Significant Increase In Proportion Research Articles

Abstract 201: DD3/PCA3 non-coding RNA regulates prostate cancer cell survival and modulates AR signaling

Abstract The prostate cancer antigen 3 (DD3/PCA3) is a non-coding RNA (ncRNA) specifically expressed in prostate tissues and overexpressed in prostate cancer (PCa) tumors. Although widely applied as a diagnostic marker for PCa, to date nothing has described about its role in PCa biology. We used herein small interfering RNA (siRNA) in order to knockdown DD3 mRNA message as an approach to elucidate DD3 functional roles in PCa cells. LNCaP cell line was been used herein as an in vitro model for DD3 functional assays. siRNA sequences were specifically designed for DD3 exon 4 mRNA sequences (siDD3), as well an scrambled siRNA (siScr), as negative control. LNCaP cells were transiently transfected with siDD3 or siScr and DD3 expression was analysed by real time PCR (qRT-PCR) using DD3 specific oligonucleotides. LNCaP cells transfected with siDD3 demonstrated a marked decrease in cell proliferation and viability, as compared to siScr transfected cells. Further, LNCaP cells in which DD3 was knocked-down presented a significant increase in proportion of cells in SubG0/G1 phase of cell cycle and presenting pyknotic nuclei, indicative of cells undergoing apoptosis. In order to investigate the putative mechanisms underlying the decrease of LNCaP cell survival as a result of DD3 knockdown, we then evaluated the involvement of DD3 on androgen receptor (AR) pro-survival signaling. DD3 expression was significantly uregulated as a result of LNCaP treatment with dihydrotestosterone (DHT), the active androgen metabolite. This effect was reverted by the addition of the AR antagonist, flutamide. Consistent to an AR activation by DHT treatment, LNCaP cells presented a significant upregulation of AR target genes. Notably, siDD3/LNCaP transfected cells significantly inhibited the expression of tested AR responsive genes. Besides, DD3 knockdown was able to counteract DHT stimulatory effects over AR target gene expression. Despite negatively modulating the transcription of AR target genes, DD3 knockdown did not alter Akt and ERK phosphorylation, suggesting that DD3 is mainly controlling the expression of signaling pathways downstream to AR activation. In summary, our findings indicate that DD3 is a ncRNA whose expression is AR regulated and is involved on the control of PCa cell survival and proliferation, in part by modulating the AR signaling pathway and its target genes. These [[Unsupported Character - fi]]ndings correspond to the first description of DD3 roles on PCa cells and could provide new insights into understanding prostate carcinogenesis, besides opening new prospects to use DD3 not only as a biomarker for PCa, but also as an specific target for therapeutic approaches aiming to inhibit PCa growth by negatively modulating AR pro-survival signal and their target genes. 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 201. doi:1538-7445.AM2012-201

Abstract A1: DD3/PCA3 non-coding RNA regulates prostate cancer cell survival and modulates AR signaling

Abstract The prostate cancer antigen 3 (DD3/PCA3) is a noncoding RNA (ncRNA) specifically expressed in prostate tissues and overexpressed in prostate cancer (PCa) tumors. Although widely applied as a diagnostic marker for PCa, to date nothing has described about its role in PCa biology. We used herein small interfering RNA (siRNA) in order to knockdown DD3 mRNA message as an approach to elucidate DD3 functional roles in PCa cells. LNCaP cell line has been used herein as an in vitro model for DD3 functional assays. siRNA sequences were specifically designed for DD3 exon 4 mRNA sequences (siDD3), as well an scrambled siRNA (siScr), as negative control. LNCaP cells were transiently transfected with siDD3 or siScr and DD3 expression was analyzed by real time PCR (qRT-PCR) using DD3 specific oligonucleotides. LNCaP cells transfected with siDD3 demonstrated a marked decrease in cell proliferation and viability, as compared to siScr transfected cells. This growth inhibition was specific for DD3 expressing cells. Further, LNCaP cells in which DD3 was knocked-down presented a significant increase in proportion of cells in SubG0/G1 phase of cell cycle and presenting pyknotic nuclei, indicative of cells undergoing apoptosis. In order to investigate the putative mechanisms underlying the decrease of LNCaP cell survival as a result of DD3 knockdown, we then evaluated the involvement of DD3 on androgen receptor (AR) pro-survival signaling. DD3 expression was significantly uregulated as a result of LNCaP treatment with dihydrotestosterone (DHT), the active androgen metabolite. This effect was reverted by the addition of the AR antagonist, flutamide. Consistent to an AR activation by DHT treatment, LNCaP cells presented a significant upregulation of AR target genes. Notably, siDD3/LNCaP transfected cells significantly inhibited the expression of tested AR responsive genes. Besides, DD3 knockdown was able to counteract DHT stimulatory effects over AR target gene expression. Despite negatively modulating the transcription of AR target genes, DD3 knockdown did not alter Akt and ERK phosphorylation, suggesting that DD3 is mainly controlling the expression of signaling pathways downstream to AR activation. Besides, DD3 mRNA expression was mainly detected in nuclei and ribosome cell compartments, indicating that in this cell niches DD3 ncRNA exert its main functional roles. In summary, our findings indicate that DD3 is a ncRNA whose expression is AR regulated and is involved on the control of PCa cell survival and proliferation, in part by modulating the AR signaling pathway and its target genes. These .ndings correspond to the first description of DD3 roles on PCa cells and could provide new insights into understanding prostate carcinogenesis, besides opening new prospects to use DD3 not only as a biomarker for PCa, but also as an specific target for therapeutic approaches aiming to inhibit PCa growth by negatively modulating AR prosurvival signal and their target genes. Citation Format: Luciana Bueno Ferreira, Antonio Palumbo, Kivviduarte de Mello, Felipe Leite, Cinthya A. Sternberg, Mauricio Caetano, Adriana Freitas Neves, Luiz Ricardo Goulart, Etel Rodrigues Pereira Gimba. DD3/PCA3 noncoding RNA regulates prostate cancer cell survival and modulates AR signaling [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A1.

