Normal Hens Research Articles

Abstract 3471: Anti-NMP autoantibodies together with serum levels of death receptor 6 (DR6) detect ovarian tumors at early stage .

Abstract Background: High rate mortality in patients with ovarian cancer (OVCA) is attributed to the lack of an effective early detection test. OVCA progression differs from other malignancies in that the tumor typically spreads in a diffuse intra-abdominal manner. Thus tumor microenvironment plays an important role both in the inhibition, as well as progression of ovarian cancer. Malignant nuclear transformation is an early step in OVCA development which sheds nuclear matrix proteins (NMP) in circulation. The immune system responds to secreted NMPs by producing anti-NMP antibodies. Conversely, the tumor evolves immunosuppressive mechanisms to escape anti-tumor immunity. Death receptor 6 (DR6) has been suggested as an immunosuppressive agent expressed in several malignancies including OVCA. Thus, serum anti-NMP antibodies and DR6 may represent early markers of OVCA. However, association of anti-NMP antibodies and DR6 expression during OVCA development is unknown. Objectives: The goal of this study was to determine if serum anti-NMP antibodies and DR6 levels are associated with malignant ovarian transformation and to examine their feasibility in detecting OVCA at early stage. Materials and Methods: Access to OVCA patients at early stage is very difficult. We used laying hen model of spontaneous OVCA. 3-4 years old normal, low or stopped egg-laying hens were selected by ultrasound scanning, sera were collected, hens were euthanized and tissues were processed for histology, immunohistochemistry (IHC) and RT-PCR. Sera were processed for ELISA for DR6 and anti-NMP antibodies. Immunoreactions were confirmed by immunoproteomics. Tumor stage and their histological types were determined by gross inspection and routine histology. Samples were divided into 3 groups namely, normal (n= 20), early (n=15) and late (n = 17) stages of OVCA. Results: Prevalence of anti-NMP antibodies was detected in approx. 75% hens at early stage and 94% hens at late stage OVCA. All serum samples from tumor hens detected NMP antigens of 30-80kDa in immunoproteomic studies. The expression of DR6 by ovarian malignant epithelium was significantly (p<0.001) higher in hens with early stage OVCA than normal hens and increased further in hens with late stage OVCA. Similar patterns were observed in immunoblotting and gene expression studies. Serum levels of DR6 were significantly (p<0.001) higher in hens with early and late stages of OVCA. Increase in serum DR6 levels were positively correlated with the prevalence of serum anti-NMP antibodies. Conclusion: Results of this study suggest a concurrent prevalence of immune responses and immunosuppression to ovarian tumors. Anti-NMP antibodies and DR6 are associated with tumor initiation and progression. Thus, serum anti-NMP antibodies together with DR6 levels may detect OVCA at early stage. This study will provide a foundation for clinical study. Support: DOD Award # W81XWH-10-1-0523 Citation Format: Animesh Barua, Jacques S. Abramowicz, Janice M. Bahr, Salvatore A. Grasso, Sameer Sharma, Sanjib Basu, Jacob Rotmensch, Pincas Bitterman. Anti-NMP autoantibodies together with serum levels of death receptor 6 (DR6) detect ovarian tumors at early stage . [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 3471. doi:10.1158/1538-7445.AM2013-3471

Abstract 4642: GRP78 in association with VEGFR-2 detects early stage ovarian cancer.

Abstract Background: The lack of an effective early detection test makes ovarian cancer (OVCA) a fatal malignancy of women. Changes in cellular metabolic processes during ovarian malignant transformation lead to the endoplasmic reticular (ER) and mitochondrial stress. Glucose-regulated protein of 78kDa (GRP78) is a marker of ER stress. Tumor-associated ER stress causes relocalization of GRP78 from ER to cell surface with an increase in its serum levels. Secreted GRP78 also stimulates tumor associated neoangiogenesis (TAN). Vascular endothelial growth factor receptor-2 (VEGFR-2) is a marker of ovarian TAN. Thus GRP78 represents a marker of ovarian malignant transformation and in association with VEGFR-2 may constitute an early detection test for OVCA. However, information on OVCA related changes in GRP78 expression and its association with ovarian TAN is unknown. Objectives: The goals of this study were to examine (i) whether GRP78 expression increases during malignant changes in the ovary and (ii) whether the frequency of TAN vessels is associated with ovarian tumor-associated GRP78 expression at early stage of OVCA in the laying hen, a spontaneous model of human OVCA. Materials and Methods: 3-4 years old White Leghorn laying hens with normal (n=25) or ovarian tumors (n=30) were selected by contrast enhanced transvaginal ultrasound scanning. Serum samples were collected, hens were euthanized, and ovarian tissues were processed for routine histology or immunohistochemistry (IHC) and protein extraction. Ovarian tumors were confirmed by gross morphology and microscopy. Expression of GRP78 by ovarian malignant cells and VEGFR-2 by TAN vessels was determined by IHC. IHC observations were confirmed by immunoproteomic studies. Results: As compared with normal ovaries (n=25), the intensity of GRP78 expression was significantly (p<0.001) higher in hens with early stage OVCA (n=12) and increased further in hens with late stage OVCA (n=18) (p<0.05). GRP78 expression was higher in hens with serous OVCA followed by endometrioid OVCA and was least in hens with mucinous and clear cell OVCA irrespective of their stages of OVCA. A band of approximately 80kDa was observed for GRP78 in all ovaries examined and the patterns of expression were similar to that of IHC. The frequency of VEGFR-2 expressing TAN vessels was significantly (p<0.001) higher in OVCA hens than in normal hens. Increase in the frequency of VEGFR-2 expressing TAN vessels was positively correlated with the intensity of GRP78 expression. Conclusion: The results of this study suggest that increase in the GRP78 expression is associated with ovarian malignant transformation, and this enhanced expression may stimulate ovarian tumor-associated neoangiogenesis. Thus GRP78 in combination with VEGFR-2 may constitute an early detection test for OVCA. These results will constitute a foundation for a clinical study to establish an early detection test for OVCA. Support: DOD Award # W81XWH-11-1-0510. Citation Format: Doungkamol Alongkronrusmee, Pincas Bitterman, Jacques S. Abramowicz, Janice M. Bahr, Sanjib Basu, Salvatore Grasso, Sameer Sharma, Jacob Rotmensch, Animesh Barua. GRP78 in association with VEGFR-2 detects early stage ovarian cancer. [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 4642. doi:10.1158/1538-7445.AM2013-4642

