Genetic background of juniper (Juniperus spp.) consumption predicted by fecal near-infrared spectroscopy in divergently selected goats raised in harsh rangeland environments.

BACKGROUND: Junipers (Juniperus spp.) are woody native, invasive plants that have caused encroachment problems in the U.S. western rangelands, decreasing forage productivity and biodiversity. A potential solution to this issue is using goats in targeted grazing programs. However, junipers, which grow in dry and harsh environmental conditions, use chemical defense mechanisms to deter herbivores. Therefore, genetically selecting goats for increased juniper consumption is of great interest for regenerative rangeland management. In this context, the primary objectives of this study were to: 1) estimate variance components and genetic parameters for predicted juniper consumption in divergently selected Angora (ANG) and composite Boer x Spanish (BS) goat populations grazing on Western U.S. rangelands; and 2) to identify genomic regions, candidate genes, and biological pathways associated with juniper consumption in these goat populations. RESULTS: The average juniper consumption was 22.4% (± 18.7%) and 7.01% (± 12.1%) in the BS and ANG populations, respectively. The heritability estimates (realized heritability within parenthesis) for juniper consumption were 0.43 ± 0.02 (0.34 ± 0.06) and 0.19 ± 0.03 (0.13 ± 0.03) in BS and ANG, respectively, indicating that juniper consumption can be increased through genetic selection. The repeatability values of predicted juniper consumption were 0.45 for BS and 0.28 for ANG. A total of 571 significant SNP located within or close to 231 genes in BS, and 116 SNP related to 183 genes in ANG were identified based on the genome-wide association analyses. These genes are primarily associated with biological pathways and gene ontology terms related to olfactory receptors, intestinal absorption, and immunity response. CONCLUSIONS: These findings suggest that juniper consumption is a heritable trait of polygenic inheritance influenced by multiple genes of small effects. The genetic parameters calculated indicate that juniper consumption can be genetically improved in both goat populations.

Woody plant encroachment modifies carbonate bedrock: field evidence for enhanced weathering and permeability.

Little is known about the effects of woody plant encroachment-a recent but pervasive phenomenon-on the hydraulic properties of bedrock substrates. Recent work using stream solute concentrations paired with weathering models suggests that woody plant encroachment accelerates limestone weathering. In this field study, we evaluate this hypothesis by examining bedrock in the Edwards Plateau, an extensive karst landscape in Central Texas. We compared a site that has been heavily encroached by woody plants (mainly Quercus fusiformis and Juniperus ashei), with an adjacent site that has been maintained free of encroachment for the past eight decades. Both sites share the same bedrock, as confirmed by trenching, and originally had very few trees, which enabled us to evaluate how encroachment impacted the evolution of hydraulic properties over a period of no more than 80 years. Using in situ permeability tests in boreholes drilled into the weathered bedrock, we found that the mean saturated hydraulic conductivity of the bedrock was higher-by an order of magnitude-beneath woody plants than in the areas where woody plants have been continuously suppressed. Additionally, woody plant encroachment was associated with greater regolith thickness, greater plant rooting depths, significantly lower rock hardness, and a 24-44% increase in limestone matrix porosity. These findings are strong indicators that woody plant encroachment enhances bedrock weathering, thereby amplifying its permeability-a cycle of mutual reinforcement with the potential for substantial changes within a few decades. Given the importance of shallow bedrock for ecohydrological and biogeochemical processes, the broader impacts of woody plant encroachment on weathering rates and permeability warrant further investigation.

Selecting goats for juniper consumption did not improve their liver Phase I detoxification.