Abstract 140: National Trends in Utilization and Outcomes of Endovascular Treatment in Acute Ischemic Stroke Patients

Background: With the availability of several new devices for mechanical thrombectomy after recent approvals by the Food and Drug Administration, the outcomes of patients undergoing endovascular treatment for acute ischemic stroke are expected to improve in United States. Objective: To evaluate trends in utilization of endovascular treatment and associated rates of death and disability among acute ischemic stroke patients over six years. Methods: We obtained data for patients admitted to hospitals in the United States between 2004 to 2009 with a primary diagnosis of ischemic stroke using a large national database. We determined the rate of utilization of endovascular treatment. Outcomes were classified as minimal disability, moderate to severe disability, and death based on discharge disposition. Results: Of the 670,069 patients with ischemic stroke, 72,342 (10.8%) received intravenous thrombolytic treatment, 18,229 (2.72%) had endovascular thrombolysis. Patients with endovascular treatment comprised 0.35% of ischemic strokes in 2004 vs 4.95% in 2009 (p < 0.001). The estimated number of patients undergoing endovascular treatment increased by 1,519% (362 to 5502) despite a 324% increase in patients receiving intravenous thrombolytic. The rates of intracranial hemorrhage remained unchanged throughout the 6 years. There was no significant difference in all age groups. The interaction between endovascular treatment among all ischemic stroke patients for predicting in-hospital mortality significantly improved (p <0.02). Conclusion: There has been a significant increase in proportion of acute ischemic stroke patients receiving endovascular treatment over the last 6 years with significant reduction in in-hospital mortality.

Abstract A28: DD3/PCA3 non-coding RNA regulates prostate cancer cell survival and modulates AR signaling

Abstract The prostate cancer antigen 3 (DD3/PCA3) is a non-coding RNA (ncRNA) specifically expressed in prostate tissues and overexpressed in prostate cancer (PCa) tumors. Although widely applied as a diagnostic marker for PCa, to date nothing has described about its role in PCa biology. We used herein small interfering RNA (siRNA) in order to knockdown DD3 mRNA message as an approach to elucidate DD3 functional roles in PCa cells. LNCaP cell line was used herein as an in vitro model for DD3 functional assays. siRNA sequences were specifically designed for DD3 exon 4 mRNA sequences (siDD3), as well an scrambled siRNA (siScr), as negative control. LNCaP cells were transiently transfected with siDD3 or siScr and DD3 expression was analysed by real time PCR (qRT-PCR) using DD3 specific oligonucleotides. LNCaP cells transfected with siDD3 demonstrated a marked decrease in cell proliferation and viability, as compared to siScr transfected cells. This growth inhibition was specific for DD3 expressing cells. Further, LNCaP cells in which DD3 was knocked-down presented a significant increase in proportion of cells in SubG0/G1 phase of cell cycle and presenting pyknotic nuclei, indicative of cells undergoing apoptosis. In order to investigate the putative mechanisms underlying the decrease of LNCaP cell survival as a result of DD3 knockdown, we then evaluated the involvement of DD3 on androgen receptor (AR) pro-survival signaling. DD3 expression was significantly upregulated as a result of LNCaP treatment with dihydrotestosterone (DHT), the active androgen metabolite. This effect was reverted by the addition of the AR antagonist, flutamide. Consistent to an AR activation by DHT treatment, LNCaP cells presented a significant upregulation of AR target genes. Notably, siDD3/LNCaP transfected cells significantly inhibited the expression of tested AR responsive genes. Besides, DD3 knockdown was able to counteract DHT stimulatory effects over AR target gene expression. Despite negatively modulating the transcription of AR target genes, DD3 knockdown did not alter Akt and ERK phosphorylation, suggesting that DD3 is mainly controlling the expression of signaling pathways downstream to AR activation. Besides, DD3 mRNA expression was mainly detected in nuclei and ribosome cell compartments, indicating that in this cell niches DD3 ncRNA exert its main functional roles. In summary, our findings indicate that DD3 is a ncRNA whose expression is AR regulated and is involved on the control of PCa cell survival and proliferation, in part by modulating the AR signaling pathway and its target genes. These findings correspond to the first description of DD3 roles on PCa cells and could provide new insights into understanding prostate carcinogenesis, besides opening new prospects to use DD3 not only as a biomarker for PCa, but also as an specific target for therapeutic approaches aiming to inhibit PCa growth through negatively modulating AR pro-survival signal and their target genes. Citation Format: Luciana Ferreira, Antonio Palumbo, Kivvi Mello, Felipe Leite, Cinthya Sternberg, Mauricio Caetano, Adriana Neves, Luiz Ricardo Goulart, Etel Gimba. DD3/PCA3 non-coding RNA regulates prostate cancer cell survival and modulates AR signaling [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A28.