Effect of Imbrasia belina (westwood), Tylosema esculentum (Burchell) Schreiber and Vigna subterranea (L) Verde as protein sources on growth and laying performance of Tswana hens raised under intensive production system

A feeding trial was conducted to determine growth and laying performance of indigenous Tswana hens fed diets formulated with four different protein sources consisting of either Tylosema esculentum (Burchell) or Vigna subterranea (L) Verde or Imbrasia belina (Westwood) and commercial diets under an intensive system in Botswana. Sixty 25 weeks old normal feathered Tswana hens were bought from a local farmer and reared up to 38 weeks on diets containing T. esculentum (Burchell) or V. subterranea (L) Verde or I. belina (Westwood) and a commercial diet (control). The hens were then randomly allocated to four treatment groups of 15 hens, with each treatment having three replicates of five hens each. Tylosema esculentum , V. subterranea and I. belina diets were formulated to meet the nutritional composition of commercial layers diet using T. esculentum (Burchell) Schreiber beans, V. subterranea (L) Verde beans and I. belina (Westwood), respectively as sources of protein before the start of egg production. Data were analyzed using PROC GLM of SAS (2002-2008) as a completely randomized design with initial body weight as a covariate. Hens fed control diet had high (P<0.05) feed intake (84.6±7.9 g) and those fed T. esculentum diet had lowest feed intake (54.6±7.9 g). Hens fed I. belina , control and V. subterranea diets had the highest (P<0.05) average daily gain (ADG) per hen of 4.77±1.31g, 3.27±1.16 g and 3.13±1.51 g, respectively. On the other hand, hens fed T. esculentum diet had the lowest (P<0.05) ADG/hen (-0.82±1.60 g) . The FCR was highest (P<0.05) for control diet with 1.76±0.16 g and lowest for hens fed I. belina diet with 1.10±0.16 g. Hen day egg production was highest (P<0.05) for control diet (90±0.87%) and lowest (41±0.87%) for T. esculentum diet. These results suggest that I. belina (Westwood) and V. subterranea (L) Verde are potentially good protein sources for formulating poultry diets.

Abstract 2455: Molecular targeted imaging of vascular endothelial growth factor receptor (VEGFR)-2 and anti-NMP autoantibodies detect ovarian tumor at early stage

Abstract Background: Although several serum markers of ovarian cancer (OVCA) have been suggested, none can detect OVCA at early stage specifically because of the lack of a corresponding marker in the ovary. Ovarian malignant cellular transformation is an early event followed by tumor-associated neoangiogenesis (TAN). Malignant cellular transformation results in shedding of nuclear matrix proteins (NMPs) in circulation and anti-NMP autoantibody production. VEGFR-2, an ovarian TAN marker, may also be an imaging marker for early OVCA detection. Thus VEGFR-2 in association with anti-NMP antibodies may constitute an early detection test for OVCA. Objectives: The goal of this study was to examine (i) whether VEGFR-2 targeted ultrasound imaging detects ovarian TAN vessels and (ii) whether the frequency of TAN vessels is associated with serum anti-NMP antibodies at early stage of OVCA in laying hens model of human OVCA. Materials and Methods: Mature White Leghorn laying hens with normal or low egg laying rates or no egg laying were scanned by transvaginal ultrasound before and after intravenous injection with VEGFR-2 targeted microbubbles. All images were archived and analyzed offline. Serum samples were collected, hens were euthanized, ovarian tissues were processed for paraffin or frozen sections and NMP extraction. Ovarian tumors were confirmed by gross morphology and routine histology. Sera were tested for anti-NMP antibodies and VEGFR-2 levels by immunoassay and 1- and 2D-Western blot (WB). The frequencies of VEGFR-2 expressing TAN vessels were determined by immunohistochemistry (IHC). Results: VEGFR-2 targeted microbubbles bounded with ovarian tumors and appeared as a ring of irregular shape on the ovarian surface. Compared with non-targeted, VEGFR-2 targeted imaging increased the visualization of TAN vessels remarkably. VEGFR-2 expressing TAN vessels confirmed targeted ultrasound imaging predictions. Serum levels of VEGFR-2 were higher in OVCA hens than in normal hens and associated with the frequencies of ovarian TAN vessels. None of the hens with normal egg laying rates was positive for anti-NMP antibodies. Immunoreactive tumor antigens (NMP) of 50-60kDa were detected by 2D-WB. The frequencies of VEGFR-2 expressing TAN vessels were higher in hens with serum anti-NMP antibodies than in normal hens. Conclusion: VEGFR-2 targeted ultrasound imaging enhanced the visualization of ovarian TAN vessels remarkably. The intensity of targeted imaging was associated with serum VEGFR-2 levels and anti-NMP antibodies. Thus, VEGFR-2 targeted imaging together 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: DOD-(OC0100192), Elmer and Sylvia Sramek Foundation, NCI-POCROC (P50 CA83636). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2455. doi:1538-7445.AM2012-2455