A sodium pentobarbital-induced sleep time study was conducted on 15 adult intact male Boer × Spanish goats selected for high (J+, n = 7) or low (J-, n = 8) juniper consumption (estimated breeding values of 13.1 ± 1.0 and -14.3 ± 0.8, respectively; mean ± standard deviation). Pentobarbital sleep time is an in vivo assay of Phase I hepatic metabolism that can be induced by exposure to barbiturates and monoterpenes. Monoterpenes and pentobarbital are initially oxidized by this pathway; thus, we hypothesized that J+ goats would have shorter sleep times than J- goats. Time to the righting reflex after pentobarbital-induced sleep was measured in all goats following a minimum period of 21 d on three different diets: 1) grazing juniper-infested rangeland (JIR), 2) forage diet with no monoterpenes (M0), and 3) forage diet with 8 g/kg added monoterpenes from camphor, sabinene, and α-pinene in a w/w ratio of 5:4:1 (M+). Fecal samples from the JIR diet were analyzed with near-infrared spectroscopy for the percentage of juniper in the diet. Fecal samples from the JIR and M+ diets were analyzed for camphor and sabinene concentrations. The percentage of juniper in the diet of J+ goats grazing rangelands was greater (P = 0.001) than J- goats (31.1% and 18.6%, respectively). Sleep time did not differ between selection lines (P = 0.36). However, the sleep time of the goats fed M+ diet was 26 min shorter (P < 0.001) than JIR or M0 diets, which were equal. The concentration of camphor and sabinene in the feces was higher (P < 0.001) for goats on the M+ diet than on the JIR diet. There were no differences between selection lines in the serum enzymes indicative of liver disease (aspartate aminotransferase, bilirubin, gamma-glutamyl transferase, and glutamate dehydrogenase; P > 0.12), and all treatment means were within the reference interval. Selecting goats for juniper consumption did not affect the Phase I detoxification system, and several alternative hypotheses for differences in juniper consumption between J+ and J- goats are discussed.

Juniper is an encroaching woody plant with high levels of essential oils and condensed tannins that can limit its consumption by herbivores. Goats were divergently selected for 15 yr to increase or decrease their juniper consumption. This study was conducted to determine if a physiological pathway for metabolism of essential oils differed between high and low juniper-consuming goat lines. The metabolic pathway for the elimination of essential oils is similar to that of the barbiturate pentobarbital. A pentobarbital-induced sleep time was used to detect differences in detoxification rates between the divergent goat lines selectively bred for either a high or low percentage of juniper in their diet. We hypothesized that high juniper-consuming goats would have shorter sleep times, indicating their detoxification pathway was more active. However, there was no difference between these lines. Additionally, there were no differences between the selection lines in blood metabolites that indicate liver tissue damage or liver weights. Therefore, higher dietary juniper preference may be associated with other detoxification mechanisms, may not be limited by essential oils, or may be a socially facilitated learned behavior.

Efficacy and Safety of Nivolumab Plus Ipilimumab vs Nivolumab Alone for Treatment of Recurrent or Metastatic Squamous Cell Carcinoma of the Head and Neck: The Phase 2 CheckMate 714 Randomized Clinical Trial.

