Tissue Supernatants Research Articles

Combined effects of vitamin E and omega-3 fatty acids on protecting ambient PM2.5-induced cardiovascular injury in rats

ObjectiveThis study aims to observe whether the combined treatment with vitamin E (vit E) and omega-3 polyunsaturated fatty acids (Ω-3 FA) could prevent the fine particulate matter (PM2.5)-induced cardiovascular injury through alleviating inflammation and oxidative stress. At the same time, the appropriate combination dosage of vit E and Ω-3 FA was explored to find an optimized protective dose to protect the injury induced by PM2.5. Methods: The SD rats were pretreated with different concentration of vit E and Ω-3 FA separately or jointly. Then the rats were exposed to ambient PM2.5 by intratracheal instillation for three times. The expression of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) in serum and supernatant of cardiac tissue were detected by ELISA kits. The levels of malondialdehyde (MDA), superoxide Dismutase (SOD) and glutathione-peroxidase (GSH-Px) in myocardium and the level of MDA in serum were measured. Meanwhile, the cardiac injury was evaluated by histopathological examination. Results: Compared with the severe injury of rats in PM2.5 exposure group, the rats in vit E or Ω-3 FA-pretreated groups had a slighter injury in heart. Meanwhile, pretreatment with vit E or Ω-3 FA induced a significantly alleviation of the inflammatory cytokines (TNF-α, IL-1β, IL-6) and the elevation of the anti-oxidative activity especially in the rats pretreated with combined vit E and Ω-3 FA. In addition, the combined protecting effects of vit E and Ω-3 FA showed a dose-dependent manner. Conclusion: Supplementation with vit E and Ω-3 FA could protect the PM2.5-induced injury, and the combination of vit E and Ω-3 FA might produce more effective effects than the separate nutrient did.

Different roles of Staphylococcus aureus enterotoxin in different subtypes of nasal polyps.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a multifactorial disease. The pathogenesis of CRSwNP remains unclear. This study was designed to investigate the role of inflammation and Staphylococcus aureus enterotoxin (SE) in this disease. The study included a total of 74 patients with CRSwNP and 6 controls. A serum Phadiatop assay was conducted to detect atopy status, and serum eosinophil cationic protein (ECP) and total immunoglobulin (Ig)E levels were determined using ELISA. SEA, SEB, total IgE, ECP and myeloperoxidase (MPO) levels in nasal tissue supernatant were measured using ELISA. The results indicated that 15 (22.1%) patients had systemic allergies. On the basis of the ECP/MPO ratio, the patients were divided into an eosinophilic CRSwNP group (n=18) and a non-eosinophilic CRSwNP group (n=56). The total ECP/MPO ratio was 0.572, with a notable bias toward neutrophilic inflammation. The supernatant ECP and MPO levels were elevated in the CRSwNP group compared with the control group, but no significant difference in the serum total IgE and ECP levels were observed between the CRSwNP and control groups. In addition, the non-eosinophilic and eosinophilic CRSwNP groups showed significant elevations in supernatant total IgE, SEA and SEB levels compared with the control group. Thus, it may be concluded that allergy is a common pathogenesis of CRSwNP, and neutrophilic inflammation is present in most Chinese CRSwNP patients. Additionally, local indicators reflect the inflammatory status more accurately than do serum indicators. SEs may act as an infection factor rather than as a superantigen in Chinese non-eosinophilic CRSwNP patients. Thus, long-term antibiotic therapy may be an option for Chinese non-eosinophilic CRSwNP patients.

Interleukin-17A (IL-17A) and IL-17F Are Critical for Antimicrobial Peptide Production and Clearance of Staphylococcus aureus Nasal Colonization.

