ABSTRACT
The clinical significance and treatment strategies for minimal acute rejection (grade A1), the most common form of acute rejection (AR), remain controversial. In this retrospective single-center cohort study of 441 lung transplant recipients, we formally evaluate the association between minimal AR and chronic lung allograft dysfunction (CLAD) and test a novel hypothesis using bronchoalveolar lavage (BAL) CXCL9 concentration during minimal AR as a biomarker of subsequent CLAD development. In univariable and multivariable models adjusted for all histopathologic injury patterns, minimal AR was not associated with CLAD development. However, minimal AR with elevated BAL CXCL9 concentrations markedly increased CLAD risk in a dose-response manner. Minimal AR with CXCL9 concentrations greater than the 25th, 50th, and 75th percentile had adjusted hazard ratios (HRs) for CLAD of 1.1 (95% confidence interval [CI] 0.8-1.6), 1.6 (95% CI 1.1-2.3), and 2.2 (95% CI 1.4-3.4), respectively. Thus we demonstrate the utility of BAL CXCL9 measurement as a prognostic biomarker that allows discrimination of recipients at increased risk of CLAD development after minimal AR. BAL CXCL9 measurement during transbronchial biopsies may provide clinically useful prognostic data and guide treatment decisions for this common form of AR, as a possible strategy to minimize CLAD development.
Subject(s)
Acute Lung Injury/diagnosis , Biomarkers/metabolism , Bronchoalveolar Lavage Fluid/cytology , Chemokine CXCL9/metabolism , Graft Rejection/diagnosis , Lung Transplantation/adverse effects , Postoperative Complications/diagnosis , Acute Lung Injury/etiology , Acute Lung Injury/metabolism , Female , Follow-Up Studies , Graft Rejection/etiology , Graft Rejection/metabolism , Graft Survival , Humans , Male , Middle Aged , Postoperative Complications/etiology , Postoperative Complications/metabolism , Prognosis , Retrospective Studies , Transplantation, HomologousABSTRACT
The impact of allograft injury time of onset on the risk of chronic lung allograft dysfunction (CLAD) remains unknown. We hypothesized that episodes of late-onset (≥6 months) allograft injury would produce an augmented CXCR3/ligand immune response, leading to increased CLAD. In a retrospective single-center study, 1894 transbronchial biopsy samples from 441 lung transplant recipients were reviewed for the presence of acute rejection (AR), lymphocytic bronchiolitis (LB), diffuse alveolar damage (DAD), and organizing pneumonia (OP). The association between the time of onset of each injury pattern and CLAD was assessed by using multivariable Cox models with time-dependent covariates. Bronchoalveolar lavage (BAL) CXCR3 ligand concentrations were compared between early- and late-onset injury patterns using linear mixed-effects models. Late-onset DAD and OP were strongly associated with CLAD: adjusted hazard ratio 2.8 (95% confidence interval 1.5-5.3) and 2.0 (1.1-3.4), respectively. The early-onset form of these injury patterns did not increase CLAD risk. Late-onset LB and acute rejection (AR) predicted CLAD in univariable models but lost significance after multivariable adjustment for late DAD and OP. AR was the only early-onset injury pattern associated with CLAD development. Elevated BAL CXCR3 ligand concentrations during late-onset allograft injury parallel the increase in CLAD risk and support our hypothesis that late allograft injuries result in a more profound CXCR3/ligand immune response.
Subject(s)
Acute Lung Injury/etiology , Graft Rejection/etiology , Lung Transplantation/adverse effects , Pulmonary Alveoli/pathology , Acute Lung Injury/diagnosis , Bronchoalveolar Lavage Fluid/chemistry , Female , Follow-Up Studies , Graft Rejection/diagnosis , Graft Survival , Humans , Male , Middle Aged , Prognosis , Retrospective Studies , Transplantation, HomologousABSTRACT
The glucose-regulated protein (GRP78/BiP) and PKR-like endoplasmic reticulum kinase (PERK) plays a crucial role in the endoplasmic reticulum (ER) stress response. GRP78/BiP is highly elevated in various human cancers. Our study is to examine the clinicopathological significance of GRP78/BiP and PERK expression in patients with tongue cancer. A total of 85 tongue cancer patients were analyzed, and tumor specimens were stained by immunohistochemistry for GRP78/BiP, PERK, GLUT1, Ki-67 and microvessel density (MVD) determined by CD34.GRP78/BiP and PERK were highly expressed in 47% and 35% of all patients, respectively. GRP78/BiP disclosed a significant relationship with PERK expression, lymphatic permeation, vascular invasion, glucose metabolism and cell proliferation. The expression of GRP78/BiP was significantly higher in metastatic sites than in primary sites (79% vs. 47%, p=0.003). We found that the high expression of GRP78/BiP was proven to be an independent prognostic factor for predicting poor outcome in patients with tongue cancer. In the analysis of PFS, PERK was identified as an independent predictor. The increased GRP78/BiP expression was clarified as an independent prognostic marker for predicting worse outcome. Our study suggests that the expression of GRP78/BiP as ER stress marker is important in the pathogenesis and development of tongue cancer.