Importance: There remains an unmet need to improve clinical outcomes in patients with recurrent or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN). Objective: To evaluate clinical benefit of first-line nivolumab plus ipilimumab vs nivolumab alone in patients with R/M SCCHN. Design, Setting, and Participants: The CheckMate 714, double-blind, phase 2 randomized clinical trial was conducted at 83 sites in 21 countries between October 20, 2016, and January 23, 2019. Eligible participants were aged 18 years or older and had platinum-refractory or platinum-eligible R/M SCCHN and no prior systemic therapy for R/M disease. Data were analyzed from October 20, 2016 (first patient, first visit), to March 8, 2019 (primary database lock), and April 6, 2020 (overall survival database lock). Interventions: Patients were randomized 2:1 to receive nivolumab (3 mg/kg intravenously [IV] every 2 weeks) plus ipilimumab (1 mg/kg IV every 6 weeks) or nivolumab (3 mg/kg IV every 2 weeks) plus placebo for up to 2 years or until disease progression, unacceptable toxic effects, or consent withdrawal. Main Outcomes and Measures: The primary end points were objective response rate (ORR) and duration of response between treatment arms by blinded independent central review in the population with platinum-refractory R/M SCCHN. Exploratory end points included safety. Results: Of 425 included patients, 241 (56.7%; median age, 59 [range, 24-82] years; 194 males [80.5%]) had platinum-refractory disease (nivolumab plus ipilimumab, n = 159; nivolumab, n = 82) and 184 (43.3%; median age, 62 [range, 33-88] years; 152 males [82.6%]) had platinum-eligible disease (nivolumab plus ipilimumab, n = 123; nivolumab, n = 61). At primary database lock, the ORR in the population with platinum-refractory disease was 13.2% (95% CI, 8.4%-19.5%) with nivolumab plus ipilimumab vs 18.3% (95% CI, 10.6%-28.4%) with nivolumab (odds ratio [OR], 0.68; 95.5% CI, 0.33-1.43; P = .29). Median duration of response for nivolumab plus ipilimumab was not reached (NR) (95% CI, 11.0 months to NR) vs 11.1 months (95% CI, 4.1 months to NR) for nivolumab. In the population with platinum-eligible disease, the ORR was 20.3% (95% CI, 13.6%-28.5%) with nivolumab plus ipilimumab vs 29.5% (95% CI, 18.5%-42.6%) with nivolumab. The rates of grade 3 or 4 treatment-related adverse events with nivolumab plus ipilimumab vs nivolumab were 15.8% (25 of 158) vs 14.6% (12 of 82) in the population with platinum-refractory disease and 24.6% (30 of 122) vs 13.1% (8 of 61) in the population with platinum-eligible disease. Conclusions and Relevance: The CheckMate 714 randomized clinical trial did not meet its primary end point of ORR benefit with first-line nivolumab plus ipilimumab vs nivolumab alone in platinum-refractory R/M SCCHN. Nivolumab plus ipilimumab was associated with an acceptable safety profile. Research to identify patient subpopulations in R/M SCCHN that would benefit from nivolumab plus ipilimumab over nivolumab monotherapy is warranted. Trial Registration: ClinicalTrials.gov Identifier: NCT02823574.

Impact of camphor on the in vitro mixed ruminal microorganism fermentation from goats selected for consumption of low and high levels of Juniperus spp. 1.

Encroaching plant species in West Texas continues to significantly reduce livestock production capacity and cause an imbalance between plant and livestock ecology. Juniperus ashei and Juniperus pinchotii are encroaching species rarely used by browsing animals, mainly due to phytochemical defenses. Juniperus spp. contain large concentrations of monoterpenes in their essential oil profiles to deter herbivory. Since 2003, two divergent bloodlines of meat goats have been selected to consume low or high (LJC or HJC) amounts of juniper foliage, through screening fecal samples with near-infrared reflectance spectroscopy. However, it remains unclear whether HJC goats expressed a physiological ability to consume a greater amount of juniper or if they were colonized by a ruminal microbial population that could detoxify juniper phytochemicals. Therefore, this study aimed to investigate the impact of 0.00 and 1.97 mM of camphor on the mixed ruminal microorganism fermentation after 0, 1, 2, 4, 12, and 24 h of incubation. Five LJC and five HJC goats were fed a juniper-free diet (n = 10) and five LJC and five HJC goats (n = 10), were fed a diet containing 30% fresh J. ashei leaves for 21 days prior to ruminal fluid collection. In vitro fermentations used LJC and HJC, ruminal fluid inoculum was added (33% v/v) to anoxic media in sealed Balch tubes. Total short-chain fatty acid (SCFA) production and acetate to propionate ratio were increased (P < 0.05), but there was no effect on pH (6.56 ± 0.09). Goats that received the juniper-free diet had higher (P < 0.05) SCFA production than juniper-containing diets. There was no consistent difference in LJC and HJC microbial fermentation end products caused by the addition of 1.97 mM of camphor, and goats receiving a juniper-free diet consistently generated more SCFAs in the presence of 1.97 mM of camphor. Furthermore, bloodline differences in juniper consumption were likely related to physiological adaption capacities within the animal and not a ruminal microbial detoxification advantage.

Impact of concentrations of camphor on the in vitro mixed ruminal microorganism fermentation from goats selected for consumption of low and high levels of Juniperus spp.-2.