Abstract 5415: Expression of immunosuppressive leukocyte inhibitory immunoglobulin like transcript 3(ILT3) receptors increases with the development and progression of ovarian tumors

Abstract Background: Immunosuppressive tumor microenvironment has been suggested as one of the barriers to anti-tumor immune response in patient with ovarian cancer (OVCA). Leukocyte Inhibitory Receptor Immunoglobulin-like Transcript 3 (ILT3) has been proposed as a factor involved in the suppression of immune response in several malignancies. However, the expression and role of ILT3 in the development and progression of ovarian tumors has not yet been studied. Objective: The goal of this study was to examine the expression and association of ILT3 in ovarian tumors in laying hen, a preclinical model of OVCA in humans. Methods: A cohort of mature White Leghorn laying hens with normal or low egg laying rates were evaluated by transvaginal ultrasound scan (15 normal hens and 30 hens with OVCA).All hens were euthanized and serum and ovarian tissues from all hens were collected and processed for immunohistochemistry (IHC), Western blotting (WB) and RT-PCR. Presence of tumors and tumor staging were confirmed by gross morphology and histology. Expression of ILT3 was examined by IHC, WB and RT-PCR. Results: Immune cell like cells in the ovarian stroma as well as epithelium of the normal ovaries and ovarian tumors expressed ILT3. The intensity of ILT3 expression was significantly higher in hens with OVCA than normal hens. Moreover, the intensity of ILT3 expression varied with histological subtypes of ovarian tumors and was significantly higher in serous OVCA than endometrioid OVCA. A band of approximately 55kDa of ILT3 protein was detected in the homogenates of normal ovarian surface epithelium or tumor epithelium as well as whole normal or ovarian tumor extracts. Similar to IHC observations, protein expression was stronger in hens with OVCA than in normal hens. Conclusion: This is the first study to show that immunosuppressive ILT3 is expressed by ovarian tumor epithelium and the intensity of expression increases as the disease progresses. Thus the results are consistent with the hypothesis that ILT3 may be involved in the suppression of anti-tumor immunity in OVCA. The laying hens may be a useful model to understand the OVCA-associated expression of ILT3. This animal model also offers the opportunity to develop and test anti-ILT3 therapy to enhance anti-tumor immunity against OVCA in humans. Support: Department of Defense (OC#93303) and Elmer Sylvia and Sramek Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5415. doi:1538-7445.AM2012-5415

Abstract 3646: Expression of death receptor 6 (DR6) increases in association with ovarian tumor development and progression

Abstract Background: Because of the lack of an early detection test, ovarian cancer (OVCA) remains a lethal malignancy of women. OVCA differs from other malignancies in its specific dissemination pattern that the tumor typically spreads in a diffuse intra-pelvic and abdominal manner. Thus tumor microenvironment plays an important role in tumor dissemination and suppression of anti-tumor immunity is a hallmark of tumor progression. Although the precise mechanism of immune evasion by the tumor is not known, expression of death receptor 6 (DR6) has been suggested to be associated with tumor progression in several malignancies. DR6 is a member of the TNF receptor superfamily and a regulator of immune function. Expression of DR6 by ovarian tumors is unknown. Access to patients at early stage OVCA is very difficult. Laying hens develop OVCA spontaneously with histopathology similar to humans. Objectives: The goal of the present study was to determine whether DR6 is expressed by ovarian tumors at early stage and to examine whether DR6 expression is associated with OVCA tumor progression in the laying hen model of human OVCA. Materials and Methods: 3 years old normal, low or stopped egg-laying hens were scanned by ultrasound, sera were collected, hens were euthanized and ovarian tissues were processed for paraffin and frozen sections, immunoblotting and mRNA extraction. Stages of OVCA were confirmed at gross morphology and routine histology. Samples were divided into 3 groups: normal (control), early and late stages of OVCA. Sera were tested for DR6 levels by ELISA and confirmed by 2D-Western blot (WB). Expression of DR6 by normal and tumor ovaries was examined by immunohistochemistry (IHC), WB and RT-PCR. Results: DR6 was expressed by the epithelium of the ovarian tumor and angiogenic microvessels in the tumor stroma. The population of DR6+ microvessels and serum levels of DR6 was significantly higher in hens with ovarian tumors than the normal hens. Immunoblotting and gene expression analysis confirmed IHC observations. There was a significant increase in DR6 expression in hens with early stage OVCA (including microscopic tumor) suggesting that serum DR6 may be a potential indicator of ovarian tumors at very early stage. The frequencies of DR6+ cells were significantly higher in hens with late stage OVCA than early stage suggesting that DR6 may be involved in ovarian tumor development and progression in laying hens. Conclusion: For the first time, this study showed that epithelial cells in ovarian tumors express DR6 and results suggest that changes in serum levels as well as tissue expression of DR6 are associated with tumor initiation and progression. Thus serum DR6 may be useful as a surrogate maker of OVCA, target for anti-tumor immunotherapy as well as molecular imaging. This study will provide a foundation for a clinical study. Support: DOD (OC#093303), NCI-POCRC (P50 CA83636) and the Elmer Sylvia and Sramek Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3646. doi:1538-7445.AM2012-3646