Occupational asthma caused by IgE-mediated sensitization to multiple woods

Wood is a natural material that is able to trigger rhinitis and asthma in exposed subjects in occupational settings. This has been described with both soft and hard woods.1,2 Involvement of both low- and high-molecular-weight allergens has been reported, and the relevance of these is related with the wood type.1 There are cases where protein may be the responsible allergen. Crossreactivity between obeche and ramin woods3 and between obeche and latex4 has been shown. However, to the best of our knowledge, this is the first report of a multiple IgE-mediated sensitization to different woods that caused occupational respiratory symptoms in the same worker.

Changes in cortical morphology resulting from long-term amygdala damage

The amygdala's contribution to emotion, cognition and behavior depends on its interactions with subcortical and cortical regions. Amygdala lesions result in altered functional activity in connected regions, but it is not known whether there might be long-term structural sequelae as well. We hypothesized that developmental bilateral amygdala lesions would be associated with specific gray matter morphometric abnormalities in the ventromedial prefrontal cortex (vmPFC), anterior cingulate cortex (ACC) and the ventral visual stream. We conducted regions of interest and vertex-based analyses of structural MRI data acquired in two patients with long-standing focal bilateral amygdala lesions (S.M. and A.P.), compared to gender- and age-matched healthy comparison subjects. Both patients showed significant proportional increases in gray matter volume of the vmPFC. Cortical thickness was increased in the vmPFC and ACC and decreased in the ventral visual stream. There were no morphometric changes in dorsolateral prefrontal cortex or dorsal visual stream cortices. These findings support the hypothesis that cortical regions strongly connected with the amygdala undergo morphometric changes with long-standing amygdala damage. This is the first evidence in humans of the remote alteration of brain morphology in association with amygdala lesions, and will help in interpreting the structural and functional consequences of amygdala pathology in neuropsychiatric disorders.

A Review of the Current Profile of Gastric Cancer Presentation in the University College Hospital Ibadan, a Tertiary Health Care Institution in the Tropics

Gastric cancer is the fourth commonest malignancy and the second leading cause of cancer-related death. Although gastric carcinoma is less common throughout Africa than in Europe, there are considerable variations in its incidence and pattern. It accounts for about 5% of cancer-related death. It is characterized with significant morbidity and mortality mainly because of late presentation in developing and poor countries. Previous studies on gastric cancer in Ibadan and other West African centres demonstrated the preponderance of distal (pyloric antrum) gastric lesions when compared to proximal (cardia, fundus) lesions. Nevertheless, recent studies in developed nations show that distal gastric lesions are on the decline while there is an increase in the proportion of proximal gastric lesions. The objective of this study is to review the pattern of presentation of patients with gastric carcinoma managed in our surgical division over a 5-year period and to determine changes in the trend in our environment. A retrospective study of all patients with gastric carcinoma between November 2004 and October 2009 was carried out. Simple descriptive analysis was used to characterize the patients' demographic parameters, symptomatology, clinical and investigative findings along with treatment and outcome modalities. There were 49 cases managed by the division over the period under review. The male to female ratio was 1.45:1 with a mean age of 56 years at presentation. Duration of symptom was less than 5 months (20 weeks) in 47.9% of the patients. Dysphagia was present in 12.2% while 52.6% had a history of suspected peptic ulcer disease. There was electrolyte derangement in 31.7% of the patients while 52.6% had anaemia at presentation. Proximal tumours of the gastro-oesophageal region, cardia and the body constituted 51% of the cases; 51.4% of the patients were blood group O as opposed to 28.6% and 20%, respectively, with blood A and B. Thirty-six patients (73.5%) had a histological diagnosis of adenocarcinoma, five patients (10%) had signet ring variant of adenocarcinoma carcinoma, while three patients (6.1%) were each had gastrointestinal stromal tumours or lymphomas. Our review shows a peak age in the sixth and seventh decades at presentation. There is a significant increase in the proportion of proximal gastric lesions and a predominance of blood group O. Gastrointestinal stromal tumour and lymphoma should be considered as differential diagnosis. Patients still present late with advanced diseases, and curative treatment is often impossible.

