Normal Hens Research Articles

Radiographic, computed tomographic, and histologic characteristics of bone for clinically normal laying hens in a free-range housing system.

Laying hens are increasingly being kept in backyard flocks and considered family pets; however, diagnostic imaging characteristics of bone for clinically normal backyard hens are currently limited. This prospective, descriptive study was to describe radiographic, computed tomographic, and histologic characteristics of bone for a group of clinically normal laying hens housed in conditions comparable to those of backyard flocks. Sixteen 60-week-old Lohmann Brown laying hens were included. Hens were housed in a free-range unit with outdoor access at a university research and teaching farm. Hens were defined as clinically normal by the farm manager and a veterinary researcher in laying hen behavior and welfare. Findings from the horizontal beam, left lateral, sternal radiographs (n=16), postmortem, and whole-body CT scans (n=4) were recorded by a veterinary radiologist and a research technician. Histologic findings for sternal, femoral, and tibiotarsal bone samples (n=5) were recorded by a veterinary pathologist. The most frequent radiographic findings for the sternal carina (keel bone) were smoothly marginated concave deviations of the ventral margin and caudal section fractures. Multiple punctate mineral opacities (PMOs) were present in radiographs and CT images for all hens and were involved in the sternal carina and multiple other bones in the axial and appendicular skeleton. No bone abnormalities were identified in any histologic sections where PMOs were radiographically detected. Authors propose that PMOs are normal radiographic and CT findings in the bones of mature, laying hens and may represent temporary calcium reservoirs formed during osteoclastic activities.

Comprehensive Proteome and Acetyl-Proteome Atlas Reveals Hepatic Lipid Metabolism in Layer Hens with Fatty Liver Hemorrhagic Syndrome.

The feeding of high-energy and low-protein diets often induces fatty liver hemorrhagic syndrome (FLHS) in laying hens. However, the mechanism of hepatic fat accumulation in hens with FLHS remains uncertain. In this research, a comprehensive hepatic proteome and acetyl-proteome analysis was performed in both normal and FLHS-affected hens. The results indicated that the upregulated proteins were primarily associated with fat digestion and absorption, the biosynthesis of unsaturated fatty acids, and glycerophospholipid metabolism, while the downregulated proteins were mainly related to bile secretion and amino acid metabolism. Furthermore, the significant acetylated proteins were largely involved in ribosome and fatty acid degradation, and the PPAR signaling pathway, while the significant deacetylated proteins were related to valine, leucine, and isoleucine degradation in laying hens with FLHS. Overall, these results demonstrate that acetylation inhibits hepatic fatty acid oxidation and transport in hens with FLHS, and mainly exerts its effects by affecting protein activity rather than expression. This study provides new nutritional regulation options to alleviate FLHS in laying hens.

PSXIII-4 Effects of Keel Bone Fracture on Behavioral Fear Responses of Laying Hens in the Late Laying Period

Abstract Keel bone fracture (KBF) of laying hens has become one of the important welfare problems for health and production performance. The present study aimed to assess the fear responses in native breed of Lindian chickens and commercial Hyline Brown laying hens with and without KBF at the late laying period. At their 56-week-age, a total of 76 Hyline Brown hens and 80 Lindian hens selected from furnished cages were housed individually in battery cages. The laying hens were divided into normal group and keel fracture group. The fear tests, including tonic immobility tests (TI), open field tests (OFT), and startle reflex tests (SRT) were measured sequentially. The fractured laying hens from two breed had significant elevated duration time in the TI test than that of normal laying hens (P < 0.05). In OFT test, the number of grids, steps, and time spent in the outer ring of fractured Lindian hens were significantly less than those of normal hens (P < 0.05), and the time spent in the inner ring was significantly greater (P < 0.05). The movement latency and the time spent in the inner circle of fractured Hyline Brown hens were significantly greater than those of normal hens (P < 0.05), but the number of grids and steps passed and the time spent in the outer ring were significantly less than those of normal hens (P < 0.05). There was no significant difference in call latency and total call between fractured and normal hens of Lindian and Hyline Brown strain (P > 0.05). The total score of startle reflex of Lindian hens with keel fracture was significantly less than that of normal laying hens (P < 0.05). Therefore, keel fracture reduced the welfare of laying hens in the later stage of laying by affecting their behavioral fear responses.

Changes in focal adhesion protein Talin-1 expression during malignant transformation leading to ovarian high-grade serous carcinoma.