Approximately 20% of the population is persistently colonized by Staphylococcus aureus in the nares. Th17-like immune responses mediated by the interleukin-17 (IL-17) family of cytokines and neutrophils are becoming recognized as relevant host defense mechanisms for resolution of S. aureus mucocutaneous infections. Since antimicrobial peptides are regulated by the IL-17 cytokines, we sought to determine the role of IL-17 cytokines in production of antimicrobial peptides in a murine model of S. aureus nasal carriage. We discovered that nasal tissue supernatants have antistaphylococcal activity, and mice deficient in both IL-17A and IL-17F lost the ability to clear S. aureus nasal colonization. IL-17A was found to be sufficient for nasal mBD-3 production ex vivo and was required for CRAMP, mBD-3, and mBD-14 expression in response to S. aureus colonization in vivo These data were confirmed in a clinical study of nasal secretions in which elevated levels of the human forms of these antimicrobial peptides were found in nasal secretions from healthy human subjects when they were colonized with S. aureus but not in secretions from noncolonized subjects. Together, these data provide evidence for the importance of IL-17A regulation of antimicrobial peptides and IL-17F in the clearance of S. aureus nasal carriage.

607. Organotypic Culture of Adult Porcine Retina as an In Vitro Screening Model for AAV-Mediated Gene Therapy in Ophthalmology

Introduction: Gene therapy studies in vivo using animal models provide critical information regarding vector specificity, therapeutic efficacy, and safety. However, long durations to observe transgene expression, high degree of variability between animals, and reduced control of experimental conditions often leads to large sample sizes and expense. Organotypic cultures of the retina can provide an efficient in vitro model with a high degree of throughput and control that can be used for pharmacological screening. These explants preserve complex intercellular processes and communications among the neural retinal layers with minimal manipulation of tissue architecture and molecular pathways, making it a useful model in target-tissue validation of adeno-associated virus (AAV) vector variants. Porcine retina has similar anatomical and physiologic features to the human retina and is therefore a suitable surrogate for preclinical testing in ophthalmology. Methods: We have developed a porcine explant culture system from retinas isolated from adult pigs as a model to screen novel rAAV vectors for transduction efficiency and cellular tropism. Pig eyes were enucleated and the neural retina dissected away from the pigment epithelium within 1-2 hours postmortem. Tissue was cut into 4 mm pieces and placed photoreceptor layer down on polycarbonate transwells, and cultured in Neurobasal-A medium supplemented with B-27. Viability of the explant cultures was examined over time by gross morphology, quantification of glucose-6-phosphate dehydrogenase release from dying cells, and TUNEL assay. Transductions with AAV vectors were performed on retinal explants on the day the cultures were started, and transgene expression was analyzed by live cell imaging and immunofluorescence (IF) staining, or ELISA from tissue supernatants. Results: Retinal explants were viable for up to 4 weeks and IF staining of retinal protein markers including β-III-tubulin, Chx10, glutamine synthetase, rhodopsin and L/M opsin suggested that most retinal cell types were preserved in vitro, with tissue architecture closely resembling that of intact in vivo retina. Transduction with different rAAV serotypes encoding GFP displayed protein expression at 3 days, with gradually rising levels during the first 2 weeks following transduction. Furthermore, IF staining using retinal cell markers revealed differential cellular transgene expression from the AAV vectors encoding various cell-specific or ubiquitous promoters under investigation. Finally, high levels of secreted soluble VEGFR-1 protein expressed from rAAV were detected from explant culture media samples with expression increasing over a period of 3 weeks. Conclusions: Our results demonstrate viability of retinal explants for up to 4 weeks and efficient rAAV transduction with various serotypes. Our organotypic porcine retinal explant model facilitates the evaluation of efficacy, cellular tropism, and promoter selectivity of rAAV vectors in a fast, reproducible, and economical manner, contributing to a reduction in the use of animals for the development of therapies in ophthalmology.

