals were 13 weeks of age, upright lateral spinal radiography was performed with use of a custom porcine lift and sagittal Cobb angles were measured in triplicate to determine the degree of kyphosis in each pig. RESULTS The experimental animals had significantly greater kyphotic sagittal Cobb angles at all time points when compared with the control animals. These hyperkyphotic deformities demonstrated no significant differences in Hounsfield units, contained a slightly lower ash content (46.7% ± 1.1% compared with 50.9% ± 1.6%; p < 0.001), and demonstrated more physeal irregularities. Linear mixed model analysis of the measured kyphosis demonstrated that maternal diet had a greater effect on sagittal Cobb angle than did nursery diet and that postnatal supplementation did not completely eliminate the risk of hyperkyphosis. CONCLUSIONS Maternal diets deficient in vitamin D increased the development of hyperkyphosis in offspring in this model. CLINICAL RELEVANCE This study demonstrates that decreased maternal dietary vitamin-D intake during pregnancy increases the risk of spinal deformity in offspring. In addition, these data show the feasibility of generating a large-animal spinal-deformity model through dietary manipulation alone.

A central issue for adoptive cellular immunotherapy is overcoming immunosuppressive signals to achieve tumor clearance. While γδ T cells are known to be potent cytolytic effectors that can kill a variety of cancers, it is not clear whether they are inhibited by suppressive ligands expressed in tumor microenvironments. Here, we have used a powerful preclinical model where EBV infection drives the de novo generation of human B cell lymphomas in vivo, and autologous T lymphocytes are held in check by PD-1/CTLA-4-mediated inhibition. We show that a single dose of adoptively transferred Vδ2+ T cells has potent antitumor effects, even in the absence of checkpoint blockade or activating compounds. Vδ2+ T cell immunotherapy given within the first 5 days of EBV infection almost completely prevented the outgrowth of tumors. Vδ2+ T cell immunotherapy given more than 3 weeks after infection (after neoplastic transformation is evident) resulted in a dramatic reduction in tumor burden. The immunotherapeutic Vδ2+ T cells maintained low cell surface expression of PD-1 in vivo, and their recruitment to tumors was followed by a decrease in B cells expressing PD-L1 and PD-L2 inhibitory ligands. These results suggest that adoptively transferred PD-1lo Vδ2+ T cells circumvent the tumor checkpoint environment in vivo.

Chronic graft-versus-host disease (cGVHD) is a significant roadblock to long-term hematopoietic stem cell (HSC) transplantation success. Effective treatments for cGVHD have been difficult to develop, in part because of a paucity of animal models that recapitulate the multiorgan pathologies observed in clinical cGVHD. Here we present an analysis of the pathology that occurs in immunodeficient mice engrafted with human fetal HSCs and implanted with fragments of human fetal thymus and liver. Starting at time points generally later than 100 days post-transplantation, the mice developed signs of illness, including multiorgan cellular infiltrates containing human T cells, B cells, and macrophages; fibrosis in sites such as lungs and liver; and thickened skin with alopecia. Experimental manipulations that delayed or reduced the efficiency of the HSC engraftment did not affect the timing or progression of disease manifestations, suggesting that pathology in this model is driven more by factors associated with the engrafted human thymic organoid. Disease progression was typically accompanied by extensive fibrosis and degradation of the thymic organoid, and there was an inverse correlation of disease severity with the frequency of FoxP3+ thymocytes. Hence, the human thymic tissue may contribute T cells with pathogenic potential, but the generation of regulatory T cells in the thymic organoid may help to control these cells before pathology resembling cGVHD eventually develops. This model thus provides a new system to investigate disease pathophysiology relating to human thymic events and to evaluate treatment strategies to combat multiorgan fibrotic pathology produced by human immune cells.