e17586 Background: Ovarian high-grade serous carcinoma (HGSC), the most lethal form of ovarian cancer, is often detected at late stages due to the lack of an early detection test resulting in low 5-year survival rates. Information on factors associated with malignant transformation and early metastasis is critical in developing interventional strategies to reduce the incidence and death due to HGSC. Talin-1, a focal adhesion protein, regulates cell–cell adhesion, and induces cytoskeletal remodeling leading to tumor cell migration and adhesion. Destabilization of the cell membrane during malignant development may shed Talin-1 in circulation which may be a potential early marker of progression of HGSC. Objective: The goal of this study was to examine the changes in Talin-1 expression during malignant transformation in ovaries and fimbria of the fallopian tube and its prevalence in serum. Difficulty in accessing patients with early-stage HGSC is a significant barrier to study malignant transformation and progression. Laying hen model of spontaneous ovarian HGSC was used together with clinical specimens. Methods: Two experiments were conducted. Exploratory study:Archived normal ovaries (n = 10), ovaries with HGSC at early (n = 10) and late (n = 10) stages from patients and their serum samples were used. Prospective study: Laying hens with no detectable abnormalities were selected and monitored using transvaginal ultrasound (TVUS) imaging at 10 week intervals for 10 months and sera were collected at each scan. Hens were euthanized at diagnosis of solid mass during scanning or at the end of the study; ovaries, fimbria and serum samples were collected. Normal hens (n = 10), hens with early (n = 7) and late-stage (n = 8) tumors were selected. Talin-1 expression in tissues and serum was determined by immunohistochemistry (IHC), Western blotting (WB), gene expression (qPCR), and immunoassay (ELISA). Significant differences in Talin-1 expression among all groups were determined by ANOVA and/or paired/unpaired t-tests, significance was taken when P< 0.05. Results: Expression of Talin-1was detected in all tissues with various intensities. Compared with normal, ovarian HGSC tissues showed intense expression of Talin-1 in IHC, WB and qPCRs ( P< 0.05). Similar patterns were observed in serum levels in ELISA ( P< 0.01) in the exploratory study. Prospective study in hens showed significant increase in serum Talin-1 levels even before the formation of a solid tumor mass in the ovary or fimbria detectable by TVUS. Conclusions: Elevated Talin-1 expression in early-stage ovarian HGSC tumors suggests its association with malignant development. Serum prevalence of Talin-1 during malignant transformation makes it a potential marker for early detection of HGSC and its progression. These results will serve as foundation for a clinical study with larger cohorts. Support: NIH R01 CA210370 (AB)

Comparative Study of Organoleptic Quality and Use Test of Pasteurized, Freeze-dried and Hot Air-dried Quail Eggs Produced in Benin

ABSTRACT This study aimed to evaluate the organoleptic quality and to carry out a use test of pasteurized, freeze-dried and hot air-dried quail eggs in order to compare with normal liquid quail and hen eggs. These egg products were reconstituted and omelets prepared using each quail egg product and the controls. Freeze-dried, hot air-dried quail eggs were mixed differently with 1% corn porridges. Single porridge served as the control. Each product type was subjected to a single round of testing by a panel of 30 untrained individuals. Reconstituted hot air-dried eggs were different (p ˂ 0.05) from reconstituted pasteurized and freeze-dried, liquid quail and hen eggs for all organoleptic parameters. While liquid, pasteurized quail eggs and liquid hen egg omelets differed (p ˂ 0.05) from freeze-dried quail egg and hot air-dried quail egg omelets. The single porridge was different (p ˂ 0.05) from freeze-dried and hot air-dried quail egg porridges.

Keel bone damage affects behavioral and physiological responses related to stress and fear in two strains of laying hens.

Keel bone damage (KBD) is more prevalent in alternative laying hen housing systems than in conventional cages, and its incidence differs from strain to strain. However, the information of KBD in Lindian chickens, a native Chinese strain, is limited. To investigate the effect of KBD on fearfulness and physiological indicators of stress in Lindian chickens and commercial laying hens, a total of two hundred 25-wk-old chickens (100 Hy-line Brown and 100 Lindian chickens) were studied for 7wk. The birds were housed in furnished cages with 10 birds per cage for each strain. At 32-wk of age, the birds in each strain were divided into normal (NK), deviated (DK), and fractured (FK) hens according to the keel bone status. Ten birds in each keel bone status per strain were subsequently selected to collect blood for the determination of stress and fear-related indicators, including corticosterone, serotonin, interleukin-1β, and interleukin-6, and measure fear responses, including novel object test (NOT), human approach test (HAT), and tonic immobility (TI) test. The results showed that egg production was lower and the incidence of keel bone fractures was higher in Lindian chickens than in Hy-line Brown hens (P < 0.05). Lindian chickens showed a significantly increased whole blood serotonin content, NOT-latency, HAT-score, and TI induction times (P < 0.05) and decreased serum interleukin-6 content and TI-duration (P < 0.05) compared with Hy-line Brown hens. Additionally, FK hens had significantly elevated whole blood corticosterone, serum interleukin-1β and interleukin-6 levels, TI-duration, and NOT-latency (P < 0.05), and a reduced whole blood serotonin content (P < 0.05) compared with NK and DK hens. Our results indicated that KBD affected stress and fear responses, and this impact was mainly reflected by FK hens compared with NK and DK hens. We suggest that keel bone fractures are the main factor impairing hen welfare. Besides, the incidence of keel bone fractures and stress and fear responses of Lindian chickens are more severe than Hy-line Brown laying hens, indicating that the strain type can affect the health and welfare of laying hens.