698 Effects of Casein and Adaptedmilk Formula on the Proliferation of Lymphocytes, Mid-Cells and Granulocytes and on Circulting Lipids

Background: The casein is the main protein that appears to be involved in childhood diseases caused by the consumption of adapted infant-milk formula (IMF). Objective: To measure the effect of casein of one of the most adapted IMF marketed in Algeria, on the proliferation of lymphocytes, granulocytes and cells MID and on the change in lipid profile. Materials and methods: Three groups of male rabbits of local breed were used in this study. The first group (n=5, age [±standard error]; 2.900±0.218 months, weight; 936±46.6 g) received different concentrations of casein, three-times daily for 3 days. The second one (n=5) received whole milk by gavage at 5 mL, three-times daily for 3 days (age; 2.7±0.289 months, weight; 932±38.26 g). The third group (n=8) was considered as controls (age; 2.625±0.286 months, weight; 892.5±60.70 g). Results: Serum levels of TG and VLDLc were significantly lower in rabbits receiving whole milk compared to controls (p=0.001 for both comparisons); however, those of LDL were significantly increased in rabbits receiving the casein solution (p=0.046). Additionally, oral administration of casein or milk caused a significant increase in rates and proportions of leucocytes, granulocytes, cells MID, and a significant decrease in the proportion of lymphocytes. Conclusions: Casein or IMF consumption can induce increased activity of phagocytes, eosinophils and basophils and inhibition of lymphocytes proliferation. Additionally, whole milk could be beneficial in the prevention of overweight and childhood obesity, while milk casein can cause an atherogenic risk at high concentrations.

Protective Effect of Guaraná (Paullinia cupana var. sorbilis) Pre-treatment on Cadmium-Induced Damages in Adult Wistar Testis

Guaraná (Paullinia cupana) is an Amazonian plant. Its antioxidant potential was demonstrated to be due to the high polyphenol concentration. On the other hand, one of the mechanisms underlying cadmium-induced cellular damage is free radical mediated, resulting in increased oxidative processes. This study investigated P. cupana's potential to attenuate cadmium-induced damages in Wistar rat testis. Adult male Wistar rats were either pre-treated with 2mg/g body weight (BW) of powdered P. cupana seed during 56days and/or injected with cadmium chloride at a dose of 1.15mg/kg BW. After cadmium exposition (48h), testes samples were evaluated by histological and stereological analyses. Both groups exposed to cadmium presented evident morphological alterations relative to control animals. A few rodents showed massive cell death in the seminiferous epithelium and intertubular space, indicating that some animals are more sensitive to cadmium. Despite the alterations observed in both groups, pre-treatment with P. cupana was effective in attenuating morphological changes in Leydig cells, as well as reducing inflammatory response, relative to animals exclusively exposed to the metal. Animals treated only with P. cupana presented a significant increase in plasma testosterone levels and a significant increase in volumetric proportions of seminiferous tubules, which are indicative of spermatogenic stimulation.

Impact of erythropoiesis-stimulating agent (ESA) reimbursement policy change on transfusion frequency for patients with chemotherapy-induced anemia (CIA).

e16507 Background: Two ESAs (darbepoetin alfa, epoetin alfa) are FDA-approved for reducing transfusions in patients (pts) with CIA. Clinical trial data have reported adverse safety signals in this population with ESA treatment targeting hemoglobin (Hb) > 12g/dL. In April 2007, UnitedHealthcare implemented a non-coverage policy for non-Medicare pts with Hb > 12g/dL. Previously, no Hb restrictions existed. The impact of this policy change on the proportion of ESA-treated pts with CIA requiring transfusion was evaluated. Methods: Retrospective claims of the i3 Innovus database from 7/1/2005 to 6/30/2008 were analyzed. Subjects were commercial enrollees ≥ 18 years with cancer and chemotherapy treatment, ≥ 2 ESA claims, and ≥ 1 qualifying treatment episode, which was defined as number of days from first to last ESA administration (no ESA claim for 90 days prior to first claim and 90 days after last claim). Subjects with claims for surgical procedures or dialysis were excluded. Proportion of pts transfused was evaluated comparing pts that initiated ESA treatment between 1/2006–9/2006 (prechange) and 7/2007–3/2008 (postchange). Results: 4,371 pts were identified; 2,775 and 1,709 had ≥ 1 ESA treatment episode in the pre-change and post- change periods, respectively. Age, gender, and tumor type were similar across both time periods. ESA treatment duration was longer in the prechange compared to postchange period (64.9 vs. 59.6 days; p < 0.001). In the prechange period, 11.5% of pts had ≥ 1 transfusions compared with 15.4% in postchange (p < 0.001). By subset analysis, the largest proportion of pts requiring transfusion was in those with lung cancer (19.8% pre, 29% post; p = 0.009). Conclusions: A small but significant increase in proportion of pts transfused and a small but significant decrease in ESA treatment duration was observed in the post-policy period. It is unknown if the increased transfusion rate was a result of policy implementation or the concurrent changes in the Centers for Medicare and Medicaid Services' coverage criteria for initiation of ESA therapy. Further study should evaluate the long-term impact of the policy on other clinical outcomes. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Centocor Ortho Biotech, United Healthcare Johnson & Johnson Johnson & Johnson