Background The relationship between lung and joint inflammation in rheumatoid arthritis is poorly understood. Lung inflammation with resultant protein citrullination may trigger anti-citrullinated protein antibodies, inflammation, and arthritis. Alternatively, lung and joint inflammation may be two manifestations of a single underlying pathology. The lung has increased citrullination and TNF-α levels are high in rheumatoid arthritis; however, it is unknown if TNF-α can induce lung protein citrullination. The citrullinating enzyme peptidylarginine deiminase 4 (PAD4) exacerbates TNF-α-induced arthritis, but a role for PAD4 in lung citrullination and TNF-α-induced lung inflammation has not been explored. Our aim was to use TNF-α-overexpressing mice to clarify the intersection of TNF-α, citrullination, PAD4, arthritis, and lung inflammation. Methods Lung protein citrullination in wild-type mice, mice that overexpress TNF-α systemically (TNF+), TNF+PAD4+/+, and TNF+PAD4-/- mice was quantified by both gel electrophoresis using a citrulline probe and western blot. Hematoxylin and eosin (H&E)-stained lung sections from TNF+PAD4+/+ and TNF+PAD4-/- mice were scored for lung inflammation. H&E-stained ankle joint sections from mice that overexpress TNF-α only in the lungs were assessed for arthritis. Results TNF+ mice have increased lung protein citrullination. TNF+PAD4-/- mice do not have significantly reduced lung protein citrullination, but do have decreased lung inflammation compared to TNF+PAD4+/+ mice. Mice that overexpress TNF-α only in the lungs do not develop arthritis. Conclusions PAD4 exacerbates lung inflammation downstream of TNF-α without having a major role in generalized protein citrullination in inflamed lungs. Also, TNF-α-induced lung inflammation is not sufficient to drive murine arthritis. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-1068-0) contains supplementary material, which is available to authorized users.

Metabolic imaging of the relative amounts of reduced NADH and FAD and the microenvironment of these metabolic electron carriers can be used to noninvasively monitor changes in metabolism, which is one of the hallmarks of carcinogenesis. This study combines cellular redox ratio, NADH and FAD lifetime, and subcellular morphology imaging in three dimensions to identify intrinsic sources of metabolic and structural contrast in vivo at the earliest stages of cancer development. There was a significant ( P < 0.05) increase in the nuclear to cytoplasmic ratio (NCR) with depth within the epithelium in normal tissues; however, there was no significant change in NCR with depth in precancerous tissues. The redox ratio significantly decreased in the less differentiated basal epithelial cells compared with the more mature cells in the superficial layer of the normal stratified squamous epithelium, indicating an increase in metabolic activity in cells with increased NCR. However, the redox ratio was not significantly different between the superficial and basal cells in precancerous tissues. A significant decrease was observed in the contribution and lifetime of protein-bound NADH (averaged over the entire epithelium) in both low- and high-grade epithelial precancers compared with normal epithelial tissues. In addition, a significant increase in the protein-bound FAD lifetime and a decrease in the contribution of protein-bound FAD are observed in high-grade precancers only. Increased intracellular variability in the redox ratio, NADH, and FAD fluorescence lifetimes were observed in precancerous cells compared with normal cells.

Objective— To assess efficacy and toxicity of a capecitabine (CAP)-based regimen for preventing rejection of renal allografts in dog erythrocyte antigen (DEA)-mismatched mongrel dogs. Study Design— Prospective, pilot study. Animals— Eight healthy, unrelated, DEA mismatched, adult mongrel dogs. Methods— All dogs received CAP, starting at 50 mg/m2 PO b.i.d. 4 days preoperatively, increasing to 200 mg/m2 PO b.i.d. by the day of surgery. All dogs received cyclosporine-A (CsA) and prednisolone starting 2 days preoperatively. Standard heterotopic renal transplantation with native nephrectomy was performed. After 90 days, surviving dogs were euthanatized and histopathologic examination was performed. Results— Two of 8 dogs developed acute neurotoxicity leading to death or euthanasia within 5 days of surgery. For the 6 remaining dogs, there were no statistically significant changes in complete blood count or serum biochemical values. No opportunistic infections developed during the study period. Five of 6 dogs had no to minimal evidence of graft rejection. Two of 6 dogs developed superficial and pigmentary keratitis. Significant histopathologic findings in all dogs included mild lymphoplasmacytic gastroenteritis, steroid hepatopathy, and corneal epithelial thinning. One dog had moderate interstitial nephritis and pyelitis. Conclusions— In this experimental model, a CAP–CsA–prednisolone immunosuppressive regimen was effective in preventing rejection of allografts in DEA-mismatched dogs. Severe, unpredictable neurotoxicity and variable ocular toxicity significantly limit clinical applications at this time. Clinical Relevance— A CAP–CsA–prednisolone protocol is an effective, oral immunosuppressive regimen for prevention of allograft rejection in DEA-mismatched mongrel dogs. For clinical application, identification of patients susceptible to toxic side effects would be necessary.