The microbial population in the gastrointestinal tract of ruminant animals aids in the utilization of forages with high levels of secondary plant compounds. Two divergent bloodlines of meat goats have been selected by screening fecal samples with near-infrared reflectance spectroscopy to assess the goat's consumption of high or low levels of Juniperus sp. leaves containing several monoterpenes, including camphor. The mechanism by which these goats can consume greater concentrations of Juniperus spp. leaves than their counterparts is unclear, and therefore, this study was designed to determine if differences existed between the ruminal microbial populations of the low and high juniper-consuming bloodlines (LJC vs. HJC) by analyzing their ruminal microbiota and fermentation end products. In the present study, concentrations (0.00, 0.5, 0.99, 1.97, or 5.91 mM) of camphor were added to mixed ruminal microorganism fermentation. Five LJC and five HJC goats were fed a juniper-free diet (n = 10), and five LJC and five HJC goats (n = 10) were fed a diet that contained 30% fresh Juniperus ashei leaves for 21 d prior to ruminal fluid collection. In vitro fermentations used LJC and HJC, ruminal fluid inoculum added (33% v/v) to anoxic media in sealed Balch tubes. Camphor increased (P < 0.05) total short-chain fatty acid (SCFA) concentrations for all but one experimental group. Between the main dietary and bloodline goat effects, the diet was significant for all SCFA results except butyrate. In contrast, bloodline was only significant for acetate and butyrate molar proportions. Rumen fluid from juniper-free-fed goats exhibited greater concentrations of Ruminococcaceae, whereas juniper-fed goats contained more Coriobacteriaceae. Results demonstrated that mixed ruminal microorganisms fermentations from HJC goats did not produce greater concentrations of SCFAs or have the ability to degrade camphor at a higher rate than did that from LJC goats. Results suggest that camphor tolerance from J. ashei, was related to hepatic catabolic mechanisms instead of ruminal microbial degradation; however, further in vivo work is warranted.

Nature, nurture, and vegetation management: Studies with sheep and goats.

Diet selection and preference by grazing animals are determined by genetic and environmental factors (i.e., nature and nurture) that interact and affect their efficacy for managing vegetation as targeted grazers. The effect of rearing environment on the consumption of leafy spurge by sheep and goats was investigated. We hypothesized that although rearing environment will affect the preference for chemically defended plants ultimately, the inherent ability to detoxify or eliminate phytotoxins will limit an animal's preference for them. The objective of this study was to determine if sheep would consume more of the invasive weed leafy spurge (Euphorbia esula) if they were raised by goat compared to sheep raised by sheep and goat raised by goat. Sheep were raised on leafy spurge-infested pastures by either their ewe (S) or a goat doe (FS) on which they were fostered within 24 hours of birth and parturition of lamb and doe, respectively. Does that fostered lambs also raised their own goat offspring (G) such that the same doe raised the FS and G animals. The rearing environment's effect on leafy spurge consumption was tested the following growing season by simultaneously grazing all animals on the same leafy spurge-infested rangeland and estimating percentage leafy spurge in their diet with either fecal near-infrared spectroscopy (f.NIR) or bite count. Goats consumed more leafy spurge as determined by either f.NIR (62.8%, P < 0.06) or bite count (71.9%, P < 0.01) than FS (35.2 % f.NIR, 39.3% bite count) or S (10.1 % f.NIR, 18.2% bite count). The FS consumed over twice as much leafy spurge as S and were numerically intermediate to G and S for leafy spurge consumption but not significantly different from the S sheep, most likely because one FS sheep did not eat leafy spurge during the evaluation period. Because leafy spurge is aversive to sheep but not goats, higher leafy spurge consumption by FS sheep is hypothesized to result from inoculation of their rumen microbes with microbes from the does capable of denaturing aversive phytotoxins in leafy spurge. The higher consumption of leafy spurge by G compared to FS shows that genetically determined physiological differences influence an animal's ability to ameliorate phytotoxins and determine the upper limit of an animal's preference for a chemically defended plant. It also indicated that in addition to the animal's genome, the genome of an animal's microbiome, which the mother may influence, can play an important role in diet selection.

Galectin-9 expression defines exhausted T cells and impaired cytotoxic NK cells in patients with virus-associated solid tumors.