Chicken Cyclo-oxygenases Response in a Pulmonary Infection Disease

Avian infectious bronchitis, associated coronavirus causes inflammation and damage to the lungs. To evaluate the molecular mechanisms behind this event, we investigated variations in the expression level of the proinflammatory factors, cyclo-oxygenase 1 (COX-1) and cyclo-oxygenases 2 (COX-2). In mammals, the increased generation of prostaglandins during the onset of inflammatory responses and activation of immune cell types has been attributed to the induction of COX-2. The recent sequencing of chicken genome and the identification of genes coding COX-1 and COX-2 allowed us to analyze their expression in lung inflammatory disease. The purpose of this study was to examine the relative expression and distribution of COX-1 and COX-2 in organs of normal hens and in hens with avian infection bronchitis. The expression levels of cyclo-oxygenase genes in several organs from uninfected chickens and infected ones were measured using a quantitative reverse transcription-PCR technique. Our results show that the expression level of COX-1 was highly up regulated in spleen, small intestine, lungs, and liver, but modestly up regulated in the pancreas. However, no significant change was observed in the expression levels of COX-2, in the small intestine, liver and spleen; suggesting a constitutive expression of this enzyme. Whereas, COX-2 mRNA expression was significantly decreased in the heart and lungs of infected chickens, ranging from 9-fold to 4-fold, respectively (p<0.05). Contrary to mammals, COX-2 is present constitutively in bird’s cells, while COX-1 is expressed primarily after inflammation.

Production Performance, Reproduction and Immunity of Sentul Hens at Different IgY Concentrations

The aim of this study was to evaluate the effect of different IgY concentrations on the production, reproduction and immunity of Sentul chicken. This research used 20 month-old Sentul chickens which consisting of 10 chickens with high IgY concentration and 10 chickens with low IgY concentration Parameters observed included prelayer performance (feed consumption, weight gain, feed conversion, morbidity and mortality), layer performance (feed consumption, egg production, egg weight, feed conversion, hatchability, morbidity and mortality), and immunity at chicks (IgY concentration of eggs and chicks serum). The experiment was conducted as a completely randomized design. The treatments were high and low IgY concentration. All treatments were repeated 10 times. The data obtained were analyzed using the SAS program t-test. Morbidity and mortality were analyzed descriptively. The results of this research indicated that the prelayer period, chickens with high IgY concentration had a higher performance than chicken with low IgY concentration. This is caused by about 30% chickens with low IgY were in sick condition for approximately seven days. Further, the layer period showed that the chickens with high IgY concentration had lower production performance than the chicken with low IgY concentration. The study concluded that chickens at low IgY concentrations are susceptible to exposure to antigens that lead to decreased productivity. In normal conditions hens, Sentul with high IgY concentration resulted in low production. The observation of chicken immunity showed that concentration of IgY from chicks and eggs did not differ between hens with high IgY concentrations and hens with low IgY concentrations.

Polycystic Ovarian Condition May Be a Risk Factor for Ovarian Tumor Development in the Laying Hen Model of Spontaneous Ovarian Cancer.

Chronic inflammation and long-standing oxidative stress are potential predisposing factors for developing malignancies, including ovarian cancer (OVCA). Information on the association of ovarian chronic abnormal conditions, including polycystic ovarian syndrome (PCOS), with the development of OVCA is unknown. The goal of this study was to examine if polycystic ovarian conditions are associated with OVCA development. In the exploratory study, 3–4-year-old laying hens were randomly selected and examined for the presence of polycystic ovaries with cancer (PCOC). In the prospective study, hens were monitored by ultrasound scanning to detect the incidence of a polycystic ovaries and subsequent development of OVCA. Tissues from normal ovaries and PCOC were examined for macrophage infiltration, expression of interleukin-16, and superoxide dismutase 2. The exploratory study detected spontaneous PCOC at early and late stages in hens. PCOC in hens were accompanied with influx of macrophages (17.33 ± 2.26 in PCOC at the early stage and 24.24 ± 2.5 in PCOC at the late stage in 20 mm2 areas of tissue as compared with 6.77 ± 1.58 in normal hens). Expression of interleukin-16 was more than 2.5-fold higher and superoxide dismutase 2 was approximately 3-fold higher in PCOC hens than normal hens. The prospective study showed the development of OVCA in some hens with polycystic ovarian condition (PCO). PCOC development in hens was associated with chronic inflammation in the ovary. Laying hens may represent a potential model for the study of spontaneous PCOS and its long-term risk of PCOC development.

Abstract 241: Vimentin offers a potential target for the prevention of ovarian tumor progression and recurrence