Association of Interleukin 16 With the Development of Ovarian Tumor and Tumor-Associated Neoangiogenesis in Laying Hen Model of Spontaneous Ovarian Cancer

Tumor-associated neoangiogenesis (TAN) is an early event in ovarian tumor development. Interleukin 16 (IL-16) is a proangiogenic cytokine that stimulates production of neoangiogenic factors. The goal of this study was to determine the association of IL-16 with tumor development and ovarian TAN in laying hens, an animal model of spontaneous ovarian cancer (OVCA). Sera and ovarian tissues from 3-year-old laying hens were collected and processed for histopathologic, immunoassay, immunohistochemistry, immunoblotting, and molecular biological studies to determine the tissue expression and serum levels of IL-16. Samples were divided into 3 groups based on the diagnosis of the histopathologic ovarian tissue examination, namely, normal (healthy control, n = 81), early (n = 23 including 11 with microscopic OVCA), and late stages (n = 16) of OVCA. Serum levels of IL-16 were significantly higher in hens with microscopic, early, and late stages of OVCA than normal hens (P < 0.0001). The frequencies of IL-16 cells in tumor-bearing ovaries were significantly higher than normal hens (P < 0.05). The expression of IL-16 protein and mRNA were stronger in tumor-bearing ovaries than normal ovaries. In addition to ovarian stroma, IL-16 was also expressed by the epithelial cells of the tumor in OVCA hens. Differences in serum levels and ovarian IL-16 expression were not significant among different histological subtypes of OVCA including serous, endometrioid, and mucinous. Similar to the serum levels and ovarian expression of IL-16, the densities of neoangiogenic microvessels were significantly higher in hens with tumor-bearing ovaries than normal hens. The results of the study suggest that changes in serum levels of IL-16 are associated with tumor development and TAN. Thus, serum IL-16 levels may be an indicator of ovarian TAN at the early stage of OVCA.

Generation of antiviral transgenic chicken using spermatogonial stem cell transfected in vivo

This study was conducted in order to generate anti-viral transgenic chickens through transfected spermatogonial stem cell with fusion gene EGFP-MMx. After injecting fusion gene EGFP-MMx into testes, tissues frozen section, polymerase chain reaction (PCR) and dot blot of testes was performed at 30, 40, 50, 60, 70 and 80 days. Few normal hens were inseminated artificially with cock semen of 60 days. Finally, PCR, reverse transcription-PCR and Western blot were used to detect the integration of exogenous gene EGFP-MMx of the offspring. Dot blot positive rate of testes genome was 72.2%. PCR and RT-PCR positive rate was 25 and 10.53% (4/38) in sperm and F1 blood genome, respectively, while Western blot positive rate of F1 blood was 7.89% (3/38). Taken together, the possibility of cultivating anti-viral transgenic chickens by the method of testes injection which could integrate foreign genes into the genome was proved. Key words : EGFP-MMx fusion protein, spermatogonial stem cell, transgenic chickens.

Generation of antiviral transgenic chicken using spermatogonial stem cell transfected in vivo

This study was conducted in order to generate anti-viral transgenic chickens through transfected spermatogonial stem cell with fusion gene EGFP-MMx. After injecting fusion gene EGFP-MMx into testes, tissues frozen section, polymerase chain reaction (PCR) and dot blot of testes was performed at 30, 40, 50, 60, 70 and 80 days. Few normal hens were inseminated artificially with cock semen of 60 days. Finally, PCR, reverse transcription-PCR and Western blot were used to detect the integration of exogenous gene EGFP-MMx of the offspring. Dot blot positive rate of testes genome was 72.2%. PCR and RT-PCR positive rate was 25 and 10.53% (4/38) in sperm and F1 blood genome, respectively, while Western blot positive rate of F1 blood was 7.89% (3/38). Taken together, the possibility of cultivating anti-viral transgenic chickens by the method of testes injection which could integrate foreign genes into the genome was proved. Key words : EGFP-MMx fusion protein, spermatogonial stem cell, transgenic chickens.