BACKGROUND: We have previously reported that the upregulation of galectin-9 (Gal-9) on CD4+ and CD8+ T cells in HIV patients was associated with impaired T cell effector functions. Gal-9 is a ligand for T cell immunoglobulin and mucin domain-3, and its expression on T cells in cancer has not been investigated. Therefore, we aimed to investigate the expression level and effects of Gal-9 on T cell functions in patients with virus-associated solid tumors (VASTs). METHODS: 40 patients with VASTs through a non-randomized and biomarker-driven phase II LATENT trial were investigated. Peripheral blood mononuclear cells and tumor biopsies were obtained and subjected to immunophenotyping. In this trial, the effects of oral valproate and avelumab (anti-PD-L1) was investigated in regards to the expression of Gal-9 on T cells. RESULTS: We report the upregulation of Gal-9 expression by peripheral and tumor-infiltrating CD4+ and CD8+ T lymphocytes in patients with VASTs. Our results indicate that Gal-9 expression is associated with dysfunctional T cell effector functions in the periphery and tumor microenvironment (TME). Coexpression of Gal-9 with PD-1 or T cell immunoglobulin and ITIM domain (TIGIT) exhibited a synergistic inhibitory effect and enhanced an exhausted T cell phenotype. Besides, responding patients to treatment had lower Gal-9 mRNA expression in the TME. Translocation of Gal-9 from the cytosol to the cell membrane of T cells following stimulation suggests persistent T cell receptor (TCR) stimulation as a potential contributing factor in Gal-9 upregulation in patients with VASTs. Moreover, partial colocalization of Gal-9 with CD3 on T cells likely impacts the initiation of signal transduction via TCR as shown by the upregulation of ZAP70 in Gal-9+ T cells. Also, we found an expansion of Gal-9+ but not TIGIT+ NK cells in patients with VASTs; however, dichotomous to TIGIT+ NK cells, Gal-9+ NK cells exhibited impaired cytotoxic molecules but higher Interferon gamma (IFN-γ) expression. CONCLUSION: Our data indicate that higher Gal-9-expressing CD8+ T cells were associated with poor prognosis following immunotherapy with anti-Programmed death-ligand 1 (PD-L1) (avelumab) in our patients' cohort. Therefore, for the very first time to our knowledge, we report Gal-9 as a novel marker of T cell exhaustion and the potential target of immunotherapy in patients with VASTs.

Efficacy and safety of avelumab treatment in patients with metastatic Merkel cell carcinoma: experience from a global expanded access program.

BACKGROUND: Avelumab, a human anti-programmed death-ligand 1 immunoglobulin G1 monoclonal antibody, showed favorable efficacy and safety in patients with metastatic Merkel cell carcinoma (mMCC) in the phase II JAVELIN Merkel 200 trial, leading to approval in multiple countries. We describe real-world experience with avelumab in patients with mMCC from an expanded access program. METHODS: Eligible patients had mMCC and progressive disease during or after chemotherapy or were ineligible for chemotherapy or clinical trial participation. Patients received an initial 3-month supply of avelumab (administered as 10 mg/kg intravenously every 2 weeks until progressive disease or unacceptable toxicity); resupply was allowed following complete response, partial response, stable disease, or clinical benefit per physician assessment. RESULTS: Between December 15, 2015, and March 4, 2019, 558 of 620 requests from 38 countries were medically approved, and 494 patients received avelumab. Among 240 evaluable patients, the objective response rate was 46.7% (complete response in 22.9%, including 3 of 16 potentially immunocompromised patients), and the disease control rate was 71.2%. The median duration of treatment in evaluable patients with response was 7.9 months (range, 1.0-41.7) overall and 5.2 months (range, 3.0-13.9) in immunocompromised patients. No new safety signals were identified. The expanded access program closed for new requests on December 31, 2018, as required after regulatory approval; benefitting patients continued to receive avelumab. CONCLUSIONS: The avelumab expanded access program for patients with mMCC demonstrated efficacy and safety in a real-world setting, consistent with the results from JAVELIN Merkel 200, and provided a treatment for patients with limited options.

Authorship conflicts: a study of awareness of authorship criteria among academic plastic surgeons.