Abstract Background: Aggressive rates of progression and frequent recurrence are the main causes of high rates of death due to ovarian cancer (OVCA). Vimentin, an intermediate filament protein, expressed by ovarian mesenchymal cells may be involved in OVCA metastasis and recurrence. Ashwagandha (Withania somnifera, ASH, a natural product) has shown to improve tumor-associated stress against several malignancies. Objectives: The goals of this study were to examine whether (1) OVCA progression is associated with vimentin expression; and whether (2) OVCA progression and vimentin expression can be prevented by dietary supplementation of Ashwagandha (ASH) in laying hen model of spontaneous OVCA. Methods: Exploratory study: Clinical specimen from normal ovaries (n=10), benign tumors (n=5) and malignant ovarian epithelial cancers at early (n=7) and late stages (n=16) were collected from patients and examined for vimentin expression. Prospective study: 4-year old laying hens with normal ovaries or ovaries with early stage OVCA were supplemented with 2% dietary ASH root powder or basal diet (24 hens in each group containing 18 healthy, 6 with early stage OVCA). Hens were provided with feed and water ad libitum for 120 days and euthanized thereafter. Tumor stages were recorded; serum, normal or cancer ovaries were collected. Expression of vimentin both in clinical and preclinical treated or untreated specimens was examined. Statistical differences in the intensity of vimentin expression among different pathological and ASH supplemented groups were determined using ANOVA and t-tests. Results: Vimentin was localized in the nucleus and cytoplasm of normal ovarian, benign and malignant cells. Compared with normal and benign tumors, the intensity of vimentin expression was significantly (P&amp;lt;0.0001) lower in early stage OVCA and increased remarkably in late stage OVCA. Similar to OVCA patients, intensity of vimentin expression was lower in hens with early OVCA than normal hens, and increased significantly (P&amp;lt;0.0001) in hens with late stage OVCA. As compared with untreated hens, tumor incidence and progression as well as vimentin expression decreased significantly in hens treated with 2% dietary ASH. Conclusions: Results of the present study suggest that vimentin expression increased during tumor metastasis. ASH treatment reduced OVCA metastasis by inhibiting vimentin expression. Therefore, vimentin may represent a potential target for ASH to prevent OVCA progression and metastasis. Support: Support: R21CA187309-02. Citation Format: Aparna Yellapa, Janice M. Bahr, Sameer Sharma, Pincas Bitterman, Sanjib Basu, Animesh Barua. Vimentin offers a potential target for the prevention of ovarian tumor progression and recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 241. doi:10.1158/1538-7445.AM2017-241

Physicochemical and functional properties of Chinese soft-shell turtle (Pelodiscus sinensis) egg

The physicochemical properties of Chinese soft-shell turtle egg were characterized for functional use in the food industry. The egg yolk of un-fertilized soft-shell turtle eggs was separated and fractionated into granules and plasma. Then, the egg yolk, albumen, granules, and plasma were freeze-dried for further analysis. Results showed that the Chinese soft-shell turtle egg typically comprised 50% egg yolk, 34% albumen, and 16% shell in average. The egg yolk composed of 61% granules and 39% plasma. The granules contained most of the protein, while the plasma contained most of the lipid in egg yolk. The albumen contained about 26% ash on a dry weight basis. Lysozyme was the major component in turtle egg albumen. The protein solubility of egg yolk, granules, and plasma was affected by the changes in pH, while that of albumen remains constant. The emulsifying and foaming properties increased when the concentration increased for all samples. Both the yolk and the albumen in turtle egg exhibited better functional properties than those in normal hen egg. These physicochemical and functional properties of Chinese soft-shell turtle egg are fundamental and essential for future study and food applications.

Estrogen Receptor Subtype Expression is Altered in the Hen Model of Ovarian Cancer

There is growing evidence that estrogens may promote tumor progression, including ovarian tumors. Estrogens exert their actions in tissues through two different receptor subtypes (ESR1 and ESR2). Studies have shown that hens develop ovarian cancer spontaneously, therefore providing a suitable animal model for the disease. Our aim was to determine the expression of mRNA and protein of the estrogen receptor subtypes in ovaries of normal hens and ovaries from hens with ovarian cancer. Ovarian tissue from normal hens and hens with ovarian cancer was collected for quantitative real-time PCR and immunofluorescence analysis. Quantitative real-time PCR results showed that the relative mRNA expression of ESR1 and the ratio of ESR1/ESR2 are significantly greater from hens with ovarian cancer when compared to normal ovarian tissue. Immunofluorescence analysis showed differential ESR1 and ESR2 protein expression in ovarian tissue sections from normal hens and hens with ovarian cancer, with results parallel to the mRNA data. There was no significant difference in plasma estradiol levels between normal hens and hens with ovarian cancer. These data suggest an increase in downstream estrogen-mediated actions in chicken ovarian tumors and, indeed, microarray analysis reveals a functionally significant estradiol-signaling pathway in chicken ovarian tumors. Interestingly, expression of a putative ovarian tumor suppressor, EPB41L3, is down regulated in this pathway. Taken together, these results suggest that, in the hen, ESR1 may be mediating a proliferative response in ovarian cancer cells. Although additional studies are required to define the role of ESR1 in tumor formation in the hen, these results support the utility of the hen for testing possible endocrine therapies.

Abstract B061: Association of leukocyte inhibitory immunoglobulin-like transcript 3 (ILT3) with ovarian cancer and prevention of its expression by dietary supplementation of Ashwagandha (Withania somnifera)