Effect of humic substances on nutrient uptake by herbage and on production and nutritive value of herbage from sown grass pastures

AbstractThe effect of humic substances on the nutrient uptake, herbage production and nutritive value of herbage from sown grass pastures was studied in six field experiments. Commercial humic substances were applied in combination with mineral fertilizer or slurry, either as a solution (HF liquid; 8·3 kg humic substances ha−1) or incorporated into the mineral fertilizer (HF incorporated; 3·6 to 6·4 kg humic substances ha−1). A series of cuts, ranging from two to five cuts, was taken during the growing season. The general response in herbage production to application of humic substances was an increase in herbage mass of dry matter (DM) at the first cut although this was only significant in two experiments for the HF incorporated treatment. Total herbage production of DM over the growing season, however, was similar for treatments with or without application of humic substances. The overall effect of HF incorporated and HF liquid on the herbage mass of DM at the first cut across the experiments was calculated using a meta‐analysis technique and it was shown that there was a significant proportional increase of 0·14 (P &lt; 0·05) with the HF incorporated treatment and a non‐significant increase of 0·08 with the HF liquid treatment compared to the control treatment. The nutritive value of the herbage at the first cut was similar across all treatments. In general nitrogen, phosphorus and potassium uptake at the first grass cut was higher after application of humic substances but only in one experiment was this increase statistically significant.

Loss of Tafazzin (TAZ) Function and Accelerated Apoptosis of Human Bone Marrow Stem and Myeloid Progenitors in Barth Syndrome.

Abstract 549Barth syndrome (BTHS) is a severe X-linked stem cell disorder characterized by neutropenia, cardio- and skeletal myopathies, and growth retardation. Barth patients have a high rate of mortality due to progressive cardiomyopathy and/or overwhelming bacterial infections. Majority of Barth patients have mutations in the tafazzin (G4.5 or TAZ) gene that appear to truncate the tafazzin protein resulting in the loss of TAZ function. Based on protein homology, tafazzin is a phospholipid acyltransferase involved in remodeling cardiolipin (CL), the main lipid of the inner mitochondrial membrane. Therefore, BTHS patients exhibit reduced levels of total CL and accumulation of monolysocardiolipin. However, the function of TAZ protein and how these metabolic defects are triggered by TAZ mutations in BTHS remain largely unknown. The cellular or mouse models of this disorder are not availabel yet, and thus, the link between TAZ mutations and severe neutropenia in Barth syndrome remains elusive. Earlier study reported increased annexin V staining but absence of apoptosis in peripheral blood neutrophils. However, the patients' bone marrow stem and myeloid progenitor cells have not been examined. We hypothesized that TAZ mutations trigger accelerated apoptosis of bone marrow stem/progenitor cells, which in turn leads to reduced production of neutrophils in the bone marrow and severe neutropenia in Barth patients. To test this hypothesis, we used TAZ-specific shRNA to knock down the expression of the tafazzin gene in human myeloid progenitor HL60 cells and examined its effect on cell survival. Transfection of human myeloid progenitor cells with two different TAZ-specific but not control scrambled shRNA results in substantial down-regulation in the tafazzin expression level as determined by Western blot and confirmed by RT-PCR using TAZ and GAPDH-specific primers. Human myeloid progenitor cells with knocked down TAZ expression exhibit significantly elevated dissipation of mitochondrial membrane potential compared with control cells with scrambled shRNA as evidenced by flow cytometry analysis of DIOC6-labeled cells ((p<0.0009, n=3). A remarkably significant increase in proportion of apoptotic annexin-positive cells was also observed in response to knock-down of TAZ expression in myeloid progenitor cells compared with control cells with scrambled shRNA (p<0.0002, n=6). The observed increase in apoptosis in response to TAZ knock-down was caspase-3 dependent as evidenced by Western blot analysis. Treatment of the cells with caspase-specific inhibitor zVAD-fmk significantly improved cell survival characteristics to near normal level as determined by flow cytometry (p<0.02, n=4). Interestingly, knock-down of TAZ expression in human lymphoid cells failed to affect their cell survival or mitochondrial membrane potential, indicating that the loss of TAZ function exhibits lineage-specific effect. Analysis of bone marrow-derived CD34+ stem cells from a Barth patient positive for TAZ mutation cultured 24h in the presence of 10% autologous serum revealed nearly 3-fold increase in proportion of apoptotic annexin V positive CD34+ cells compared with the same cell subpopulation from a healthy volunteer (36% in Barth vs 13% in ctrl). More differentiated CD15+ neutrophil precursors also exhibit substantially increased rate of apoptosis compared with control (Barth 27% vs ctrl 17%). CD33+ myeloid-committed progenitor cells from Barth patient do not show increased annexin V binding compared with corresponding cell subpopulation from a healthy volunteer, but demonstrate approximately 2-fold increase in dissipation of mitochondrial membrane potential compared with control donor cells. Thus, these data demonstrate that tafazzin functions as an anti-apoptotic protein, the loss of function of the TAZ gene is cytotoxic to hematopoietic cells, and that severe neutropenia in patients with Barth syndrome is due to accelerated apoptosis of bone marrow myeloid progenitor cells. Our data also reveal that caspase-specific inhibitors may represent potentially therapeutic agents capable of restoring normal production of myeloid cells in Barth Syndrome. Disclosures:No relevant conflicts of interest to declare.