First isolation and identification of Zika virus in China

Objective To establish a method for the isolation of Zika virus and to gather experiences for viral isolation. Methods Suckling mice at age 1-3 days were inoculated with serum samples positive for Zika virus through intracranial injection. All mice were sacrificed 6 days after the injection. Viral nucleic acids were extracted from brain, heart, liver, spleen, lung, kidney, muscle, skin and intestine tissue samples and analyzed by real-time RT-PCR. The supernatants of brain tissues positive for Zika virus were used for subculturing. Nested PCR was performed to amplify the NS5 gene of the isolated virus. The sequences of NS5 gene were analyzed by using MEGA6.0 software. Results All of the tissue samples were positive for Zika virus. Higher viral loads were detected in heart and brain tissue samples with cycle threshold (Ct) values of 24.4 and 25.3, respectively. The second generation of Zika virus was identified in suckling mice brain tissues 2 days after infection by using real-time RT-PCR. The amplified product of nested PCR was 972 bp in length. Sequencing analysis showed that the isolated Zika virus (GDZ16002 strain) belonged to the Asian lineage. Conclusion A strain of Zika virus was successfully isolated in China by using intracranial injection via a suckling mouse model. The isolated Zika virus belonged to the Asian lineage. Key words: Zika virus; Viral isolation; Arbovirus; Suckling mice

Organic acid component from Taraxacum mongolicum Hand.-Mazz alleviates inflammatory injury in lipopolysaccharide-induced acute tracheobronchitis of ICR mice through TLR4/NF-κB signaling pathway

Inflammation plays an important role in the pathogenesis of acute tracheobronchitis. Taraxacum mongolicum Hand.-Mazz (TMHM) is a dietic herb for heat-clearing and detoxifying functions as well as swell-reducing and mass-resolving effect in Traditional Chinese Medicine. Studies have shown that its major ingredient organic acid component (OAC) possesses favorable anti-inflammatory activity. However, the protective effect of OAC from TMHM (TMHM-OAC) on inflammatory injury of acute tracheobronchitis and its possible mechanism remains poorly understood. In this study, HPLC-DAD was used to analyze the components of TMHM-OAC. Lipopolysaccharide of 1mg/ml was used to induce respiratory inflammation in ICR mice at the dose of 5mg/kg by intratracheally aerosol administration. Enzyme-linked immunosorbent assay (ELISA) was employed to detect the levels of inflammation factors such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and nitric oxide in serum and supernatant of trachea tissue. Western blotting (WB) and Immunohistochemistry analysis (IHC) were conducted in parallel to determine TNF-α, IL-6, inducible nitric oxide synthase (iNOS), Toll-like receptors 4(TLR4) protein expressions and nuclear factor-kappa B p65 (NF-κB p65) phosphorylation. Hematoxylin-Eosin staining (HE) was applied to evaluate pathological lesions of trachea tissue. Experimental results showed that TMHM-OAC significantly reduced the levels of the TNF-α, IL-6 and NO in serum and supernatant of tracheal of LPS-induced ICR mice. The protein expression levels of TNF-α, IL-6 and iNOS in tracheal tissue were also down-regulated significantly by the treatment of TMHM-OAC. Moreover, TMHM-OAC downregulated phosphorylation of NF-κB p65 and protein expression of TLR4. Our results indicated that TMHM-OAC could improve LPS-induced histopathological damage of tracheal tissues through the regulation of TLR4/NF-κB signaling pathway and could be beneficial for the treatment of acute tracheobronchitis.

CARD9 knockout ameliorates myocardial dysfunction associated with high fat diet-induced obesity

Obesity is associated with chronic inflammation which plays a critical role in the development of cardiovascular dysfunction. Because the adaptor protein caspase recruitment domain-containing protein 9 (CARD9) in macrophages regulates innate immune responses via activation of pro-inflammatory cytokines, we hypothesize that CARD9 mediates the pro-inflammatory signaling associated with obesity en route to myocardial dysfunction.C57BL/6 wild-type (WT) and CARD9−/− mice were fed normal diet (ND, 12% fat) or a high fat diet (HFD, 45% fat) for 5months. At the end of 5-month HFD feeding, cardiac function was evaluated using echocardiography. Cardiomyocytes were isolated and contractile properties were measured. Immunofluorescence was performed to detect macrophage infiltration in the heart. Heart tissue homogenates, plasma, and supernatants from isolated macrophages were collected to measure the concentrations of pro-inflammatory cytokines using ELISA kits. Western immunoblotting analyses were performed on heart tissue homogenates and isolated macrophages to explore the underlying signaling mechanism(s). CARD9 knockout alleviated HFD-induced insulin resistance and glucose intolerance, prevented myocardial dysfunction with preserved cardiac fractional shortening and cardiomyocyte contractile properties. CARD9 knockout also significantly decreased the number of infiltrated macrophages in the heart with reduced myocardium-, plasma-, and macrophage-derived cytokines including IL-6, IL-1β and TNFα. Finally, CARD9 knockout abrogated the increase of p38 MAPK phosphorylation, the decrease of LC3BII/LC3BI ratio and the up-regulation of p62 expression in the heart induced by HFD feeding and restored cardiac autophagy signaling.In conclusion, CARD9 knockout ameliorates myocardial dysfunction associated with HFD-induced obesity, potentially through reduction of macrophage infiltration, suppression of p38 MAPK phosphorylation, and preservation of autophagy in the heart.