Abstract Background: Tumor-induced immunosuppression may be a reason for aggressive metastasis of ovarian cancer (OVCA). OVCA is a fatal malignancy of women with the highest case to death ratio among gynecological cancers. Most of the OVCA patients are postmenopausal women affected severely by the toxicity of current chemotherapeutics and danger of radiotherapy. Thus immunotherapy may be an alternative option for OVCA patients. Because OVCA mainly disseminates in the peritoneal cavity, immune functions in the tumor environment plays a crucial role in its progression. However, the mechanism(s) of immunosuppression by ovarian tumors are not well understood. Expression of leukocyte inhibitory immunoglobulin-like transcript 3 (ILT3, a member of inhibitory receptor family) by the tumors is one of the mechanisms in tumor-induced immunosuppression. Prevention or blocking of ILT3 expression may enhance anti-tumor immune responses. Information on ILT3 expression in OVCA and its prevention is not available. Herbs have long been used in traditional medicine against many human diseases including cancers. Ashwagandha or Winter Cherry (ASH, Withania somnifera), a herb known for its anti-tumor property, may enhance anti-tumor immunity by reducing ILT3 expression. Objectives: The goal of this study was to determine whether ovarian malignant tumors express ILT3 and whether dietary supplementation of ASH root powder reduces ILT3 expression in OVCA. Methods: Two experiments were performed. In the first one, archived specimen from normal ovaries of postmenopausal women (n=10), benign tumors (cystadenomas/adenofibromas, n=10) and ovarian malignant tumors (papillary serous) at early (n=7) and late stages (n=10) were examined for ILT3 expression. In the second group, laying hens, a preclinical model of spontaneous OVCA, were used. Healthy hens with normal ovaries or hens with early stage OVCA (selected by transvaginal ultrasound scanning) were divided into 3 groups and provided with basal diet (control) or diet supplemented with either 1% or 2% ASH root powder for 90 days. Each group had 20 hens (15 healthy, 5 with early stage OVCA). Hens were euthanized thereafter and gross morphology including normal and tumor stages were recorded, serum, ovarian tissues with or without cancer were collected and processed for ILT3 expression by immunohistochemistry (IHC), Western blotting or gene expression analysis. Differences in ILT3 expression among different pathological or supplemented groups were analyzed by one-way ANOVA and paired t-test for group-wise comparison. Results: Strong expression of ILT3 was detected in ovarian malignant tumors at early stage. A very weak or no expression of ILT3 was observed in normal ovaries, while it was weak to moderate for benign tumors. The expression was stronger as the OVCA progressed to late stages. Immunoblotting detected a band of approximately 55kDa. Similarities in the patterns of ILT3 expression was observed between OVCA hens and patients. As compared with untreated and 1% ASH-treated hens, tumor incidence and progression as well as ILT3 expression was decreased significantly in hens supplemented with 2% dietary ASH. Conclusions: The present study suggests that ILT3 is expressed by ovarian papillary serous cancers, and the expression of ILT3 increases with the progression of OVCA to late stages. Dietary supplementation of OVCA hens with 2% ASH reduced ILT3 expression and decreased the rate of tumor progression. Decrease in tumor progression was associated with the decrease in ILT3 expression. Thus ILT3 represents a potential target for enhancing the anti-tumor immune function in OVCA patients.ASH may be used to reduce ILT3 expression and prevention of OVCA progression. Support: Swim across America. Citation Format: Natasha Gupta, Sameer Sharma, Seby L. Edassery, Janice M. Bahr, Lindsey Franklin, Erika Paulsen, Pincas Bitterman, Animesh Barua. Association of leukocyte inhibitory immunoglobulin-like transcript 3 (ILT3) with ovarian cancer and prevention of its expression by dietary supplementation of Ashwagandha (Withania somnifera). [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B061.

Abstract AS14: Contrast enhanced interleukin 16 targeted imaging detects ovarian tumor at early stage

Abstract Background: The high rate of death of ovarian cancer (OVCA) patients can be reduced if it is detected at early stage. Neither suggested serum markers nor the currently available traditional transvaginal ultrasound (TVUS) imaging or their combinations can detect OVCA at early stage. No imaging target in the ovary for TVUS imaging corresponding to the suggested serum markers has been defined. Moreover, due to the limited resolution, TVUS imaging cannot detect OVCA at early stage. Thus, new imaging target(s) together with improvement in resolution is necessary for early OVCA detection by TVUS imaging. Interleukin 16 (IL-16), a proinflammatory cytokine, associated with longstanding unresolved inflammation due to frequent ovulation, has been reported to be increased during OVCA development. IL-16 is expressed by the ovarian malignant cells and tumor associated neoangiogenic (TAN) microvessels. Thus IL-16 represents a potential marker of early OVCA which can be detected in vivo by TVUS imaging provided a molecular targeted contrast enhanced imaging agent can be developed. Objective: The goal of this study was to develop and test the efficacy of molecular (IL-16)-targeted ultrasound imaging probe for the detection of early OVCA. Materials and Methods: 3-years old (n=150) White Leghorn laying hens with normal or low egg laying rates or stopped-egg laying were scanned by TVUS before and after intravenous injection with IL-16-targeted microbubbles. IL-16-targeted imaging agents were prepared by conjugating anti-chicken IL-16 antibodies with Targetster® containing microbubbles (Targeson Inc, San Diego). All images were archived and analyzed offline. Serum samples were collected, hens were euthanized, ovarian tissues were processed for paraffin or frozen sections and nuclear matrix protein (NMP) extraction. Ovarian tumors were confirmed by gross morphology and routine histology. Sera were tested for anti-NMP antibodies (a marker of malignant nuclear transformation) and IL-16 levels by immunoassay and 1- &amp; 2D-Western blot (WB). The frequencies of IL-16 expressing cells were determined by immunohistochemistry (IHC). Results: IL-16-targeted microbubbles bounded with ovarian tumors and appeared as shining mass of irregular-shape. Compared with non-targeted, IL-16-targeted imaging increased the visualization of ovarian tumors remarkably. All hens with suspected tumor mass (n=23, 7 early and 16 late stages) were detected by IL-16-targeted imaging and confirmed by gross examination. The frequency of IL-16 expressing cells detected by IHC confirmed the prediction of targeted ultrasound imaging. Serum levels of IL-16 were higher in OVCA hens than in normal hens and correlated with the frequencies of IL-16 expressing cells and ovarian TAN vessels. Prevalence of anti-NMP antibodies were not detected in normal hens while all hens with OVCA were positive. Immunoreactive tumor antigens (NMPs) of 50-80kDa were detected by 2D-WB. Conclusion: IL-16-targeted ultrasound imaging enhanced the visualization of ovarian tumors remarkably. The enhanced intensity of IL-16-targeted imaging was correlated with serum IL-16 levels and the prevalence of anti-NMP antibodies. Thus, IL-16-targeted imaging in association with serum anti-NMP antibodies may improve OVCA detection at early stage. These results will form a foundation for a clinical study. Support: Dept. of Defense award # W81XWH-12-1-0460. Citation Format: Animesh Barua, Aparna Yellapa, Pincas Bitterman, Janice M Bahr, Sanjib Basu, Sameer Sharma, Jacques S. Abramowicz. Contrast enhanced interleukin 16 targeted imaging detects ovarian tumor at early stage [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS14.