The relationship of various muscular and skeletal scores and ultrasound measurements in the live animal, and carcass classification scores with carcass composition and value of bulls

The relationships of live animal muscular and skeletal scores and ultrasound measurements and carcass conformation and fat scores with carcass composition and value were determined using 74 bulls. The animals consisted of 53 late-maturing breed crosses and 21 Holstein–Friesian slaughtered at 13 to 17 months of age. They were offered concentrates ad-libitum and 1 kg of grass silage dry matter per head daily for the final 139 day finishing period. Live animal muscular and skeletal scores and ultrasonic muscle and fat depth measurements of the M . longissimus dorsi were recorded at 8 to 12 months of age and pre-slaughter. Following slaughter, carcasses were classified for conformation and fatness and the right side of each carcass was dissected into meat, fat and bone. Carcass conformation and fat scores, (scale 1 to 15) ranged from 4.7 to 14.4 and 2.7 to 11.5, respectively. Pre-slaughter muscular scores showed significant positive correlations with kill-out proportion ( r = 0.82), carcass meat proportion ( r = 0.72), conformation score ( r = 0.94), carcass value ( r = 0.72), and the proportion of high-value meat cuts in the carcass ( r = 0.49), and significant negative correlations with carcass bone ( r = − 0.89) and fat ( r = − 0.32) proportions. The associations between pre-slaughter muscular scores and proportion of high-value cuts in meat, perinephric plus retroperitoneal fat and fat score were not significant. Corresponding correlations with muscular scores at 8 to 12 months of age were generally lower than those recorded pre-slaughter. Correlations of ultrasound muscle depth with carcass traits showed similar trends but lower values to those obtained using the muscular scoring procedure. Ultrasound fat depth pre-slaughter was positively correlated with carcass fat proportion ( r = 0.56) and fat score ( r = 0.54), and negatively correlated with carcass meat proportion, proportion of high-value cuts and carcass value. Correlations with other carcass traits were not significant. Correlations of live animal skeletal scores with carcass traits were generally non-significant. A one unit (scale 1–15) increase in carcass conformation score was associated with significant increases in kill-out proportion, meat yield and carcass value of 11.9 g/kg, 11.9 g/kg and 5.8 cent/kg, respectively. Corresponding effects for a one unit change in fat score were − 2.9 g/kg, − 11.1 g/kg and − 4.9 c/kg. In conclusion, live animal muscular scores and ultrasound measurements and carcass conformation and fat scores were shown to be useful predictors of carcass composition and value.

The microbiota on different oral surfaces in healthy children

Knowledge of the early oral colonization patterns could provide a better understanding of oral biofilm development and disease initiation that in turn could be the basis for early preventive programmes. Microbial samples were collected from five different oral habitats from a total of 93 children (age 3-12 years), attending the Dental School of the University of Athens, who were split into three age groups. A total of 38 microbial species were sought out by the checkerboard DNA-DNA hybridization technique. All of the test species, except Parvimonas micra and Porphyromonas gingivalis, differed significantly among sample locations providing quite distinct microbial profiles for the different oral surfaces. Supragingival and subgingival plaque had similar profiles and exhibited higher proportions of Actinomyces species and Green complex while soft tissue samples were dominated by streptococci of the Yellow complex. The profiles of the tongue dorsum and saliva were also similar. Many of the species were in similar proportions in all three age groups for a given location. Periodontal pathogens showed increases in proportions with increasing age. Specifically, the Red complex species (Tannerella forsythia, P. gingivalis, Treponema denticola) showed a significant increase in proportion with age (P < 0.05) in all sample locations. The results showed a pattern of colonization in children similar to that previously found in adults. Differences in the profile between age groups suggest a gradual maturation of the oral microbiota, with it being made up of an increasing number of Orange and Red complex species.

Abstract No. 354: Does Tip of the Implantable Port Catheter Really Migrate to the Cephalic Direction Dragged by Downward Force of the Implantable Venous Access Port in Upright Position?