Subject(s)
Endoplasmic Reticulum Stress/physiology , Heat-Shock Proteins/metabolism , Tongue Neoplasms/metabolism , Tongue Neoplasms/pathology , eIF-2 Kinase/metabolism , Biomarkers, Tumor/metabolism , Cell Proliferation , Endoplasmic Reticulum Chaperone BiP , Female , Humans , Immunohistochemistry , Male , PrognosisABSTRACT
Primary graft dysfunction (PGD) is a possible risk factor for bronchiolitis obliterans syndrome (BOS) following lung transplantation; however, the mechanism for any such association is poorly understood. Based on the association of TGF-ß with acute and chronic inflammatory disorders, we hypothesized that it might play a role in the continuum between PGD and BOS. Thus, the association between PGD and BOS was assessed in a single-center cohort of lung transplant recipients. Bronchoalveolar lavage fluid concentrations of TGF-ß and procollagen collected within 24 h of transplantation were compared across the spectrum of PGD, and incorporated into Cox models of BOS. Immunohistochemistry localized expression of TGF-ß and its receptor in early lung biopsies posttransplant. We found an association between PGD and BOS in both bilateral and single lung recipients with a hazard ratio of 3.07 (95% CI 1.76-5.38) for the most severe form of PGD. TGF-ß and procollagen concentrations were elevated during PGD (p < 0.01), and associated with increased rates of BOS. Expression of TGF-ß and its receptor localized to allograft infiltrating mononuclear and stromal cells, and the airway epithelium. These findings validate the association between PGD and the subsequent development of BOS, and suggest that this association may be mediated by receptor/TGF-ß biology.
Subject(s)
Biomarkers/metabolism , Bronchiolitis Obliterans/diagnosis , Graft Rejection/diagnosis , Lung Transplantation/adverse effects , Primary Graft Dysfunction/diagnosis , Transforming Growth Factor beta/metabolism , Aged , Bronchiolitis Obliterans/etiology , Bronchiolitis Obliterans/metabolism , Case-Control Studies , Female , Follow-Up Studies , Graft Rejection/blood , Graft Rejection/etiology , Graft Survival , Humans , Immunoenzyme Techniques , Lung Diseases/surgery , Male , Middle Aged , Postoperative Complications , Primary Graft Dysfunction/etiology , Primary Graft Dysfunction/metabolism , Prognosis , Prospective Studies , Retrospective Studies , Risk Assessment , Risk Factors , Severity of Illness IndexABSTRACT
Staphylococcus aureus is the most commonly isolated gram-positive bacterium after lung transplantation (LT) and has been associated with poor posttransplant outcomes, but its effect on bronchiolitis obliterans syndrome (BOS) and death in the context of the allograft inflammatory environment has not been studied. A three-state Cox semi-Markovian model was used to determine the influence of allograft S. aureus and the ELR+ CXC chemokines on the survival rates and cause-specific hazards for movement from lung transplant (State 1) to BOS (State 2), from transplant (State 1) to death (State 3), and from BOS (State 2) to death (State 3). Acute rejection, pseudomonas pneumonia, bronchoalveolar lavage fluid (BALF) CXCL5 and its interaction with S. aureus all increased the likelihood of transition from transplant to BOS. Transition to death from transplant was facilitated by pseudomonas infection and single lung transplant. Movement from BOS to death was affected by the interaction between aspergillus, pseudomonas and CXCL5, but not S. aureus. S. aureus isolation had state specific effects after LT and only in concert with elevated BALF CXCL5 concentrations did it augment the risk of BOS. Pseudomonas and elevated BALF concentrations of CXCL5 continued as significant risk factors for BOS and death after BOS in lung transplantation.