Selected Blood Chemistry Values in Mobility-Impaired Broiler Breeder Hens with Suspected Calcium Tetany Using the i-STAT® Handheld Clinical Analyzer

Calcium tetany is a poorly defined disease of broiler breeder hens that results from acute hypocalcemia. It is characterized by impaired mobility, increased mortality, and absence of gross lesions that would explain the impaired mobility. To evaluate if hens with impaired mobility had calcium tetany or other abnormalities, blood values from normal and affected hens were determined using the i-STAT handheld clinical analyzer. Three flocks were evaluated weekly prior to peak production (range 25-30 wk of age) comparing normal hens to hens with clinically apparent calcium tetany. Calcium tetany suspect (CaTS) hens from four additional flocks were also evaluated. Significant hypocalcemia (P < 0.001) was observed in CaTS hens (average = 1.14 mmol/L ionized calcium [iCa]) compared to normal hens (average = 1.53 mmol/L iCa) in only one of three flocks sampled weekly. Clinically affected hens from one of the other four flocks also had hypocalcemia. Blood value abnormalities in mobility-impaired hens without hypocalcemia included hypernatremia. Findings in this study indicate calcium tetany is one cause of impaired mobility in breeder hens, but mobility impairment without hypocalcemia can also occur. Calcium tetany should be confirmed by finding significantly decreased levels of iCa in the blood, as diagnosis based on clinical presentation and necropsy results can be inaccurate. The i-STAT handheld clinical analyzer is an efficient, relatively low-cost method to determine iCa and other blood chemistry values that may be associated with impaired mobility in broiler breeder hens.

Salovum Egg Yolk Containing Antisecretory Factor as An Adjunct Therapy in Severe Cholera in Adult Males: A Pilot Study

Cholera involves stimulation of intestinal secretory process in response to cholera toxin leading to profuse watery diarrhoea that might cause death due to dehydration unless timely rehydration therapy is initiated. Efforts to identify and test potential antisecretory agents are ongoing. Antisecretory factor (AF) is a naturally-occurring protein produced in the human secretory organs, including the intestine, with antisectory properties demonstrated in animal and human models of secretory diarrhoea. Salovum egg yolk powder contains antisecretory proteins in a much higher (500 times) concentration than that of normal hen eggs. This is achieved by feeding hens with specially-processed cereals, capable of inducing antisecretory proteins in the yolk. The aim of the study was to examine the effect of Salovum egg yolk powder containing AF in the treatment of adult cholera patients. In an open, randomized controlled trial (pilot study), 40 adult male patients with severe cholera were studied: 20 received standard treatment (oral rehydration solution, antibiotic, and usual hospital diet) plus Salovum egg yolk powder (study group) and 20 received standard treatment alone (control group). All the patients received tablet doxycycline (300 mg) once immediately after randomization. Written informed consent was obtained from each subject before enrollment. The main outcome measures were stool weight and duration of diarrhoea. The demographic and baseline clinical characteristics of the study patients were comparable between the groups. No significant differences were found in the mean stool weight, g/kg of body-weight during the first 24 hours [study vs control group, mean +/- standard deviation (SD), 218 +/- 119 vs 195 +/- 136], second 24 hours (mean +/- SD, 23 +/- 39 vs 22 +/- 34), and cumulative up to 72 hours (mean +/- SD, 245 +/- 152 vs 218 +/- 169). The duration (hours) of diarrhoea after admission in the hospital was also similar in both the groups (mean +/- SD, 33 +/- 14 vs 32 +/- 10). No adverse effect was observed. Salovum egg powder containing AF as an adjunct therapy in the treatment of severe cholera could not demonstrate any beneficial effect. Further studies with higher doses of Salovum egg yolk powder might be considered in future to establish its antisecretory effect.

Use of contrast-enhanced ultrasound imaging with microbubbles targeted to αvβ3 integrins to enhance detection of early-stage ovarian tumors.

5076 Background: Lack of an early detection test makes ovarian cancer (OVCA) a lethal gynecological malignancy. Tumor associated neo-angiogenesis (TAN) is an early event in tumor development and represents a potential target for early detection. Traditional transvaginal ultrasound (TVUS) cannot detect TAN at early stage. The goal of this pilot study was to examine the enhancement of TVUS using αvβ3 integrins targeted microbubble ultrasound contrast agent (UCA) in laying hens, a preclinical spontaneous model of human OVCA. Methods: 3-4 years old hens (n=50) were selected randomly and scanned continuously by TVUS before, during and after UCA (Visister-integrins, Targeson Inc. CA) injection at brachial veins. UCA was visualized using a low mechanical index contrast imaging pulse sequence. All pre- and post-contrast injection images were archived and analyzed off-line. Gross diagnosis was recorded at euthanasia and tissues were processed for histology. Tumors and their types were confirmed by routine histology. TAN markers (SMA and αvβ3 integrins) were detected by immunohistochemistry. TAN vessels were counted and correlation with ultrasound prediction was examined. Results: αvβ3-integrins targeted UCA enhanced the visualization of ovarian tumors significantly in laying hens. At gray scale, UCA bounded areas appeared as a ring on the ovarian surfaces of 8 of 50 hens and were suspected for ovarian tumors. Tumors were confirmed in all hens predicted to have OVCA. In 7 hens tumors were early stage and in one hen the tumor metastasized to the oviduct. A microscopic lesion was found in one hen which was not detected by ultrasound imaging. Thus 9 of 50 hens had ovarian tumors and UCA detected approximately 88% at early stages. The frequency of immunopositive TAN vessels (SMA and αvβ3 integrins positive) were significantly higher in OVCA hens than normal hens (P<0.05) and was positively correlated (0.86, P< 0.05) with ultrasound prediction. Conclusions: Our results demonstrate that UCA targeting αvβ3 integrin enhanced TVUS detection of early stage ovarian tumors in a pre-clinical model and may form the foundation for a clinical study. Support: Department of Defense (#09-3303), Sramek Foundation.