Humans' closest living relatives are bonobos (Pan paniscus) and chimpanzees (Pan troglodytes), yet these great ape species differ considerably from each other in terms of social behavior. Bonobos are more tolerant of conspecifics in competitive contexts and often use sexual behavior to mediate social interactions. Chimpanzees more frequently employ aggression during conflicts and actively patrol territories between communities. Regulation of emotional responses is facilitated by the amygdala, which also modulates social decision-making, memory and attention. Amygdala responsiveness is further regulated by the neurotransmitter serotonin. We hypothesized that the amygdala of bonobos and chimpanzees would differ in its neuroanatomical organization and serotonergic innervation. We measured volumes of regions and the length density of serotonin transporter-containing axons in the whole amygdala and its lateral, basal, accessory basal and central nuclei. Results showed that accessory basal nucleus volume was larger in chimpanzees than in bonobos. Of particular note, the amygdala of bonobos had more than twice the density of serotonergic axons than chimpanzees, with the most pronounced differences in the basal and central nuclei. These findings suggest that variation in serotonergic innervation of the amygdala may contribute to mediating the remarkable differences in social behavior exhibited by bonobos and chimpanzees.

Prostatic acid phosphatase (PAP) is a prostate tumor antigen currently being investigated as a target antigen in several human vaccine trials, some with evidence of clinical benefit. We have previously demonstrated that plasmid DNA vaccines encoding either human or rat PAP can elicit antigen-specific cellular and humoral immunity in rat models. The current study was performed to determine the safety and potential immunological efficacy in rodents of large and repetitive doses of a GMP-grade plasmid DNA vaccine encoding human PAP, pTVG-HP. Fifty-four male Lewis rats were immunized intradermally at 2-week intervals with 100, 500, or 1,500 microg pTVG-HP with 5 microg recombinant rat GM-CSF protein given as a vaccine adjuvant. An additional 12 male Lewis rats served as controls with groups immunized with 1,500 microg of a parental DNA vector not encoding human PAP, and a group that received GM-CSF protein only without plasmid DNA. Groups of animals (n=3-6) were euthanized after two, four, or six immunizations with collections of tissues and blood for toxicity assessment and immunological analysis. No significant toxicities were observed in terms of animal weights, histopathology, hematological changes, or changes in serum chemistries. Six of fifty-four were found to have subtle evidence of possible renal toxicity, however these findings were not statistically different from control animals. The vaccine was found to be effective in eliciting PAP-specific CD4 and CD8 T cells, predominantly Th1 in type, in all immunized animals at all doses and numbers of immunizations. PAP-specific IgG were detected in a dose-dependent fashion, with titers increasing after multiple immunizations. These studies demonstrate that, in rats, immunization with the pTVG-HP vaccine is safe and effective in eliciting PAP-specific cellular and humoral immune responses. These findings support the further clinical evaluation of pTVG-HP in patients with prostate cancer.

Background and Objectives The first objective of this study was to evaluate the performance of fluorescence spectroscopy for diagnosing pre-cancers in stratified squamous epithelial tissues in vivo using two different probe geometries with (1) overlapping versus (2) non-overlapping illumination and collection areas on the tissue surface. Probe (1) and probe (2) are preferentially sensitive to the fluorescence originating from the tissue surface and sub-surface tissue depths, respectively. The second objective was to design a novel, angled illumination fiber-optic probe to maximally exploit the depth-dependent fluorescence properties of epithelial tissues. Study Design/Materials and Methods In the first study, spectra were measured from epithelial pre-cancers and normal tissues in the hamster cheek pouch and analyzed with a non-parametric classification algorithm. In the second study, Monte Carlo modeling was used to simulate fluorescence measurements from an epithelial tissue model with the angled illumination probe. Results An unbiased classification algorithm based on spectra measured with probes (1) and (2), classified pre-cancerous and normal tissues with 78 and 94% accuracy, respectively. The angled illumination probe design provides the capability to detect fluorescence from a wide range of tissue depths in an epithelial tissue model. Conclusions The first study demonstrates that fluorescence originating from sub-surface tissue depths (probe (2)) is more diagnostic than fluorescence originating from the tissue surface (probe (1)) in the hamster cheek pouch model. However in general, it is difficult to know a priori the optimal probe geometry for pre-cancer detection in a particular epithelial tissue model. The angled illumination probe provides the capability to measure tissue fluorescence selectively from different depths within epithelial tissues, thus obviating the need to select a single optimal probe design for the fluorescence-based diagnosis of epithelial pre-cancers. Lasers Surg. Med. 34:25–38, 2004. © 2004 Wiley-Liss, Inc.