Flaxseed reduces the pro-carcinogenic micro-environment in the ovaries of normal hens by altering the PG and oestrogen pathways in a dose-dependent manner.

The objective of the present study was to find the optimum dose of flaxseed that would decrease PG and alter oestrogen pathway endpoints implicated in ovarian cancer. In the study, four groups of fifty 1.5-year-old chickens were fed different amounts of flaxseed (0, 5, 10 or 15% of their total diet) for 4 months and were then killed to collect blood and tissues. Levels of flaxseed lignan metabolites, Enterolactone (EL) and Enterodiol (ED) were measured in the serum, liver and ovaries by liquid chromatography-MS/MS, and n-3 and n-6 fatty acid (FA) levels were measured by GC. The effects of the varied flaxseed doses were assessed by measuring levels of PGE2 and oestrogen metabolites (16-hydroxyestrone (16-OHE1) and 2-hydroxyestrone (2-OHE1)) as well as by analysing the expression of the oestradiol metabolising enzymes CYP3A4 (cytochrome p450, family 3, subfamily A, polypeptide 4), CYP1B1 (cytochrome p450, family 1, subfamily B, polypeptide 1) and CYP1A1 (cytochrome p450, family 1, subfamily A, polypeptide 1) and that of oestrogen receptor α (ERα) in the ovaries. The ratio of n-3:n-FA increased with an increase in flaxseed supplementation and corresponded to a dose-dependent decrease in cyclo-oxygenase-2 protein and PGE2 levels. EL and ED increased in the serum, liver and ovaries with increased concentrations of flaxseed. Flaxseed decreased the expression of ERα in the ovaries. The ratio of 2-OHE1:16-OHE1 in the serum increased significantly in the 15% flaxseed diet, and there was a corresponding increase in CYP1A1 in the liver and decrease in CYP3A4 in the ovaries. CYP1B1 mRNA also decreased with flaxseed diet in the ovaries. The 15% flaxseed-supplemented diet significantly decreased inflammatory PGE2, ERα, CYP3A4, CYP1B1 and 16-OHE1, but it increased CYP1A1 and 2-OHE1, which thus reduced the inflammatory and pro-carcinogenic micro-environment of the ovaries.

VEGFR2-Targeted Ultrasound Imaging Agent Enhances the Detection of Ovarian Tumors at Early Stage in Laying Hens, a Preclinical Model of Spontaneous Ovarian Cancer.

Tumor-associated neoangiogenesis (TAN) is an early event in ovarian cancer (OVCA) development. Increased expression of vascular endothelial growth factor receptor 2 (VEGFR2) by TAN vessels presents a potential target for early detection by ultrasound imaging. The goal of this study was to examine the suitability of VEGFR2-targeted ultrasound contrast agents in detecting spontaneous OVCA in laying hens. Effects of VEGFR2-targeted contrast agents in enhancing the intensity of ultrasound imaging from spontaneous ovarian tumors in hens were examined in a cross-sectional study. Enhancement in the intensity of ultrasound imaging was determined before and after injection of VEGFR2-targeted contrast agents. All ultrasound images were digitally stored and analyzed off-line. Following scanning, ovarian tissues were collected and processed for histology and detection of VEGFR2-expressing microvessels. Enhancement in visualization of ovarian morphology was detected by gray-scale imaging following injection of VEGFR2-targeted contrast agents. Compared with pre-contrast, contrast imaging enhanced the intensities of ultrasound imaging significantly (p < 0.0001) irrespective of the pathological status of ovaries. In contrast to normal hens, the intensity of ultrasound imaging was significantly (p < 0.0001) higher in hens with early stage OVCA and increased further in hens with late stage OVCA. Higher intensities of ultrasound imaging in hens with OVCA were positively correlated with increased (p < 0.0001) frequencies of VEGFR2-expressing microvessels. The results of this study suggest that VEGFR2-targeted contrast agents enhance the visualization of spontaneous ovarian tumors in hens at early and late stages of OVCA. The laying hen may be a suitable model to test new imaging agents and develop targeted therapeutics.