Retinoic acid decreases the severity of Salmonella enterica serovar Typhimurium mediated gastroenteritis in a mouse model

Gastroenteritis is a global burden; it’s the major cause of morbidity and mortality both in adults and children of developing countries. Salmonella is one of the leading causes of bacteria-mediated gastroenteritis and due to its increasing multidrug antibiotic resistance; Salmonella-mediated gastroenteritis is difficult to control. Retinoic acid, the biologically active agent of vitamin A has an anti-inflammatory effect on experimental colitis. In this study we have shown All trans retinoic acid (ATRA) treatment down regulates Salmonella-mediated colitis in a murine model. Macroscopic signs of inflammation such as decrease in body weight and cecum weight, shorter length of proximal colon and pathological score of colitis were observed less in ATRA treated mice than in a vehicle control group. ATRA treatment not only reduced pro-inflammatory cytokine responses, such as TNF-α, IL-6, IL-1β, IFN-γ and IL-17 production but also increased IL-10 response in the supernatant of intestinal tissue. Results also suggested that ATRA treatment enhances the number of FoxP3-expressing T regulatory cells in MLN and also decreases bacterial load in systemic organs. We concluded that ATRA treatment indeed reduces Salmonella Typhimurium-mediated gastroenteritis in mice, suggesting it could be an important part of an alternative therapeutic approach to combat the disease.

Abstract A124: Tertiary lymphoid structures: Predictors of effective antitumor immunity in human breast cancer?