To determine the level of in ovo methylmercury (MeHg) exposure that results in detrimental effects on fitness and survival of loon embryos and hatched chicks, we conducted a field study in which we injected eggs with various doses of MeHg on day 4 of incubation. Eggs were collected following about 23 days of natural incubation and artificially incubated to observe hatching. Reduced embryo survival was evident in eggs injected at a rate of ≥1.3 μg Hg/g wet-mass. When maternally deposited Hg and injected Hg were considered together, the median lethal concentration of Hg (LC(50)) was estimated to be 1.78 μg Hg/g wet-mass. Organ mass patterns from eggs of chicks injected at a rate of 2.9 μg Hg/g differed from that of controls and chicks from the 0.5 μg Hg/g treatment, largely related to a negative relation between yolk sac mass and egg mercury concentration. Chicks from eggs in the 2.9 μg Hg/g treatment were also less responsive to a frightening stimulus than controls and chicks from the 0.5 μg Hg/g treatment. We also found that the length of incubation period increased with increasing egg mercury concentration. Tissue Hg concentrations were strongly associated (r(2) ≥ 0.80) with egg Hg concentration.

Previously, dietary conjugated linoleic acid [(CLA), an equal mixture of cis-9, trans-11 (c9t11) and trans-10, cis-12 (t10c12) CLA isomers], was found to reduce inflammation in the murine collagen antibody-induced arthritis model, but less so in the murine collagen-induced arthritis (CIA) model, an arthritic model dependent upon acquired immunity. Because CLA is known to alter the acquired immune response, it was hypothesized that feeding CLA after the establishment of arthritis would reduce paw swelling in the CIA model. In this study, upon the establishment of arthritic symptoms, mice were randomized to the following dietary treatments: corn oil (CO) control (n = 6), 0.5% c9t11-CLA (n = 8), 0.5% t10c12-CLA (n = 6), or 1% combined CLA (1:1 c9t11:t10c12-CLA, n = 6). Paws were scored for severity of arthritis and measured for changes in thickness during an 84-d study period. Dietary c9t11- and combined-CLA similarly decreased the arthritic score (29%, P = 0.036, P = 0.049, respectively, when normalized to initial score) and paw thickness (0.11 mm, P = 0.027, P = 0.035, respectively) compared with CO. Dietary t10c12-CLA reduced the arthritic score (41%, P = 0.007 when normalized) and paw thickness (0.12 mm, P = 0.013) relative to CO. Reduced interleukin-1beta on d 7 and 21 for all CLA treatments (n = 3) relative to CO suggested that antiinflammatory effects of CLA isomers might work by common mechanisms of known pathways involved in chronic inflammation. In conclusion, dietary CLA reduced inflammation associated with CIA, and both c9t11-CLA and t10c12-CLA exhibited antiinflammatory effects.

When administered in overtly toxic doses to postweanling male rats, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces adverse effects on the reproductive system including a decrease in spermatogenesis. Because the male reproductive system may be particularly susceptible to toxic insult during the perinatal period, the effects of in utero and lactational TCDD exposure on its development were examined. Male rats born to dams given TCDD (0.064, 0.16, 0.40, or 1.0 μg/kg, po) or vehicle on Day 15 of gestation were evaluated at various stages of development; effects on spermatogenesis and male reproductive capability are reported herein. Testis, epididymis, and cauda epididymis weights were decreased in a dose-related fashion at 32, 49, 63, and 120 days of age, that is, when males were at the juvenile, pubertal, postpubertal, and mature stages of sexual development, respectively. When measured on Days 49, 63, and 120, daily sperm production by the testis was reduced at the highest maternal TCDD dose to 57–74% of the control rate. Cauda epididymal sperm reserves in 63- and 120-day-old males were decreased to as low as 25 and 44%, respectively, of control values, although the motility and morphology of these sperm appeared to be unaffected. The magnitude of the effects described above tended to lessen with time; nevertheless, the decreases in epididymis and cauda epididymis weights, daily sperm production, and cauda epididymal sperm number were statistically significant at the lowest maternal dose tested (0.064 μg TCDD/kg) on Day 120 and at most earlier times. To determine if in utero and lactational TCDD exposure also affects male reproductive capability, rats were mated at approximately 70 and 120 days of age with control females. Little if any effect on fertility was seen, and the survival and growth of offspring was unaffected. These results are not inconsistent with the pronounced reductions in daily sperm production and cauda epididymal sperm reserves caused by perinatal TCDD exposure since rats produce and ejaculate far more sperm than are required for normal fertility. The TCDD-induced reduction in spermatogenesis cannot be accounted for by concurrent effects on plasma follicle-stimulating hormone or androgen concentrations or by undernutrition. To investigate the nature of the spermatogenic lesion, leptotene spermatocyte to Sertoli cell ratios were determined. The finding that in utero and lactational TCDD exposure did not affect these ratios in 49-, 63-, and 120-day-old rats suggests that the decrease in spermatogenesis is caused by impaired division and/or increased attrition of cells during the conversion of leptotene spermatocytes to spermatozoa and/or by a reduction in Sertoli cell number. Regardless of mechanism, results from this study show that spermatogenesis is much more susceptible to TCDD when exposure occurs perinatally than when it follows weaning. The reduction in spermatogenesis occurs at doses of TCDD that are among the lowest reported to cause toxicity in the rat.