ATL: A Preclinical Model of Spontaneous Ovarian Cancer

Because of the lack of an effective early detection test, ovarian cancer (OVCA) in most cases is detected at late stages and remains a fatal gynecological malignancy. Molecular imaging provides information on the changes associated with the development of a disease at molecular levels. Because angiogenesis is an early event in tumor development, increased expression of αvβ3 integrins by ovarian tumor-associated angiogenic microvessels provides a target for noninvasive ultrasound imaging to detect early-stage OVCA. The goal of this study was to examine the feasibility of αvβ3 integrin-targeted molecular imaging agent in enhancing the detection of spontaneous ovarian tumor in laying hens, a preclinical model of OVCA. The study was conducted in 2 phases, including a cross-sectional exploratory followed by a prospective monitoring of hens for 45 weeks with targeted ultrasound imaging. Changes in ultrasound signal intensity were determined before and after the injection of αvβ3 integrin-targeted imaging agent in hens with spontaneous OVCA. All images were digitally stored. After scanning, ovarian tissues from all hens were collected and processed for histopathologic and immunohistochemical studies. Ultrasound signal intensity was significantly (P < 0.001) higher in hens with early-stage OVCA than in normal hens and increased further in late-stage OVCA. Compared with that in normal cases, ultrasound signal intensities increased approximately 19-fold in early stage and 26-fold in late-stage OVCA. Differences in signal enhancement were not observed among different histologic subtypes of OVCA. Higher signal intensities from targeted imaging of ovarian tumors were associated with increased number of αvβ3 integrin-expressing ovarian microvessels. Prospective monitoring of hens with αvβ3 integrin-targeted imaging agent detected OVCA at early stage. These results suggest that αvβ3 integrin-targeted imaging agent enhanced the visualization of ovarian tumor-associated angiogenic microvessels in hens with early-stage OVCA and may form a foundation for clinical studies.

Production of Pure Hinai-dori with Normal Reproductive Capability from Transferred Primordial Germ Cells

The production method of germline chimeras by transfer of primordial germ cells (PGCs) can be used in gene banking of chickens. However, little is known about the reproductive capability of offspring obtained by mating of male and female germline chimeras. To cryopreserve chicken genetic resources at the cellular level in future, it is necessary to demonstrate whether offspring originating from donor-derived PGCs have normal reproductive capability. In this study, we attempted to obtain the pure Hinai-droi offspring by mating of male and female germline chimeras produced by the transplantation of Hinai-dori PGCs into the Akita White Leghorn line (WL) embryos. Hinai-dori offspring from germline chimeras were screened using the Hinai-dori-specific autosomal microsatellite marker, namely, ADL0315, which allows distinction between the Hinai-dori breed and Akita WL. After sexual maturity of the Hinai-dori offspring originating from donor-derived Hinai-dori PGCs, the reproductive capability (egg production rate, fertilization rate and hatchability) was examined. Percentage hen-day egg production was investigated between 21 and 60 wk of age, and fertilization rate and hatchability were investigated between 43 and 60 wk of age, respectively. Among the 105 progeny obtained by mating of male and female germline chimeras, a single brown-colored female hatchling was obtained. The results from molecular genotyping using the Hinai-dori-specific microsatellite marker ADL0315 were consistent with results from morphological identification, and the brown-colored female hatchling was identified as the pure Hinai-dori breed. The mean percentage hen-day egg production, fertilization rate and hatchability of the Hinai-dori hen were 36.4%, 58.1% and 53.2%, which were similar to those of normal Hinai-dori hens. These results indicated that the pure Hinai-dori offspring originating from donor-derived Hinai-dori PGCs has normal reproductive capability.

Abstract A64: Association of interleukin 16 with early metastasis of ovarian tumors