BACKGROUND: Authorship is an important yardstick in academic medicine. The purpose of this study was to assess the prevalence of authorship conflicts among academic plastic surgeons and determine any change in authorship awareness over an 8-year period. METHODS: In 2003, members of the American Association of Plastic Surgeons were surveyed using an anonymous, 15-item, one-page questionnaire. In 2011, members were resurveyed using a similar questionnaire. In both surveys, nonresponders were contacted by telephone at 2 weeks to encourage response. RESULTS: The authors obtained a response rate of 80.4 percent (258 of 321) in 2003 and 81.6 percent (486 of 595) in 2011. In both cohorts, one-third of respondents felt that they had not been appropriately acknowledged as authors at some point during their career. Furthermore, in 2003, 29 percent of respondents admitted to being involved in a dispute with a colleague over authorship issues. This had decreased slightly to 22 percent by 2011. Interestingly, 64 percent of respondents in 2003, compared with only 37 percent of respondents in 2011, stated that they had included someone as an author who had not fulfilled any authorship criteria. In 2003, only 16 percent of respondents were aware of any journal authorship criteria. This had increased to 59 percent by 2011. CONCLUSIONS: The authors found an increase in awareness of authorship criteria among academic plastic surgeons in 2011 compared with those in 2003. In addition, academic plastic surgeons surveyed more recently reported more rigorous justification for including individuals as authors, supporting a trend toward increasing transparency and accountability.

Development of a contagious ecthyma vaccine for goats.

OBJECTIVE: To identify a strain of contagious ecthyma virus from goats that possesses the appropriate characteristics for an effective vaccine for goats. ANIMALS: 25 goat kids used for vaccine development and 100 goat kids used for evaluation of vaccine efficacy. PROCEDURES: 5 strains of contagious ecthyma virus were tested in a vaccination-challenge study to identify the best strain to be the seed strain for a contagious ecthyma vaccine. The vaccine derived from the chosen viral stain was tested at 2 concentrations for efficacy in a vaccination-challenge study. RESULTS: 2 of 5 viral strains induced moderate to severe scabs following infection, and 3 viral strains protected the goats from wild-type virus challenge following vaccination. Viral strain 47CE was selected as the seed source for the production of a contagious ecthyma vaccine because of the larger vaccine-to-challenge scab formation ratio. Vaccine 47CE protected all goat kids (48/48) following challenge with the wild-type contagious ecthyma virus; all goat kids (32/32) in the control group had scab formation following challenge with the wild-type contagious ecthyma virus, which indicated no protection following administration of vaccine diluent. CONCLUSIONS AND CLINICAL RELEVANCE: A vaccine containing a caprine strain of contagious ecthyma virus used in goats appeared to provide the characteristics needed for an effective vaccine, including good scab production and protection from wild-type infection. This vaccine may potentially provide better protection for goats from contagious ecthyma than currently available vaccines labeled for sheep.

Epithelial barrier disruption allows nondisease-causing bacteria to initiate and sustain IBD in the IL-10 gene-deficient mouse.

BACKGROUND: In the IL-10 gene-deficient mouse model, development of intestinal inflammation is associated with a defect in epithelial barrier integrity that is thought to allow sufficient passage of bacteria or bacterial antigens to initiate a mucosal immune response. Microbial monoassociation experiments into axenic animals have shown that some, but not all, endogenous bacteria will initiate an intestinal inflammatory response. For instance, Bacteroides vulgatus does not initiate intestinal inflammation in axenic IL-10 gene-deficient mice. We investigated whether B. vulgatus requires concomitant disruption of the intestinal epithelial barrier integrity in order to initiate an inflammatory response. METHODS: We first identified a dose of the indomethacin that would cause a primary disruption of the epithelial barrier without causing intestinal inflammation. IL-10 axenic mice were then administered this dose of indomethacin in their drinking water for 7 days and concomitantly monoassociated, by oral gavage, with B. vulgatus. RESULTS: Indomethacin treatment (2 microg/g/d) for 7 days resulted in disruption of epithelial barrier integrity, but it caused neither a systemic inflammatory response nor a mucosal inflammatory response in the colon or cecum. Monoassociation with B. vulgatus alone did not lead to a mucosal inflammatory response, despite a measurable systemic response. In contrast, administration of indomethacin plus B. vulgatus-monoassociation resulted in a marked intestinal inflammatory response in colon and cecum. CONCLUSIONS: Our data show that, in a genetically predisposed animal model, the nondisease-causing endogenous bacteria, B. vulgatus, is able to cause an intestinal inflammatory response provided that disruption of the intestinal epithelial barrier has occurred.