Abstract The clinical relevance of tumor infiltrating lymphocytes (TIL) in breast cancer (BC) is now broadly accepted; however, the relationship between protective immunity, observed in some patients, and critical features of lymphoid subset composition and organization are largely unknown. Our recent work revealed that tertiary lymphoid structures (TLS), principally composed of T and B cells, present in the peri-tumoral regions of BC are associated with increased TIL infiltration in the tumor bed and good clinical outcomes in both the neo-adjuvant and adjuvant settings. To gain insight into the immune components linked with a significant TIL and TLS presence, we initiated a prospective study of T and B cell TIL. Breast tissues from tumors (current n=483) and matched non-adjacent non-tumor tissues (NANT) as well as normal tissue from mammary reductions (n=49) were systematically immunophenotyped by flow cytometry on the day of surgery. Primary tumor supernatants derived from the tissue homogenates (non-enzymatic) and plasma from all patients were stored for cytokine/chemokine and immunoglobulin analysis. TIL organization and spatial distribution was analyzed on paraffin sections using immunohistochemistry (IHC) and immunofluorescence (IF). The fresh tissue analyses revealed that TIL distribution in BC forms a continuum. The infiltration levels detected in normal tissue and NANT were used to define cutoffs that discriminate between TIL positive (TILpos) and negative (TILneg) tumors. TILneg tumors are defined as those with a TIL density <99th percentile of the lymphocyte density in normal breast tissue. TILpos tumors include TIL-high (TILhi) where TIL density is >99th percentile of lymphocyte density in NANT and TIL-intermediate (TILint). Applying these thresholds to the TIL infiltration levels identified approximately 25% of tumors as TILneg with infiltrating lymphocytes similar to normal breast tissue. The TILpos tumors (75%) are subdivided into 36% TILint and 39% TILhi tumors. TILpos tumors are characterized by an increase in CD4+ T cells and CD19+/CD20+ B cells. The median CD4/CD8 ratio was >1 in TILpos compared to <1 in TILneg tumors and NANT. CD4+ and CD8+ T cells were predominantly CD45RO+ memory cells with a significant proportion expressing PD-1. All stages of B cell differentiation were detected in the infiltrate; however, memory B cells were significantly increased in BC (50%) compared to normal tissues (<15%) with the highest levels seen in TILhi tumors. An increase in centroblasts and centrocytes, the germinal center (GC) B cells, was associated with Tfh cells, both resident in peri-tumoral TLS. Their presence was positively correlated with antibody-secreting cells, suggesting local humoral immune responses are generated in TLS, which is supported by the increased immunoglobulin levels detected in tumor supernatants compared to NANT/normal tissue supernatants. Closer examination of Tfh cells infiltrating BC indicates that CXCL13-producing Tfh TIL vary from Tfh cells in secondary lymphoid organs such as tonsils. Tfh TIL express the highest levels of some GC Tfh cell markers (PD-1, CD200 and TIGIT) but intermediate levels of others (ICOS and BCL6) and are essentially negative for CXCR5. Closer examination of Tfh cells in the tumor bed revealed that they directly contact CD20+ B cells, IgG+ and IgA+ plasma cells as well as CXCL13–CD8+ TIL. Taken together, our data suggest that active cross-talk between adaptive immune cells in the tumor microenvironment may be key to establishing immunological memory sufficiently robust to control residual BC over the long term. These data further signify that TLS and/or the presence of GC-associated Tfh and B cells may be important biomarkers that identify effective anti-tumor immunity, which can ultimately be used to independently grade therapeutic responses. Citation Format: Soizic Garaud, Laurence Buisseret, Chunyan Gu-Trantien, Gert Van den Eynden, Alexandre de Wind, Edoardo Migliori, Roberto Salgado, Denis Larsimont, Martine Piccart, Karen Willard-Gallo. Tertiary lymphoid structures: Predictors of effective antitumor immunity in human breast cancer? [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A124.

Pharmacokinetic Behaviors of Intravenously Administered siRNA in Glandular Tissues.

The pharmacokinetics of small interfering RNAs (siRNAs) is a pivotal issue for siRNA-based drug development. In this study, we comprehensively investigated the behavior of siRNAs in vivo in various tissues and demonstrated that intravenously-injected naked siRNA accumulated remarkably in the submandibular gland, bulbourethral gland, and pancreas, with a respective half-life of ~22.7, ~45.6, and ~30.3 h. This was further confirmed by gel separation of tissue homogenates and/or supernatants. In vivo imaging and cryosectioning suggested that delivery carriers significantly influence the distribution and elimination profiles of siRNA. Gene-silencing assays revealed that neither naked nor liposome-formulated siRNA resulted in gene knockdown in the submandibular and bulbourethral glands after systemic administration, suggesting that these glands function as drug reservoirs that enable slow siRNA release into the circulation. But robust gene-silencing was achieved by local injection of liposome-encapsulated siRNA into the submandibular gland. Our results enhance understanding of the pharmacokinetic properties of siRNAs and we believe that they will facilitate the development of siRNA therapy, especially for the submandibular gland.

Abstract 13458: Caspase Recruitment Domain-Containing Protein 9 Deficiency Mitigates Obesity-Induced Cardiac Dysfunction