Subject(s)
Bronchiolitis Obliterans/microbiology , Chemokine CXCL5/metabolism , Chemokines, CXC/metabolism , Staphylococcus aureus/pathogenicity , Bronchiolitis Obliterans/surgery , Bronchoalveolar Lavage Fluid , Humans , Lung Transplantation , Treatment OutcomeABSTRACT
BACKGROUND: Amino-acid transporters are necessary for the tumour cell growth and survival, and have a crucial role in the development and invasiveness of cancer cells. But, it remains unclear about the prognostic significance of L-type amino-acid transporter 1 (LAT1), system ASC amino-acid transporter-2 (ASCT2), and xCT expression in patients with tongue cancer. We conducted the clinicopathological study to investigate the protein expression of these amino-acid transporters in tongue cancer. METHODS: Eighty-five patients with surgically resected tongue cancer were evaluated. Tumour sections were stained by immunohistochemistry for LAT1, ASCT2, xCT, 4F2hc/CD98hc (4F2hc), Ki-67, and microvessel density (MVD) determined by CD34, and p53. RESULTS: L-type amino-acid transporter 1 and 4F2hc were highly expressed in 61% (52 out of 85) and 45% (38 out of 47), respectively. ASC amino-acid transporter-2 and xCT were positively expressed in 59% (50 out of 85) and 21% (18 out of 85), respectively. The expression of both LAT1 and ASCT2 was significantly associated with disease staging, lymph-node metastasis, lymphatic permeation, 4F2hc expression and cell proliferation (Ki-67). xCT expression indicated a significant association with advanced stage and tumour factor. By univariate analysis, disease staging, lymphatic permeation, vascular invasion, LAT1, ASCT2, 4F2hc, and Ki-67 had a significant relationship with overall survival. Multivariate analysis confirmed that LAT1 was an independent prognostic factor for predicting poor prognosis. CONCLUSIONS: L-type amino-acid transporter 1 and ASCT2 can serve as a significant prognostic factor for predicting worse outcome after surgical treatment and may have an important role in the development and aggressiveness of tongue cancer.
Subject(s)
Amino Acid Transport System ASC/analysis , Amino Acid Transport System y+/analysis , Biomarkers, Tumor/analysis , Carcinoma, Squamous Cell/chemistry , Large Neutral Amino Acid-Transporter 1/analysis , Neoplasm Proteins/analysis , Tongue Neoplasms/chemistry , Adult , Aged , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Squamous Cell/blood supply , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/surgery , Chemotherapy, Adjuvant , Combined Modality Therapy , Disease-Free Survival , Docetaxel , Drug Combinations , Female , Fusion Regulatory Protein 1, Heavy Chain/analysis , Humans , Kaplan-Meier Estimate , Ki-67 Antigen/analysis , Lymphatic Metastasis , Male , Middle Aged , Minor Histocompatibility Antigens , Neoplasm Staging , Oxonic Acid/administration & dosage , Prognosis , Taxoids/administration & dosage , Tegafur/administration & dosage , Tongue Neoplasms/blood supply , Tongue Neoplasms/drug therapy , Tongue Neoplasms/surgery , Treatment Outcome , Tumor Suppressor Protein p53/analysisABSTRACT
Hepatopulmonary syndrome (HPS) is characterized by impaired oxygenation due to pulmonary vascular dilatation in patients with end-stage liver disease. At our center, we identified 29 patients who were listed for liver transplantation (LT) with a model for end-stage liver disease exception for HPS between 2001 and 2012. Five of these patients were found to have concurrent interstitial lung disease (ILD). The chest high-resolution computed-tomography demonstrated ground-glass opacities and subpleural reticulation, most consistent with nonspecific interstitial pneumonia (NSIP). All four of our patients who underwent LT experienced prolonged hypoxemia postoperatively, with one surgery-related death. However, the three surviving patients had eventual resolution of their hypoxemia with no evidence of ILD progression. In conclusion, we report a high prevalence of ILD, most consistent with NSIP, among patients with HPS. Although there may be increased perioperative risks, the finding of ILD in patients with HPS should not be considered an absolute contraindication to LT.
Subject(s)
Hepatopulmonary Syndrome/complications , Lung Diseases, Interstitial/complications , Adult , Aged , Biopsy , California , Fatal Outcome , Female , Hepatopulmonary Syndrome/diagnosis , Hepatopulmonary Syndrome/surgery , Humans , Hypoxia/diagnosis , Hypoxia/etiology , Liver Transplantation/adverse effects , Lung Diseases, Interstitial/diagnostic imaging , Male , Middle Aged , Patient Selection , Risk Assessment , Risk Factors , Tomography, X-Ray Computed , Treatment Outcome , Waiting ListsABSTRACT
Aspergillus colonization after lung transplantation may increase the risk for bronchiolitis obliterans syndrome (BOS), a disease of small airways. We hypothesized that colonization with small conidia Aspergillus species would be associated with a greater risk of BOS, based upon an increased likelihood of deposition in small airways. We studied adult primary lung recipients from two large centers; 298 recipients at University of California, Los Angeles and 482 recipients at Duke University Medical Center. We grouped Aspergillus species by conidia diameter≤3.5 µm. We assessed the relationship of colonization with outcomes in Cox models. Pre-BOS colonization with small conidia Aspergillus species, but not large, was a risk factor for BOS (p=0.002, HR 1.44, 95% CI 1.14-1.82), along with acute rejection, single lung and Pseudomonas. Colonization with small conidia species also associated with risk of death (p=0.03, HR 1.30, 95% CI 1.03-1.64). Although other virulence traits besides conidia size may be important, we have demonstrated in two large independent cohorts that colonization with small conidia Aspergillus species increases the risk of BOS and death. Prospective evaluation of strategies to prevent Aspergillus colonization of small airways is warranted, with the goal of preserving lung allograft function as long as possible.