To reassess whether catheter tip of the implantable venous access port really migrates to the cephalic direction dragged by downward force of the port in upright position. A total of 87 patients who underwent placement of the port through internal jugular vein under fluoroscopic guidance were included in this study. There were 36 men and 51 women, aged 54.4 years (range, 23-76 years). The catheter position was evaluated on the image obtained in the supine position during the procedure and chest radiograph in the upright position just after the procedure. In each position, we calculated the vertical distance from the highest point of the catheter to the catheter tip (long segment) and point of port and catheter (short segment) junction. We drew a horizontal line crossing the upper margin of the transverse process of the first thoracic spine and measured the vertical distance from the line to the highest point of the catheter. Difference in magnification on both images was corrected by measurement of the height of the first 3 thoracic vertebral bodies including disc spaces. Length differences of matching two segments in each position were attained. Correlation analysis was used to demonstrate statistical significance. Vertical lengths from the first thoracic spine to the long and short segments increased by 10.3mm±7.37 and 15.3mm±2.12, respectively. They showed statistically significant (r=-0.425, p<0.01) proportional increase in length. The highest point of the catheter moved downward an average of 12.75mm±1.78 in the upright position. This tendency did not change according to sex or side of insertion. The length of the implantable port catheter tip from the highest point actually increased in the upright position rather than being dragged and shortened by the downward migration force of the port. We presume that both segments of the catheter simply appear to have moved downward in the upright position due to gravitational effects and redundant soft tissue of the anterior chest wall.

FOXP3 expression accurately defines the population of intratumoral regulatory T cells that selectively accumulate in metastatic melanoma lesions

AbstractRegulatory T (Treg) cells are often found in human tumors; however, their functional characteristics have been difficult to evaluate due to low cell numbers and the inability to adequately distinguish between activated and Treg cell populations. Using a novel approach, we examined the intracellular cytokine production capacity of tumor-infiltrating T cells in the single-cell suspensions of enzymatically digested tumors to differentiate Treg cells from effector T cells. Similar to Treg cells in the peripheral blood of healthy individuals, tumor-infiltrating FOXP3+CD4 T cells, unlike FOXP3− T cells, were unable to produce IL-2 and IFN-γ upon ex vivo stimulation, indicating that FOXP3 expression is a valid biological marker for human Treg cells even in the tumor microenvironment. Accordingly, we enumerated FOXP3+CD4 Treg cells in intratumoral and peritumoral sections of metastatic melanoma tumors and found a significant increase in proportion of FOXP3+CD4 Treg cells in the intratumoral compared with peritumoral areas. Moreover, their frequencies were 3- to 5-fold higher in tumors than in peripheral blood from the same patients or healthy donors, respectively. These findings demonstrate that the tumor-infiltrating CD4 Treg cell population is accurately depicted by FOXP3 expression, they selectively accumulate in tumors, and their frequency in peripheral blood does not properly reflect tumor microenvironment.

Knock-Down of TAZ Gene Expression: A Model of Barth Syndrome with Accelerated Apoptosis of Myeloid Progenitor Cells Improved upon Treatment with Caspase-Specific Inhibitor

Barth syndrome (BTHS) is an X-linked recessive disorder characterized by neutropenia, cardiac and skeletal myopathies, and growth retardation. The mortality is high due to progressive cardiomyopathy and/or overwhelming bacterial infections. The incidence of BTHS is estimated to be as high as 1 in 100,000, but it is still a poorly recognized disease. The majority of Barth patients have mutations in the tafazzin (G4.5 or TAZ) gene, most of which appear to truncate the tafazzin protein likely resulting in the loss of its function. Tafazzin is a phospholipid acyltransferase involved in remodeling cardiolipin, the main lipid of the inner mitochondrial membrane. As a result, BTHS patients exhibit reduced levels of total CL and accumulation of monolysocardiolipin. A drosophila model of the Barth syndrome was recently reported, but cellular or mouse models of this disorder are not yet available. The link between these metabolic defects triggered by TAZ mutations and neutropenia remains largely unknown. We hypothesized that TAZ mutations lead to the loss of function of tafazzin protein causing impaired cell survival of neutrophil precursors, reduced production of neutrophils and neutropenia. To test this hypothesis, we knocked down the expression of the tafazzin gene in human myeloid progenitor HL60 cells using TAZ-specific shRNA and examined its effect on cell survival. Four shRNAs specific to exons 4 through 7 of the TAZ gene were used for transfection of human myeloid progenitor HL60 cells that were later examined by flow cytometry and Western blot analyses. At least 2 of the shRNA constructs resulted in substantial down-regulation in the expression level of the tafazzin gene in transfected human myeloid progenitor cells as determined by Western blot and confirmed by RT-PCR using gene-specific and GAPDH-specific primers. Flow cytometry analysis of DIOC6-labeled cells revealed that knock-down of the TAZ gene expression was associated with a significantly elevated dissipation of mitochondrial membrane potential compared with control cells with scrambled shRNA ((p<0.0009, n=3). Apoptosis studies using flow cytometry analyses revealed a significant increase in proportion of apoptotic annexin-positive cells compared with control cells transfected with scrambled shRNA (p<0.0002, n=6). The observed increase in apoptosis in response to TAZ knock-down was caspase3-dependent as evidenced by Western blot analysis. Treatment of the cells with caspase-specific inhibitor zVAD-fmk significantly improved cell survival characteristics to near normal level as determined by flow cytometry (p<0.02, n=4), suggesting that caspase-specific inhibitors may represent potentially therapeutic agents for patients with Barth syndrome. Thus, these data demonstrate that the loss of function of the TAZ gene is cytotoxic to hematopoietic cells and suggest that severe neutropenia is due to accelerated apoptosis of myeloid progenitor cells.