Abstract 3190: Interleukin (IL-16) and tumor associated neo-angiogenesis detects ovarian cancer at early stage

Abstract Background: The lack of an early detection test is one of the reasons of high mortality rate of women due to epithelial ovarian cancer (OVCA). Understanding the pathogenesis and progression of OVCA is essential to establish an effective early detection test. Tumor associated -immuno-chemotaxis (TAI) and -neoangiogenesis (TAN) are the two earlier events in tumor development. CD8+ T cells in tumor vicinity produce immune-chemotactic IL-16 cytokines which stimulates the production of pro-angiogenic cytokines responsible for TAN. Ovarian TAI and TAN represent potential target for an effective early detection test. Access to patients at early stage OVCA is very difficult and laying hens develop spontaneous OVCA with histopathology similar to humans. Objectives: The goal of the present study was to determine the feasibility of markers of ovarian TAI and TAN in detecting early stage OVCA in laying hens. Materials and Methods: 3 years old laying hens (normal, low or stopped egg-laying) were scanned by Ultrasound, sera were collected, hens were euthanized and ovarian tissues were processed for paraffin and frozen sections, and mRNA extraction. Ovarian tumor stages were confirmed at gross and routine histology. Samples were divided into 4 groups namely normal (control), early stage OVCA [microscopic or tumors limited to the ovaries], late stage OVCA, with non-tumor ovarian abnormalities (atrophied ovaries). Sera were tested for IL-16 levels by ELISA and confirmed by 2D-Western blot (WB). Ovarian expression of TAN markerS (VEGF) and, neoangiogenic microvessel density (MVD) as well as IL-16 mRNA was determined. Results: The population of CD8+ T cells and the serum IL-16 levels were significantly higher in tumor hens (P &amp;lt; 0.05) than normal hens. Significant increase in IL-16 levels in hens with microscopic tumor (undetectable at gross examination) suggesting that serum IL-16 may be a potential indicator of ovarian tumors at very early stage. Differences in serum IL-16 levels were not observed between hens with non-tumor ovarian pathology and normal hens suggesting that increased serum IL-16 levels in OVCA hens are tumor specific. Two immunoreactive bands (12 &amp; 50kDa) for IL-16 were identified in the ovary while only one band (50 kDa) was detected in sera suggesting that IL-16 may be the active in tetramerized form. IL-16 protein and mRNA expression as well as the frequency of MVD were significantly higher in hens with OVCA than healthy hens. VEGF expressing microvessels were localized in the ovarian stroma preceding the tumor indicating that ovarian TAN precedes tumor progression. Conclusion: The results of the study suggest that changes in serum levels of IL-16 are positively correlated with tumor initiation and progression. Thus serum IL-16 level together with marker of ovarian TAN may constitute a feasible test for the early detection of OVCA. Support: Prevent Cancer Foundation, Sramak Foundation and DOD (OC#093303). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3190. doi:10.1158/1538-7445.AM2011-3190

Contrast-Enhanced Sonography Depicts Spontaneous Ovarian Cancer at Early Stages in a Preclinical Animal Model

Our goal was to examine the feasibility of using laying hens, a preclinical model of human spontaneous ovarian cancer, in determining the kinetics of an ultrasound contrast agent indicative of ovarian tumor-associated neoangiogenesis in early-stage ovarian cancer. Three-year-old White Leghorn laying hens with decreased ovarian function were scanned before and after intravenous injection of a human serum albumin-perflutren contrast agent at a dose of 5 μL/kg body weight. Gray scale morphologic characteristics, Doppler indices, the arrival time, peak intensity, and wash-out of the contrast agent were recorded and archived on still images and video clips. Hens were euthanized thereafter; sonographic predictions were compared at gross examination; and ovarian tissues were collected. Archived clips were analyzed to determine contrast parameters and Doppler intensities of vessels. A time-intensity curve per hen was drawn, and the area under the curve was derived. Tumor types and the density of ovarian microvessels were determined by histologic examination and immunohistochemistry and compared to sonographic predictions. The contrast agent significantly (P < .05) enhanced the visualization of microvessels, which was confirmed by immunohistochemistry. Contrast parameters, including the time of wash-out and area under the curve, were significantly different (P < .05) between ovaries of normal hens and hens with ovarian cancer and correctly detected cancer at earlier stages than the time of peak intensity. The laying hen may be a useful animal model for determining ovarian tumor-associated vascular kinetics diagnostic of early-stage ovarian cancer using a contrast agent. This model may also be useful for testing the efficacy of different contrast agents in a preclinical setting.