We conducted a dose-response laboratory study to quantify the level of exposure to dietary Hg, delivered as methylmercury chloride (CH3HgCl), that is associated with suppressed immune function in captive-reared common loon (Gavia immer) chicks. We used the phytohemagglutinin (PHA) skin test to assess T-lymphocyte function and the sheep red blood cell (SRBC) hemagglutination test to measure antibody-mediated immunity. The PHA stimulation index among chicks receiving dietary Hg treatment did not differ significantly from those of chicks on the control diet (p = 0.15). Total antibody (immunoglobulin [Ig] M [primary antibody] + IgG [secondary response]) production to the SRBC antigen in chicks treated with dietary methylmercury (MeHg), however, was suppressed (p = 0.04) relative to chicks on control diets. Analysis indicated suppression of total Ig production (p = 0.025 with comparisonwise alpha level = 0.017) between control and 0.4 microg Hg/g wet food intake treatment groups. Furthermore, the control group exhibited a higher degree of variability in antibody response compared to the Hg groups, suggesting that in addition to reducing the mean response, Hg treatment reduced the normal variation attributable to other biological factors. We observed bursal lymphoid depletion in chicks receiving the 1.2 microg Hg/g treatment (p = 0.017) and a marginally significant effect (p = 0.025) in chicks receiving the 0.4 microg Hg/g diet. These findings suggest that common loon chick immune systems may be compromised at an ecologically relevant dietary exposure concentration (0.4 microg Hg/g wet wt food intake). We also found that chicks hatched from eggs collected from low-pH lakes exhibited higher levels of lymphoid depletion in bursa tissue relative to chicks hatched from eggs collected from neutral-pH lakes.

Objectives We evaluated role of sFlt-1 and VEGF in a preeclampsia model driven by RAS activation. We hypothesized as transgenic crossbreeds develop preeclampsia, features of hypertension, proteinuria, kidney and placenta end-organ damage will occur concurrently with (1) sFlt1 rising higher in preeclamptic mice, (2) VEGF remaining high in preeclamptic mice but equivalent to controls; and, (3) preeclamptic kidneys demonstrating increased VEGF binding. Methods Transgenic hAGT and hREN, or wild type (WT) mice were used. Non-gravid females underwent telemetric transmitter implantation for blood pressure (BP) measurements. Mice were cross-bred: hAGT × hREN for RAS preeclamptic model and pregnant controls (WT × WT). Blood and urine collected on days (GD) 12, 15, and 18. Vascular reactivity in mesenteric arteries investigated by wire myography using KCl, acetylcholine (ACH), phenylephrine (PE). ELISA performed on urine for albumin and on plasma for VEGF and sFlt-1. Necropsies were performed GD 18-19. Kidneys and placenta were sectioned for HE and immunostain. Results 11 RAS pregnancies and 9 WT pregnancies (WTP) compared. 12,414 h of BP data analyzed. RAS mice demonstrated higher BP and proteinuria. RAS mice had glomerular endotheliosis (80% vs. 11%; p = 0.02), and placental necrosis (60% vs. 0%; p Conclusions While RAS model of preeclampsia recapitulates human preeclamptic state with high fidelity, renal injury may actually be mediated by increased VEGF binding. Disclosures J.M. Denney: None. C.E. Shaw: None. A. Gendron-Fitzpatrick: None. I. Bird: None. D. Shah: None.

This study characterizes the morphologic features and the endogenous fluorescence in the stratified squamous epithelia of the 7,12-dimethylbenz(a)anthracene-treated hamster cheek pouch model of carcinogenesis using multiphoton laser scanning microscopy (MPLSM). MPLSM allows high-resolution, three-dimensional image data to be collected deeper within thick tissue samples with reduced phototoxicity compared with single-photon imaging. Three-dimensional image stacks of normal (n = 13), precancerous (dysplasia, n = 12; carcinoma in situ, n = 9) and cancerous tissue [nonpapillary squamous cell carcinoma (SCC), n = 10, and papillary SCC, n = 7] sites in the hamster cheek pouch were collected in viable, unsectioned tissue biopsies at a two-photon excitation wavelength of 780 nm. Five features were quantified from the MPLSM images. These included nuclear density versus depth, keratin layer thickness, epithelial thickness, and the fluorescence per voxel in the keratin and epithelial layers. Statistically significant differences in all five features were found between normal and both precancerous and cancerous tissues. The only exception to this was a lack of statistically significant differences in the keratin fluorescence between normal tissues and papillary SCCs. Statistically significant differences were also observed in the epithelial thickness of dysplasia and carcinoma in situ, and in the keratin layer thickness of dysplasia and SCCs (both nonpapillary and papillary). This work clearly shows that three-dimensional images from MPLSM of endogenous tissue fluorescence can effectively distinguish between normal, precancerous, and cancerous epithelial tissues. This study provides the groundwork for further exploration into the application of multiphoton fluorescence endoscopy in a clinical setting.