Abstract Introduction: Ovarian cancer (OVCA) differs from other malignancies in its specific dissemination pattern that the tumor typically spreads in a diffuse intra-abdominal fashion rather than systemic circulation. Interactions among different cell types and their secretory products in the tumor microenvironment may contribute to the tumor development and metastasis. Interleukin 16 (IL-16) is a chemoattractant and pro-angiogenic cytokine involved in multiple immunopathobiological processes. IL-16 is expressed by several cell types including CD8 T cells and macrophages and occasionally by malignant cells. IL-16 expression is reported to be increased in several tumors including OVCA. Thus, IL-16 may be involved in the development and metastasis of ovarian tumors, if so, how IL-16 enhances tumor metastasis is not known. CD9, a member of tetraspanin family, has been implicated in the regulation of cell proliferation, invasion and motility. Emerging studies reported CD9 as a receptor for IL-16. Objectives: The goal of this study was to determine (1) whether IL-16 is associated with ovarian tumor metastasis in the vicinity of tumors including the omentum and (2) if so, to determine how IL-16 is associated with ovarian tumor metastasis. In this study we examined association of IL-16 and its receptor (CD9) expression with respect to ovarian tumor development and progression. The association of CD8+T cells and IL-16 expression during tumor initiation was examined in laying hens, an animal model of spontaneous OVCA. Methods: Experiment-1: Tumor specimen from patients with serous OVCA (10 early and 20 late stages), serous benign ovarian tumors (n=10) and normal (n=5, from patients underwent hysterectomies for non-ovarian disease) and omental tissues (n= 5, each of OVCA and benign) were used. Changes in IL-6 and CD9 expression in tumor tissues (ovaries and omentum) were examined by immunohistochemistry, proteomics and by quantitative polymerase chain reaction (qPCR). Experiment-II: Laying hens with normal ovaries (n=10) or microscopic OVCA (n=10) were examined for CD8 T cells and IL-16 expressing cells as mentioned above. The correlation between the IL-16 and CD8+ T cells were examined. In addition, to examine the effects of IL-16 on CD9 expression, normal ovarian surface epithelial (OSE) cells were treated with recombinant IL-16 protein. Results: Compared with normal ovaries and benign tumors, the frequencies of IL-16 expressing cells were significantly high in early stage OVCA (P&amp;lt;0.01) and increased further in late stage OVCA. Compared with primary ovarian tumors, the population of IL-16 expressing cells was remarkably high in omental tissues containing tumor (P&amp;lt;0.01). Similarly, compared with normal, intense staining was observed for CD9 expression both in malignant ovarian and omental tissues. CD9 staining intensities were positively correlated with the increased frequency of IL-16 expressing cells. Recombinant IL-16 treatment of OSE enhanced CD9 expression suggesting the stimulatory effects of IL-16 on CD9 expression. Compared with normal hens, frequency of CD8+ T cells increased significantly in hens with microscopic OVCA. Similarly, compared with normal hens, the population of IL-16 expressing cells increased significantly in hens with microscopic OVCA. The frequency of IL-16 expressing cells was positively correlated with the frequency of CD8+ T cells. Conclusions: The results of the present study suggest that increased IL-16 expression may be associated with enhanced CD9 expression which may be a factor for ovarian tumor progression and metastasis to the omental tissues. Increased CD8+ T cells in the tumor vicinity suggests that CD8+ T cells may be a source of enhanced IL-16 expression in developing ovarian tumors. Support: DOD award # W81XWH-11-1-0510 Citation Format: Aparna Yellapa, Pincas Bitterman, Jacques S. Abramowicz, Janice M. Bahr, Sameer Sharma, Sanjib Basu, Animesh Barua. Association of interleukin 16 with early metastasis of ovarian tumors. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A64.

Abstract 4641: IL-16 in association with VEGFR-2 detect early tumor associated changes in the ovary.

Abstract Background: Ovarian cancer (OVCA) is a lethal malignancy of women with high rate of death among gynecological cancers. Due to the lack of an effective early detection test, OVCA in most cases are detected at late stage. Interleukin (IL)-16 is a cytokine involved in multiple immunopathobiological processes including inflammatory responses and production of proangiogenic cytokines. IL-16 is expressed by malignant epithelium and secreted in serum. Ovarian malignant transformation is followed by neovascularization and vascular endothelial growth factor receptor-2 (VEGFR-2) is a marker of tumor associated neovascularization (TAN). Thus, IL-16 and VEGFR-2 represent markers of OVCA at early stage may constitute an early detection test for OVCA. Objectives: The goal of this study was to determine whether IL-16 expression is associated with ovarian malignant transformation and to examine association between serum IL-16 levels and VEGFR-2 expression by TAN vessels during ovarian tumor development. Materials and methods: Normal (n =15) White Leghorn laying hens (3-4 years old) or hens with ovarian tumors (n=32) were selected by contrast enhanced transvaginal ultrasound scanning. Following serum collection, hens were euthanized, tissues were processed for routine histology, immunohistochemistry (IHC), protein and gene expression studies. Ovarian tumors were confirmed by gross morphology and microscopy. Expression of IL-16 by ovarian malignant epithelium and VEGFR-2 by ovarian TAN vessels were determined by IHC, immunoblotting and quantitative PCR. Serum IL-16 levels were determined by immunoassay. Correlation between serum IL-16 levels and frequencies of VEGFR-2 expressing microvessels were examined. Results: The intensity of IL-16 expression by normal ovarian epithelium was very weak (n=15). In contrast, the staining intensity of IL-16 by malignant epithelium increased significantly (p&amp;lt;0.001) in hens with early stage OVCA (n=15) and late stage OVCA (n=17). Compared with normal hens, serum levels of IL-16 were significantly higher in hens with early (n=15) and late stages (n=17) of OVCA (P&amp;lt; 0.0001). The expression of IL-16 protein and mRNA were stronger in malignant ovaries than normal ovaries. The population of VEGFR-2 expressing microvessels increased significantly (p&amp;lt;0.001) in hens with early stage OVCA than normal hens (p&amp;lt;0.001) and increased further in late stage OVCA. The increase in serum IL-16 was positively correlated with the population of VEGFR-2 expressing TAN vessels. Conclusion: IL-16 expression by ovarian malignant epithelium and its serum levels are associated with ovarian malignant transformation and correlated with VEGFR-2 expression. Thus IL-16 may be a useful serum marker and in combination with VEGFR-2 may detect OVCA at early stage. These findings will facilitate clinical studies to establish a non-invasive early OVCA detection test. Support: DOD Award # W81XWH-12-1-0460. Citation Format: Aparna Yellapa, Pincas Bitterman, Jacques S. Abramowicz, Janice M. Bahr, Sanjib Basu, Sameer Sharma, Animesh Barua. IL-16 in association with VEGFR-2 detect early tumor associated changes in the ovary. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4641. doi:10.1158/1538-7445.AM2013-4641