Unilateral tibio-tarsal rotation in a 14-month old ostrich hen destined for slaughter

The authors would like to report an unusual incidence of severe tibio-tarsal rotation in a 14-month old ostrich hen designed for slaughter and housed on an ostrich farm in Poland (51o43´N, 15o33´E, October 2007). The ambient indoor and exterior temperature from 0600-1800 was recorded at 2-hr intervals as 20.40 (mean) ± 0.12 (SEM) and 14.98 ± 1.35oC, respectively. The exterior relative humidity recorded likewise was 50.00 ± 0.67%. The pathology expressed itself in the left limb with an outward angle of rotation in the foot of 92o (Figures 1-3). The perpendicular height of the hen, and length of the right femur, tibia, metatarsals, toe and claw lengths were 120 vs . 140, 59 vs . 56, 36 vs . 42, 10 vs . 5.5, 6 vs . 11 and 3 vs . 4.5 cm, respectively by comparison with an age-matched normal hen.

Gene expression profiling reveals differentially expressed genes in ovarian cancer of the hen: support for oviductal origin?

Ovarian cancer has a high mortality rate due, in part, to the lack of early detection and incomplete understanding of the origin of the disease. The hen is the only spontaneous model of ovarian cancer and can therefore aid in the identification and testing of early detection strategies and therapeutics. Our aim was to combine the use of the hen animal model and microarray technology to identify differentially expressed genes in ovarian tissue from normal hens compared with hens with ovarian cancer. We found that the transcripts up-regulated in chicken ovarian tumors were enriched for oviduct-related genes. Quantitative real-time PCR and immunohistochemistry confirmed expression of oviduct-related genes in normal oviduct and in ovaries from hens with early- and late-stage ovarian tumors, but not in normal ovarian surface epithelium. In addition, one of the oviduct-related genes identified in our analysis, paired box 2 has been implicated in human ovarian cancer and may serve as a marker of the disease. Furthermore, estrogen receptor 1 mRNA is over-expressed in early-stage tumors, suggesting that expression of the oviduct-related genes may be regulated by estrogen. We have also identified oviduct-related genes that encode secreted proteins that could represent putative serum biomarkers. The expression of oviduct-related genes in early-stage tumors is similar to what is seen in human ovarian cancer, with tumors resembling normal Müllerian epithelium. These data suggest that chicken ovarian tumors may arise from alternative sites, including the oviduct.

Abstract A144: Anti-NMP antibodies in association with tumor-associated neoangiogenesis detects ovarian cancer at early stage

Abstract Background: The lack of an effective early detection test is one of the main reasons of high mortality due to ovarian cancer (OVCA). Changes in nuclear morphology including nuclear matrix proteins (NMPs) followed by the establishment of tumor associated neoangiogenesis (TAN) are the two earlier events in malignant transformation and progression of OVCA. NMPs are released in circulation, elicit autoimmune responses and anti-NMP antibodies are produced. Anti-NMP antibodies and ovarian TAN represent the potential marker of early stage OVCA. Identification of Patients at early stage OVCA is very difficult and laying hens have been shown to develop spontaneous OVCA with histopathology similar to humans. Objectives: The goals of the study were to determine the prevalence of anti-NMP antibodies and the feasibility of Doppler ultrasound (DUS) imaging in detecting ovarian TAN related to early stage OVCA in laying hens. Materials and Methods: Experiment-1: White Leghorn laying hens (3 years old with normal, low or stopped egg-laying) were scanned by DUS, sera were collected, hens were euthanized, ovarian tissues were processed for paraffin and frozen sections, and NMP extraction. Ovarian tumors with their stages were confirmed at gross and routine histology. Samples were divided into 3 groups: hens with normal ovaries (n= 8, control), with early stage OVCA [microscopic or tumors limited to the ovaries, n =7], with late stage OVCA (n = 5), with non-tumor ovarian abnormalities (atrophied ovaries, n = 2) and with non-ovarian tumors (gastrointestinal tumors, GI, n = 2). Sera were tested for the presence of anti-NMP antibodies against extracted ovarian NMP antigens by immunoassay and immunoreactivities were confirmed by 2D-Western blot (WB). Ovarian expression of TAN markers (VEGF and αvβ-integrins) was determined by immunohistochemistry (IHC). Experiment-2: Hens without any detectable ovarian abnormality were selected by DUS and divided into hens with (n =6) or without circulating anti-NMP antibodies (n = 3). Hens were monitored by DUS scanning at 15 week interval until they developed ovarian TAN or up to 45weeks and confirmed by gross, histology and IHC. Results: Sera from normal hens were negative for anti-NMP antibodies while 72% hens with early and 80% with late stage OVCA were positive against ovarian NMP antigens. Hens with ovarian atrophy or with GI tumors were negative for anti-NMP antibodies suggesting that these antibodies in OVCA hens are ovarian tumor specific. While no reactivity of sera from normal hens was observed in 2D-WB, sera from hens with early OVCA or late stage OVCA reacted against several ovarian NMP antigens with molecular weight range between 15–25 and 40–50 (kDa). In longitudinal study, 5 out 6 hens developed ovarian TAN between 30–45 weeks from initial scan. Conclusion: Anti-NMP antibodies appear in circulation in association with preneoplastic and microscopic ovarian lesions followed by the development of ovarian TAN before the tumor becomes detectable by ultrasound imaging. Anti-NMP antibodies together with ovarian TAN may constitute an early detection tool for OVCA. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A144.