Endogenous fluorescence offers promise for diagnosing oral cancers. This is the first study to use multiphoton imaging to characterize the spatial and depth distribution of endogenous fluorescence in neoplastic and non-neoplastic epithelial tissues.

Interferon regulatory factor 1-deficient (IRF-1−/−) mice infected with virulentBrucella abortus2308 at 5 × 105CFU developed acute hepatitis similar to many natural hosts but, unlike natural hosts, IRF-1−/−mice were unable to resolve infection and died. In contrast, IRF-1−/−mice survived when infected at 5 × 105CFU with several attenuatedBrucellastrains (S19, RB51, cbp, and cyd). The survival of infected IRF-1−/−mice is likely a function of the level of virulence of eachBrucellastrain and the extent of retained immunity. Further, these findings suggest that adaptive immunity may be important to the survival of IRF-1−/−mice since attenuatedBrucellastrains can protect IRF-1−/−mice against lethal challenge with virulentBrucella. Using the IRF-1−/−mouse model, the following set of criteria were identified to defineBrucellavirulence: (i) the day of death for 50% of mice infected with 5 × 105CFU ofBrucella, (ii) the extent of liver toxicity, and (iii) the minimum immunizing dose ofBrucellato protect against challenge with virulent S2308. Thus, IRF-1−/−mice are important to determining the level ofBrucellavirulence, to evaluatingBrucellamutants for attenuation, and to investigating adaptive immunity in brucellosis.

IFN-γ is a key cytokine controlling Brucella infection, and the diverse functions of this cytokine are mediated by IFN regulatory factors (IRFs) such as IRF-1, IRF-2, and IFN consensus sequence binding protein (ICSBP). However, the roles of these three IRFs in Brucella infection have not been investigated. The infection of each IRF-deficient mouse strain provides an opportunity to determine not only the significance of each IRF molecule but also the crucial immune components necessary for host defense during in vivo infection, because respective IRF-deficient mouse strains contain unique immunodeficient phenotypes. Brucella abortus S2308-infected IRF-1−/− mice were dead within 2 wk postinfection, while IRF-2−/− mice contained less splenic Brucella CFU than wild-type mice at the early stage of infection. Infected ICSBP−/− mice maintained a plateau of splenic Brucella CFU throughout the infection. Additional infection of IL-12p40-, NO synthase 2-, and gp91phox-deficient mice indicates that these immune components are crucial for Brucella immunity and may contribute to the susceptibility of IRF-1−/− and ICSBP−/− mice. Immunologic and histopathological analyses of infected IRF-1−/− mice indicate that the absence of IL-12p40 induction and serious hepatic damage are involved in the death of IRF-1−/− mice. These results indicate that 1) IRF-1 and ICSBP are essential transcriptional factors for IFN-γ-mediated protection against Brucella; 2) IL-12, reactive nitrogen intermediates, and reactive oxygen intermediates are crucial immune components against Brucella, and their absence may contribute to the susceptibility of IRF-1−/− and ICSBP−/− mice; and 3) hepatic damage caused by Brucella virulence contributes to the death of IRF-1−/− mice.

Recurrent urinary tract infections (UTIs) are a significant clinical problem for many women; however, host susceptibility factors have not been completely defined. The mouse model of induced UTI provides an experimental environment in which to identify specific host characteristics that are important in initial bacterial colonization of the urinary tract and in resolution of an infection. This study examined initial susceptibility, bacterial clearance, and host defense mechanisms during induction and resolution of Escherichia coli UTIs in genetically distinct strains of mice. Of the ten inbred strains tested, six (BALB/c, C3H/HeN, C57BL/6, DBA.1, DBA.2, and AKR) showed progressive resolution of bladder infections over a 14-day period. A constant, low-level bladder infection was observed in SWR and SJL mice. High bladder infection levels persisted over the 14-day study period in C3H/HeJ and C3H/OuJ mice. Kidney infection levels generally correlated with bladder infection levels, especially in C3H/HeJ and C3H/OuJ mice, the two most susceptible strains, in which infections became more severe with time after challenge. The degree of inflammation in bladder and kidneys, as well as antibody-forming cell responses, positively correlated with infection intensity in all strains except C3H/HeJ, which had minimal inflammation despite high infection levels. These results demonstrate two important aspects of host defense against UTI. First, the innate immune response to an infection in the bladder or kidneys consists primarily of local inflammation, which is followed by an adaptive response characterized in part by an antibody response to the infecting bacteria. Second, a UTI will be spontaneously resolved in most cases; however, in mice with specific genetic backgrounds, a UTI can persist for an extended length of time. The latter result strongly suggests that the presence or absence of specific host genes will determine how effectively an E. coli UTI will be resolved.