Bacterial antigens alone can influence intestinal barrier integrity, but live bacteria are required for initiation of intestinal inflammation and injury.

Intestinal flora plays a critical role in the initiation and perpetuation of inflammatory bowel disease. This study examined whether live fecal bacteria were necessary for the initiation of this inflammatory response or whether sterile fecal material would provoke a similar response. Three preparations of fecal material were prepared: (1) a slurry of live fecal bacteria, (2) a sterile lysate of bacterial antigens, and (3) a sterile filtrate of fecal water. Each preparation was introduced via gastric gavage into the intestines of axenic interleukin-10 gene-deficient mice genetically predisposed to develop inflammatory bowel disease. Intestinal barrier integrity and degrees of mucosal and systemic inflammations were determined for each preparation group. Intestinal barrier integrity, as determined by mannitol transmural flux, was altered by both live fecal bacterial and sterile lysates of bacterial antigens, although it was not altered by sterile filtrates of fecal water. However, only live fecal bacteria initiated mucosal inflammation and injury and a systemic immune response. Fecal bacterial antigens in the presence of live bacteria and sterile fecal bacterial antigens have different effects on the initiation and perpetuation of intestinal inflammation.

Bioproduction of conjugated linoleic acid by probiotic bacteria occurs in vitro and in vivo in mice.

Probiotics have been shown to reduce the incidence of colon cancer in animal models. The mechanisms responsible for this activity are poorly defined. Conjugated linoleic acids (CLA) are a group of isomers of linoleic acid (LA) possessing anti-inflammatory and anticarcinogenic properties, which can be produced from LA by certain bacterial strains. In this study, the ability of probiotic bacteria to exert anticarcinogenic effects through the production of CLA was assessed. Incubation of probiotic bacteria (VSL3, Lactobacillus acidophilus, L. bulgaricus, L. casei, L. plantarum, Bifidobacterium breve, B. infantis, B. longum, and Streptococcus thermophilus) in the presence of LA yielded CLA production as measured by gas chromatography. Conditioned medium, containing probiotic-produced CLA, reduced viability and induced apoptosis of HT-29 and Caco-2 cells, as assessed by MTT assay and DNA laddering, respectively. Western blotting demonstrated an increased expression of PPARgamma in cells treated with conditioned medium compared with LA alone. Incubation of murine feces with LA after administering VSL3 yielded 100-fold more CLA than feces collected prior to VSL3 feeding. This study supports a role for supplemental probiotics as a strategy both for attenuating inflammation and for preventing colon cancer.

AMP-activated protein kinase is a positive regulator of poly(ADP-ribose) polymerase.

AMPK acts as a cellular fuel gauge and responds to decreased cellular energy status by inhibiting ATP-consuming pathways and increasing ATP-synthesis. The aim of this study was to examine the role of AMPK in modulating poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in maintaining chromatin structure and DNA repair. HT-29 cells infected with constitutively active AMPK demonstrated increased PARP automodification and an increase in bioNAD incorporation. AMPK and PARP co-immunoprecipitated under basal conditions and in response to H(2)O(2), suggesting a physical interaction under both resting and stress-induced conditions. Incubation of PARP with purified AMPK resulted in the phosphorylation of PARP; and the inclusion of AMP as an AMPK activator potentiated PARP phosphorylation. Using immobilized PARP, the incorporation of bioNAD by PARP was dramatically increased following the addition of AMPK. These data suggest a novel role for AMPK in regulating PARP activity through a direct interaction involving phosphorylation.

Serum amyloid A activates NF-kappaB and proinflammatory gene expression in human and murine intestinal epithelial cells.