Obesity has become an epidemic and is associated with cardiovascular anomalies. Obesity-induced low-grade chronic inflammation and macrophage infiltration into the heart plays a critical role in the development of cardiac dysfunction. Because the adaptor protein caspase recruitment domain-containing protein 9 (CARD9) regulates the innate immunity in macrophages via activation of pro-inflammatory cytokines, we hypothesized that CARD9 mediates chronic inflammation and plays a detrimental role in obesity-associated cardiac dysfunction. Male C57BL/6 wild-type (WT) and CARD9 knockout (CARD9-/-) mice were fed normal chow (ND, 12% fat) or a Western diet (WD, 45% fat) for 5 months starting at 4 weeks of age. At the end of 5-month WD feeding, cardiac geometry and function were evaluated using echocardiography. Immunofluorescence assay was performed to detect macrophage infiltration in the heart. Heart tissue homogenates, plasma, and supernatants from cultured macrophages were collected to measure the activities of pro-inflammatory cytokines by ELISA kits. Western immunoblotting analyses were performed on isolated peritoneal macrophages and heart tissue to dissect the pro-inflammatory signaling pathways. WD feeding induced insulin resistance and glucose intolerance in the WT mice and the effects were attenuated in the CARD9-/- mice. Myocardial fractional shortening and ejection fraction were significantly compromised in the WD-fed WT mice and CARD9 knockout preserved cardiac function. CARD9 knockout also significantly decreased the number of infiltrated macrophages in the heart. In addition, myocardium-, plasma- and macrophage-derived cytokines IL-6, IL-1β and TNFα were attenuated in the CARD9-/- mice compared to the WT mice. WD feeding resulted in increased protein phosphorylation levels of p38 MAPK in the heart and macrophages which were suppressed in the CARD9-/- mice. Taken together, these results suggest that CARD9 knockout ameliorates obesity-induced cardiac dysfunction, potentially through reduction of macrophage infiltration and suppression of p38 MAPK phosphorylation in the heart.

Avian influenza virus H9N2 infections in farmed minks.

BackgroundThe prevalence of avian H9N2 viruses throughout Asia, along with their demonstrated ability to infect mammals, puts them high on the list of influenza viruses with pandemic potential for humans. In this study, we investigated whether H9N2 viruses could infect farmed minks.MethodsFirst, we conducted a serological survey for avian influenza virus antibodies on a random sample of the field-trial population of farmed minks. Then we inoculated farmed minks with A/Chicken/Hebei/4/2008 H9N2 viruses and observed the potential pathogenicity of H9N2 virus and virus shedding in infected minks.ResultsH9 influenza antibodies could be detected in most farmed minks with a higher seropositivity, which indicated that farmed minks had the high prevalence of exposure to H9 viruses. After infection, the minks displayed the slight clinical signs including lethargy and initial weight loss. The infected lungs showed the mild diffuse pneumonia with thickened alveolar walls and inflammatory cellular infiltration. Influenza virus detection showed that viruses were detected in the allantoic fluids inoculated supernatant of lung tissues at 3 and 7 days post-infection (dpi), and found in the nasal swabs of H9N2-infected minks at 3–11 dpi, suggesting that H9N2 viruses replicated in the respiratory organ, were then shed outwards. HI antibody test showed that antibody levels began to rise at 7 dpi.ConclusionsOur data provided the serological and experimental evidences that strongly suggested farmed minks under the natural state were susceptible to H9N2 viral infection and might be the H9N2 virus carriers. It is imperative to strengthen the H9N2 viral monitoring in farmed minks and pay urgent attention to prevent and control new influenza viruses pandemic prevalence.

EXPERIMENTAL STUDY OF GASTROPROTECTIVE ACTIVITY OF TRANSDERMAL ETHYLMETHYLHYDROXYPYRIDINE SUCCINATE

We have carried out an experimental study of dose-dependent gastroprotective activity of transdermal plaster with ethylmethylhydroxypyridine succinate (mexidol), the purpose of which was the histological study of gastric mucosa, determination of lipid peroxidation (LP) in gastric tissue supernatant, weight study of thymus, spleen, and adrenals. Ethylmethylhydroxypyridine succinate has gastroprotective action in transdermal implementation at 20-75 mg/kg dose on the model of 18-hour immobilization of animals which had been starving for 24 hours.