Effects of DHA-enriched hen egg yolk and L-cysteine supplementation on quality of cryopreserved boar semen.

The objective of the present study was to determine the effects of docosahexaenoic acid (DHA)-enriched hen egg yolks and L-cysteine supplementation on the qualities of the cryopreserved boar semen. A total of 15 ejaculates from 5 Pietrain boars were divided into 4 groups according to the compositions of the freezing extenders used, that is, normal hen egg yolk (group I), DHA-enriched hen egg yolk (group II), normal hen egg yolk with 5 mmol L(-1) of cysteine supplementation (group III) and DHA-enriched hen egg yolk with 5 mmol L(-1) of cysteine supplementation (group IV). The semen was cryopreserved using controlled rate freezer and was thawed at 50 degrees C for 12 s. Progressive motility, sperm viability, acrosome integrity and functional integrity of sperm plasma membrane of the post-thawed semen were evaluated. The supplementation of L-cysteine in the freezing extender alone (group III) improved progressive motility (P < 0.05), and the supplementation of L-cysteine in combination with DHA-enriched hen egg yolk (group IV) improved both progressive motility (P < 0.05) and acrosome integrity (P < 0.01). The use of DHA-enriched hen egg yolk alone (group II) did not enhance any of the post-thawed semen qualities (P > 0.05). In conclusion, the supplementation of antioxidant L-cysteine alone or in combination with DHA-enriched hen egg yolk significantly improved the post-thawed semen qualities, especially progressive motility and acrosome integrity.

Prevalence of Antitumor Antibodies in Laying Hen Model of Human Ovarian Cancer

Antitumor antibodies are associated with tumors in human cancers. There is relatively little information on the timing and progression of antibody response to tumors. The objective of the study was to determine if spontaneous ovarian cancer in the egg-laying hen is associated with antitumor antibodies. Antibodies were detected by immunoassay and immunoblotting using proteins from normal ovary and ovarian tumors. Candidate antigens were identified by mass spectrometry of immunoreactive spots cut from 2-dimensional gels and Western blot. Antitumor (serum reacting against tumor ovarian extract) and antiovarian (serum reacting against normal ovarian extract) antibodies were significantly associated with ovarian cancer (67%; P <or= 0.001) compared with normal control hens. Hens with abnormal histology but no gross tumor had antitumor antibodies (63%; P <or= 0.025) but not antiovarian antibodies. There were common as well as different immunoreactions against normal ovary and homologous and heterologous tumor proteins in 2-dimensional Western blots. The candidate antigens included those commonly associated with human cancers and other diseases such as vimentin, apolipoprotein A1, Annexin II, enolase, DJ-1, and so on. The results suggest that antitumor antibodies are associated with ovarian cancer in hens, similar to human ovarian cancer. The egg-laying hen may be a model for understanding the antitumor humoral immune response, particularly at early tumor stages that are not readily accessible in human ovarian cancer.

E-cadherin expression in ovarian cancer in the laying hen, Gallus domesticus, compared to human ovarian cancer

Objective Epithelial ovarian carcinoma (EOC) is a leading cause of cancer deaths in women. Until recently, a significant lack of an appropriate animal model has hindered the discovery of early detection markers for ovarian cancer. The aging hen serves as an animal model because it spontaneously develops ovarian adenocarcinomas similar in histological appearance to the human disease. E-cadherin is an adherens protein that is down-regulated in many cancers, but has been shown to be up-regulated in primary human ovarian cancer. Our objective was to evaluate E-cadherin expression in the hen ovary and compare its expression to human ovarian cancer. Methods White Leghorn hens aged 185 weeks (cancerous and normal) were used for sample collection. A human ovarian tumor tissue array was used for comparison to the human disease. E-cadherin mRNA and protein expression were analyzed in cancerous and normal hen ovaries by immunohistochemistry (IHC), Western blot, and quantitative real-time PCR (qRT-PCR). Tissue fixed in neutral buffered formalin was used for IHC. Protein from tissue frozen in liquid nitrogen was analyzed by Western blot. RNA was extracted from tissue preserved in RNAlater and analyzed by qRT-PCR. The human ovarian tumor tissue array was used for IHC. Results E-cadherin mRNA and protein expression were significantly increased in cancerous hen ovaries as compared to ovaries of normal hens by qRT-PCR and Western blot. Similar expression of E-cadherin was observed by IHC in both human and hen ovarian cancer tissues. Similar E-cadherin expression was also observed in primary ovarian tumor and peritoneal metastatic tissue from cancerous hens. Conclusions Our findings suggest that the up-regulation of E-cadherin is an early defining event in ovarian cancer and may play a significant role in the initial development of the primary ovarian tumor. E-cadherin also appears to be important in the development of secondary tumors within the peritoneal cavity. Our data suggest that E-cadherin may prove to be an important target in the preventative treatment of metastatic ovarian cancer and further confirm that the laying hen is a good model for the study of human epithelial ovarian carcinoma.