Serum amyloid A (SAA) is an acute-phase protein whose levels positively correlate with disease activity in inflammatory bowel diseases. In this study we investigated the impact of SAA on NF-kappaB signaling and proinflammatory gene expression in intestinal epithelial cells (IEC). Human HT-29 and Caco-2 monolayers were stimulated with recombinant SAA and NF-kappaB activation/NF-kappaB-dependent gene expression measured. Adenoviral dominant negative mutants IkappaB-alpha (Ad5IkappaBAA) were utilized to determine the contribution of NF-kappaB signaling pathway to SAA-dependent gene expression. Intestinal explant and primary IEC derived from kappaB-EGFP transgenic mice were exposed to SAA and NF-kappaB-dependent enhanced green fluorescent protein (EGFP) fluorescence measured. SAA induced IkappaB-alpha degradation, RelA serine 536 (S536) phosphorylation, NF-kappaB transcriptional activity, RelA recruitment to the IL-8 gene promoter and endogenous gene expression (IL-8, COX-2) in HT-29 cells. Further, Ad5IkappaBAA abrogated SAA-induced RelA nuclear translocation, NF-kappaB transcriptional activity and IL-8 gene expression. SAA-dependent IL-8 gene expression required activation of the MAPK ERK, p38 and JNK in HT-29 cells. Finally, SAA induced EGFP expression in intestinal explants isolated from kappaB-EGFP transgenic mice and enhanced RelA and IkappaBalpha phosphorylation in primary IEC. This indicates that SAA potentially participate in the inflammatory process by virtue of its ability to activate proinflammatory signaling in IEC.

Maternal pulmonary adenocarcinoma metastatic to the fetus: first recorded case report and literature review.

We report the first maternal pulmonary adenocarcinoma metastatic to the fetus as well as an updated literature review. Review of the literature revealed that there have been only 67 cases of maternal malignancy metastatic to the products of conception. These were mostly malignant melanoma and hematopoietic tumors. A 46-year old multiparous woman with metastatic pulmonary adenocarcinoma, diagnosed at 23 weeks gestation, delivered a male infant who appeared normal at birth. The mother died 2 days after delivery. The child developed multiple scalp tumors a 2 weeks of age. The tumors recurred rapidly after initial resection. Wide local excision of the involved scalp and skin graft coverage was performed at 14 weeks of age. Histopathology of these tumors was identical to that of the maternal tumor. The maternal origin of these tumors was confirmed by fluorescence in situ hybridization (FISH). The child is now 5 years old and free of disease.

Somatic mutation of vascular endothelial growth factor receptors in juvenile hemangioma.

Juvenile hemangiomas are the most common tumors of infancy, occurring in as many as 10% of all births. These benign vascular lesions enlarge rapidly during the first year of life by hyperplasia of endothelial cells and attendant pericytes and then spontaneously involute over a period of years, leaving loose fibrofatty tissue. Several hypotheses have been put forth concerning hemangiogenesis, including the possibility that the tumor is the result of somatic mutation in one or more components of critical vascular growth-regulatory pathways. To test this hypothesis, we obtained 15 proliferative-phase hemangiomas after surgical resection and dissected them to enrich for the lesional (endothelial and pericytic) components of each specimen. To determine whether hemangiomas represent a clonal expansion from a single progenitor cell, we assayed X-inactivation patterns for each lesion by using the polymorphic X-linked human androgen receptor gene. Twelve of 14 informative hemangiomas showed a significant degree of allelic loss after methylation-based and transcription-based polymerase chain reaction clonality assays, suggesting a nonrandom X-inactivation pattern and, thus, a monoclonal origin. We then sequenced genes encoding the receptors of the vascular endothelial growth factors (VEGFs) as candidates for potential somatic mutation. Mutations were found in two of the 15 hemangioma specimens: a missense mutation (P1147S) in the kinase domain of the VEGFR2 (FLK1/KDR) gene in one specimen and a missense mutation (P954S) in the kinase insert of the VEGFR3 (FLT4) gene in another specimen. In each case, the mutation was detected in tumor tissue but not in adjacent normal tissue. These results suggest that one potential mechanism involved in hemangioma formation is the alteration of the VEGF signaling pathway in endothelial and/or pericytic cells.