The Effects of Smoking on the Osmotic Pressure of Human Dental Pulp Tissue

Objective: We aimed to investigate the effect of smoking on the osmotic pressure (OP) of human dental pulp tissue. Materials and Methods: Sixty male dental patients (smokers and nonsmokers) scheduled for root canal treatment for prosthodontics were included in the study. Fifteen patients (1 premolar tooth/patient) were allocated to each of the following groups according to their smoking habits, i.e. group 1: ≤10 cigarettes/day, group 2: 11-20 cigarettes/day, group 3: >20 cigarettes/day and group 4: nonsmoking controls. Apical pulp tissues were removed via coronal access. Pulp tissue supernatants were obtained to measure the pulpal OP by means of a semimicro digital osmometer. One-way analysis of variance and the post hoc Duncan test were used to analyze the differences in OP between groups. Regression analysis was used to determine the relationship between the number of cigarettes smoked daily and the pulpal OP. Results: The mean (± SD) OP value decreased as cigarette consumption increased: group 4 (268.00 ± 10.09 mosm/kg) > group 1 (259.20 ± 7.16 mosm/kg) > group 2 (248.90 ± 2.23 mosm/kg) > group 3 (239.90 ± 7.40 mosm/kg). The OP differed significantly between groups (p < 0.01), and a significant negative correlation was found between cigarette consumption and pulpal OP (r = -0.809, p < 0.01). Conclusion: In this study, the OP decreased as the number of cigarettes smoked increased. In clinical examination, there may be misdiagnosis of pulpal conditions in smokers (even in healthy pulp tissue) due to the effect of altered OP on pulpal tissue reactions.

Assay of Arginyltransferase Activity by a Fluorescent HPLC Method.

Syntheses of fluorescent substrate and product for arginyltransferase, N-aspartyl-N'-dansylamido-1,4-butanediamine (Asp-4DNS), and N-arginylaspartyl-N'-dansylamido-1,4-butanediamine (ArgAsp-4DNS), respectively, including their precursor 4-dansylamidobutylamine (4DNS), are described. Then, HPLC conditions are summarized for a baseline separation of the three compounds in 10 min. The present method, which permits the simultaneous determination of Asp-4DNS, 4DNS, and ArgAsp-4DNS (in eluting order), is advantageous in measuring arginyltransferase activity and detecting the unfavorable enzyme(s) in 105,000 × g supernatant of tissues to ensure accurate determination.

Effects of Hyperoxia Exposure on Free Radicals Accumulation in Relation to Ultrastructural Pathological Changes of Diaphragm

COPD are associated with an increased load on the diaphragm leading to accumulation of reactive oxygen species (ROS) and the subsequent cellular damages and death. The pathological alterations inducted by ROS in the diaphragm during oxygen breathing are not known. The purpose of the present study was to examine the effects of hyperoxia exposure (HP) on free radicals (FR) accumulation in relation to the ultrastructural pathological alterations in the diaphragm. Twenty adult male rats were randomly assigned to two groups; control (C); and hyperoxia (HP). Animals of the HP were breathing 100% O2 for 72 hr continuously. Both serum and diaphragm tissue supernatant analysis showed significantly higher (p<0.05) FR in HP group, as compared with control group. Ultrastructure examinations showed that HP resulted in variety of pathological alterations in the mitochondria and endoplasmic reticulum that were associated with disarrangement of myofibrils, loss of I-banding for myosin, focal myolysis of the myofilaments, complete fragmentation of myosin, tearing of myofilamments from Z plates and tearing of the endothelial cell of the interstitial blood capillaries. Based on the results of the present study, it can be concluded that hyperoxia-induced acceleration ROS formation damaged the contractile apparatuses of the diaphragm and related endomembrane proteins that could involve intracellular calcium channels proteins.

Effect of carnosine supplementation on apoptosis and irisin, total oxidant and antioxidants levels in the serum, liver and lung tissues in rats exposed to formaldehyde inhalation