A single immunization with a dry powder anthrax vaccine protects rabbits against lethal aerosol challenge

Here we confirm that intranasal (IN) dry powder anthrax vaccine formulations are able to protect rabbits against aerosol challenge 9 weeks after a single immunization. The optimum dose of rPA in our dry powder anthrax vaccine formulation in rabbits was experimentally determined to be 150μg and therefore was chosen as the target dose for all subsequent experiments. Rabbits received a single dose of either 150μg rPA, 150μg rPA+150μg of a conjugated 10-mer peptide representing the Bacillus anthracis capsule (conj), or 150μg of conj alone. All dry powder formulations contained MPL and chitosan (ChiSys®). Significant anti-rPA titers and anthrax lethal toxin neutralizing antibody (TNA) levels were seen with both rPA containing vaccines, although rPA-specific IgG and TNA levels were reduced in rabbits immunized with rPA plus conj. Nine weeks after immunization, rabbits were exposed to a mean aerosol challenge dose of 278 LD50 of Ames spores. Groups immunized with rPA or with rPA+conj had significant increases in survivor proportions compared to the negative control group by Logrank test (p=0.0001 and 0.003, respectively), and survival was not statistically different for the rPA and rPA+conj immunized groups (p=0.63). These data demonstrate that a single immunization with our dry powder anthrax vaccine can protect against a lethal aerosol spore challenge 9 weeks later.

Effects of in vivo hyperthermia on natural killer cell activity, in vitro proliferative responses and blood mononuclear cell subpopulations

This work was designed to test the hypothesis that elevations in body temperature of humans induce immunostimulation. Eight healthy volunteers were immersed in a water bath (water temperature 39.5 degrees C) for 2 h, during which their rectal temperature rose to 39.5 degrees C. On a later day they served as their own controls, being immersed into thermoneutral water (34.5 degrees C) for 2 h. Blood samples were collected before immersion, at body temperatures of 38 degree C, 39 degree C and 39.5 degree C, and 2 h after water immersion. The interleukin-2 (IL-2) enhanced natural killer (NK) cell activity (lysis per fixed number of mononuclear cells), as well as the proportion and total number of NK cells (CD16+ cells), increased significantly during hyperthermia compared with control values. The lymphocyte proliferative responses did not differ significantly between hyperthermia and thermoneutral conditions. The proportion of pan-T (CD3+) cells was maximally depressed 2 h after water immersion. The decreased proportion of CD3+ cells was mainly due to a decreased percentage of CD4+ cells (not significant). The proportion of B cells (CD19+ cells) did not fluctuate significantly, while a marked and significant increase in monocyte proportion (CD14+ cells) was found 2 h after hyperthermia. Two hours after hot water immersion the lymphocyte concentration declined while the neutrophil and monocyte concentrations were augmented. Induced hyperthermia causes significantly increased serum cortisol, plasma norepinephrine and plasma epinephrine concentrations compared to controls. It is possible that the altered immune functions induced by elevated body temperature can be ascribed to altered composition and function of blood mononuclear cells induced by elevated levels of stress hormones.

Effects of Arginine Vasopressin on Oxygenation and Haemodynamics during One-Lung Ventilation in an Animal Model

In a case of arterial hypotension during one-lung ventilation, haemodynamic support may be required to maintain adequate mean arterial pressure. Arginine vasopressin, a potent systemic vasoconstrictor with limited effects on the pulmonary artery pressure, has not been studied in this setting. Twelve female pigs were anaesthetised and ventilated and arterial, central venous and pulmonary artery catheters were inserted. A left-sided double lumen tube was placed via tracheostomy and one-lung ventilation was initiated. The animals were in the left lateral position, with the left lung ventilated and right lung collapsed. Respiratory and haemodynamic values were recorded before and during a continuous infusion of arginine vasopressin sufficient to double the mean arterial pressure. The arginine vasopressin caused a decrease in cardiac output (3.8+/-1.1 vs. 2.7+/-0.7 l/min, P <0.001) and mixed-venous oxygen tension (39.1+/-5.8 vs. 34.4+/-5 mmHg, P=0.003). Pulmonary artery pressure was unchanged (24+/-2 vs. 24+/-3 mmHg, P=0.682). There was no effect of the arginine vasopressin on arterial oxygen tension (226+/-106 vs. 231+/-118 mmHg, P=0.745). However, there was a significant decrease in shunt fraction (28.3+/-6.2 vs. 24.3+/-7.8%, P=0.043) and a significant proportional increase in perfusion of the ventilated lung (78.8+/-9.5 vs. 85.5+/-7.9%, P=0.036). In our animal model of one-lung ventilation, doubling mean arterial pressure by infusion of arginine vasopressin significantly affected global haemodynamics, but had no influence on systemic arterial oxygen tension.