Host defense against bacterial urinary tract infections (UTI) includes both inflammatory and immune responses to infecting bacteria. The cellular events leading up to local inflammation are thought to be under genetic control and initiated by lipopolysaccharides (LPS) of gram-negative bacteria such as Escherichia coli. It has been previously reported that mice which lack functional Lps genes are more susceptible to induced E. coli UTI than mice with normal mitogenic responses to LPS. In contrast to these findings, data in this report demonstrate that LPS-responder and nonresponder C3H mouse strains are equally susceptible to E. coli UTI. When C3H/OuJ (Lps(n)/Lps(n)) and C3H/HeJ (Lps(d)/Lps(d)) were intravesically inoculated with equal numbers of uropathogenic E. coli organisms, neither strain was able to effectively resolve the induced UTI. The inability of C3H/OuJ mice to combat the infection was not due to an impaired response to LPS, nor could defect in the local inflammatory response be identified. The results indicate that factors other than LPS responsiveness are also important in determining hose resistance to UTI.

Sixteen wild Peromyscus leucopus, trapped for the establishment of a breeding colony, developed signs of neurological damage (trembling, incoordination, circling, head tilt, and lameness of the rear legs) 2–47 days after capture in southern Wisconsin. Spirochetes were cultured from the brain of 5/11 mice, and Borrelia burgdorferi was cultured from 1 brain. A spirochete was isolated from the bladder of 1 mouse. The spirochete was identified by fluorescent antibody staining with the monoclonal antibody specific for B. burgdorferi, H5332. Serum antibodies to the spirochete were found in 14/15 mice. Negative results were obtained in all tests for viruses and bacteria, including Listeria (2/2), Mycoplasma (2/2), mouse hepatitis virus (10/10), Theilers's encephalomyelitis virus (GD VII) (8/8), REO 3 virus (2/2), and lymphocytic choriomeningitis virus (4/4). There was no bacterial growth from brains cultured on eosin methylene blue or blood agar (3/3). Histologic lesions included nonsuppurative cellular infiltrates in the brain, kidney, liver, and lung. Three outbred Swiss-Webster mice were inoculated orally with a suspension of the brain in BSKII medium, and 3 were inoculated with unpassed B. burgdorferi cultured from the brain of a P. leucopus with motor dysfunction. Five of the inoculated mice developed antibody titers of 1:128; one mouse was positive at 1:256. Motor signs of neurologic damage developed in 3/6 mice 2–24 weeks post-inoculation, and B. burgdorferi was detected in the brains of 2 mice by isolation and by fluorescent antibody.

Interpretation of studies comparing the efficacy of different dietary fat sources in promoting 7,12-dimethylbenz[a]-anthracene (DMBA)-induced rat mammary tumorigenesis often ignores the fact that about 4% (wt/wt) linoleic acid (18:2n-6) is required for optimal tumor promotion. We therefore fed DMBA-intubated or placebo-intubated female, Sprague-Dawley rats 20% fat diets containing 18:2n-6 (wt/wt) from either high-linoleic safflower oil (SL, 14.6% 18:2n-6), high-oleic safflower oil (SO, 3.4% 18:2n-6), olive oil (OO, 1.1% 18:2n-6), or OO supplemented with 18:2n-6 (OL, 3.4% 18:2n-6) for 16 weeks. Results indicated that OO-fed rats had longer tumor-free time, fewer tumors per rat, and lower tumor incidence compared with SO and OL. Addition of 2.3% 18:2n-6 to OO enhanced tumor promotion (p less than 0.04); SL, SO, and OL demonstrated similar tumor-enhancement effect. About 74% of observed mammary tumors were adenocarcinomas; a greater number of tumors appeared in the thoracic and inguinal than in the cervical and abdominal regions irrespective of diet. These results indicate that once an optimal amount of linoleic acid is provided in the diet, oleic- or linoleic-rich oils have similar effects on promotion of mammary tumors in the rat.