The main objective of the study has been to show whether carnosine has positive effects on liver and lung tissues of rats exposed to a range of formaldehyde concentrations, and to explore how irisin expression and antioxidant capacity are altered in these tissues by carnosine supplementation. Sprague-Dawley type male rats were divided into 8 groups with 6 animals in each: (I) Control; no chemical supplementation); (II) sham (100mg/kg/day carnosine); (III) low dose formaldehyde (LDFA) for 5 days/week; (IV) LDFA for 5 days/week and carnosine); (V) moderate dose formaldehyde (MDFA) for 5 days/week); (VI) MDFA for 5 days/week and carnosine; (VII) high dose formaldehyde (HDFA) for 5 days/week; (VIII) and HDFA for 5 days/week and carnosine. Sham and control groups were exposed to normal air. Irisin levels of the serum, liver and lung tissue supernatants were analyzed by ELISA, while the REL method was used to determine total oxidant/antioxidant capacity. Irisin production by the tissues was detected immunohistochemically. Increasing doses of FA decreased serum/tissue irisin and total antioxidant levels relative to the controls, as also to increases in TUNEL expressions, total oxidant level, oxidant and apoptosis index. Irisin expression was detected in hepatocyte and sinusoidal cells of the liver and parenchymal cells of the lung. In conclusion, while FA exposure reduces irisin and total oxidant in the serum, liver and lung tissues in a dose-dependent manner and increases the total antioxidant capacity, carnosine supplementation reduces the oxidative stress and restores the histopathological and biochemical signs.

A simple and rapid protocol to non-enzymatically dissociate fresh human tissues for the analysis of infiltrating lymphocytes.

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45(+) cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45(+) cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

Prolonged exposure of cholestatic rats to complete dark inhibits biliary hyperplasia and liver fibrosis.

Biliary hyperplasia and liver fibrosis are common features in cholestatic liver disease. Melatonin is synthesized by the pineal gland as well as the liver. Melatonin inhibits biliary hyperplasia of bile duct-ligated (BDL) rats. Since melatonin synthesis (by the enzyme serotonin N-acetyltransferase, AANAT) from the pineal gland increases after dark exposure, we hypothesized that biliary hyperplasia and liver fibrosis are diminished by continuous darkness via increased melatonin synthesis from the pineal gland. Normal or BDL rats (immediately after surgery) were housed with light-dark cycles or complete dark for 1 wk before evaluation of 1) the expression of AANAT in the pineal gland and melatonin levels in pineal gland tissue supernatants and serum; 2) biliary proliferation and intrahepatic bile duct mass, liver histology, and serum chemistry; 3) secretin-stimulated ductal secretion (functional index of biliary growth); 4) collagen deposition, liver fibrosis markers in liver sections, total liver, and cholangiocytes; and 5) expression of clock genes in cholangiocytes. In BDL rats exposed to dark there was 1) enhanced AANAT expression/melatonin secretion in pineal gland and melatonin serum levels; 2) improved liver morphology, serum chemistry and decreased biliary proliferation and secretin-stimulated choleresis; and 4) decreased fibrosis and expression of fibrosis markers in liver sections, total liver and cholangiocytes and reduced biliary expression of the clock genes PER1, BMAL1, CLOCK, and Cry1. Thus prolonged dark exposure may be a beneficial noninvasive therapeutic approach for the management of biliary disorders.

Depressive symptoms and cytokine levels in Serum and Tumor Tissue in patients with an Astrocytoma: a pilot study.

BackgroundPreoperative depressive symptoms are associated with poor outcomes in patients with an astrocytoma. Cytokines are associated with depressive symptoms in the general population and are important mediators of tumor growth and progression.The aims of this study were to: (1) characterize depressive symptoms, other treatment-related symptoms and biological mediators; and (2) determine whether preoperative depressive symptoms were associated with the selected biological mediators.MethodsA prospective, exploratory study was carried out among 22 patients with a high-grade astrocytoma. Self-report questionnaires and peripheral blood samples were collected on the day of surgery. Tumor tissue was collected intraoperatively. Self-report questionnaires were assessed at 3, 6, 9, and 12-months postoperatively.ResultsIn circulation, serum IL-8 was inversely correlated with depressive symptoms while IL-17 measured in tumor tissue supernatant was inversely correlated with depressive symptoms. Depressive symptoms showed a significant increase at 12 months from baseline levels and were positively associated with treatment-related symptoms at 3 months and symptom distress at 12 months post-surgery.ConclusionsIn this pilot study, depressive symptoms were negatively associated with IL-8 in serum and IL-17 in tumor tissue. The changes among depressive symptoms, treatment-related symptoms and symptom distress highlight the need for multi-faceted symptom management strategies over the treatment trajectory in this patient population.