We explored the use of diffuse reflectance spectroscopy in the ultraviolet-visible (UV-VIS) spectrum for the diagnosis of epithelial precancers and cancers in vivo. A physical model (Monte Carlo inverse model) and an empirical model (principal component analysis, (PCA)) based approach were compared for extracting diagnostic features from diffuse reflectance spectra measured in vivo from the dimethylbenz[alpha]anthracene-treated hamster cheek pouch model of oral carcinogenesis. These diagnostic features were input into a support vector machine algorithm to classify each tissue sample as normal (n=10) or neoplastic (dysplasia to carcinoma, n=10) and cross-validated using a leave one out method. There was a statistically significant decrease in the absorption and reduced scattering coefficient at 460 nm in neoplastic compared to normal tissues, and these two features provided 90% classification accuracy. The first two principal components extracted from PCA provided a classification accuracy of 95%. The first principal component was highly correlated with the wavelength-averaged reduced scattering coefficient. Although both methods show similar classification accuracy, the physical model provides insight into the physiological and structural features that discriminate between normal and neoplastic tissues and does not require a priori, a representative set of spectral data from which to derive the principal components.

Multiphoton fluorescence lifetime imaging microscopy (FLIM) is a noninvasive, cellular resolution, three-dimensional functional imaging technique. This study investigates the potential for in vivo pre-cancer diagnosis with metabolic imaging via multiphoton FLIM of the endogenous metabolic co-factor, NADH. The dimethylbenz[α]anthracene (DMBA)-treated hamster cheek pouch model of oral carcinogenesis and MCF10A cell monolayers were imaged using multiphoton FLIM at 780 nm excitation. The cytoplasm of normal hamster cheek pouch epithelial cells had short (0.29 ± 0.03 ns) and long lifetime components (2.03 ± 0.06 ns), attributed to free and protein-bound NADH, respectively. Low grade pre-cancers (mild to moderate dysplasia) and high grade pre-cancers (severe dysplasia and carcinoma in situ) were discriminated from normal tissues by their decreased protein-bound NADH lifetime (p

We developed and evaluated a surgical procedure for implanting intra-abdominal radiotransmitters with external whip antennas in captive mallards (Anas platyrhynchos). Transmitters were implanted in the abdominal cavity and the antennas exited through the caudal abdominal wall and skin. Birds with implanted transmitters developed mild to moderate localized air sac reactions. These reactions involved adhesions of the right anterior abdominal air sac to the liver with contractions around the transmitters and antenna catheters. The adhesions were reinforced by a proliferation of connective tissue and lined by multi-nucleated giant cells (foreign body reaction). Casual observation indicated that neither behavior nor activity of the birds was altered by the histological reaction to the transmitter implant. No increase in systemic lesions (particularly liver or kidney) could be correlated with the histological reactions. Our evaluations indicate that the procedure is a reliable method for radiomarking ducks and the technique has been successfully used in 2 field studies.

Fertilized eggs of ring-necked pheasants (Phasianus colchicus) were injected into the albumin or yolk with vehicle or graded doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (0.01, 0.1, 1, 10, 100, 1,000, 10,000, or 100,000 pg TCDD/g egg) on day 0 of embryonic development and toxicity was assessed in 1-d-old hatchlings and 28-d-old chicks. The most sensitive effect of in ovo TCDD exposure was induction of hepatic ethoxyresorufin-O-deethylase (EROD) activity in 1-d-old hatchlings. The ED50 for this response was 312 pg TCDD/g egg. Embryo mortality was the most sensitive sign of toxicity. The TCDD dose that caused 50% mortality above control (LD50) when injected into the egg albumin or yolk was 1,354 and 2,182 pg TCDD/g egg, respectively. At egg TCDD doses up to and including 1,000 pg TCDD/g egg, no effect was detected in 1-d-old hatchlings and 28-d-old chicks in body growth, organ weights, carcass morphometrics, incidence of edema, or incidence of histological alterations in the liver, spleen, heart, Bursa of Fabricius, or thymus. Egg TCDD doses as high as 1,000 pg TCDD/g egg also had no effect on cardiac morphometrics or incidence of cardiac malformations in 1-d-old hatchlings, or on antibody-mediated immunity in 28-d-old chicks. We conclude that embryo mortality is the most sensitive sign of TCDD toxicity in the ring-necked pheasant following in ovo exposure. The ring-necked pheasant embryo is less sensitive than the chicken (Gallus domesticus) embryo and more sensitive than the eastern bluebird (Sialia sialis) embryo to TCDD toxicity.