Subject(s)
Aspergillosis/complications , Aspergillus/pathogenicity , Bronchiolitis Obliterans/etiology , Lung Transplantation/adverse effects , Aged , Aspergillosis/diagnosis , Bronchiolitis Obliterans/microbiology , California , Cohort Studies , Female , Humans , Male , Middle Aged , North Carolina , Proportional Hazards Models , Pseudomonas Infections/diagnosis , Respiratory Function Tests , Risk Factors , Spores, Fungal/pathogenicityABSTRACT
The identification and characterization of excitatory and inhibitory neurons are significant steps in understanding neural network functions. In this study, we investigated the intrinsic electrophysiological properties of neurons in the prepositus hypoglossi nucleus (PHN), a brainstem structure that is involved in gaze holding, using whole-cell recordings in brainstem slices from vesicular GABA transporter (VGAT)-Venus transgenic rats, in which inhibitory neurons express the fluorescent protein Venus. To characterize the intrinsic properties of these neurons, we recorded afterhyperpolarization (AHP) profiles and firing patterns from Venus-expressing [Venusâº] and Venus-non-expressing [Venusâ»] PHN neurons. Although both types of neurons showed a wide variety of AHP profiles and firing patterns, oscillatory firing was specific to Venus⺠neurons, while a firing pattern showing only a few spikes was specific to Venusâ» neurons. In addition, AHPs without a slow component and delayed spike generation were preferentially displayed by Venus⺠neurons, whereas a firing pattern with constant interspike intervals was preferentially displayed by Venusâ» neurons. We evaluated the mRNAs expression of glutamate decarboxylase (GAD65, GAD67) and glycine transporter 2 (GlyT2) to determine whether the recorded Venus⺠neurons were GABAergic or glycinergic. Of the 67 Venus⺠neurons tested, GlyT2 expression alone was detected in only one neuron. Approximately 40% (28/67) expressed GAD65 and/or GAD67 (GABAergic neuron), and the remainder (38/67) expressed both GAD(s) and GlyT2 (GABA&GLY neuron). These results suggest that most inhibitory PHN neurons use either GABA or both GABA and glycine as neurotransmitters. Although the overall distribution of firing patterns in GABAergic neurons was similar to that of GABA&GLY neurons, only GABA&GLY neurons exhibited a firing pattern with a long first interspike interval. These differential electrophysiological properties will be useful for the identification of specific types of PHN neurons.
Subject(s)
Brain Stem/metabolism , Neurons/cytology , Neurons/metabolism , Animals , Bacterial Proteins , Brain Stem/cytology , Glycine/metabolism , In Situ Hybridization , Luminescent Proteins , Organ Culture Techniques , Patch-Clamp Techniques , Rats , Rats, Transgenic , Reverse Transcriptase Polymerase Chain Reaction , gamma-Aminobutyric Acid/metabolismABSTRACT
Successful offspring production after intracytoplasmic injection of freeze-dried sperm has been reported in laboratory animals but not in domesticated livestock, including pigs. The integrity of the DNA in the freeze-dried sperm is reported to affect embryogenesis. Release of endonucleases from the sperm is one of the causes of induction of sperm DNA fragmentation. We examined the effects of chelating agents, which inhibit the activation of such enzymes, on DNA fragmentation in freeze-dried sperm and on the in vitro and in vivo developmental ability of porcine oocytes following boar sperm head injection. Boar ejaculated sperm were sonicated, suspended in buffer supplemented with (1) 50 mM EGTA, (2) 50 mM EDTA, (3) 10 mM EDTA, or (4) no chelating agent and freeze-dried. A fertilization medium (Pig-FM) was used as a control. The rehydrated spermatozoa in each group were then incubated in Pig-FM at room temperature. The rate of DNA fragmentation in the control group, as assessed by the TUNEL method, increased gradually as time after rehydration elapsed (2.8% at 0 min to 12.2% at 180 min). However, the rates in all experimental groups (1-4) did not increase, even at 180 min (0.7-4.1%), which were all significantly lower (p < 0.05) than that of the control group. The rate of blastocyst formation after the injection in the control group (6.0%) was significantly lower (p < 0.05) than those in the 50 mM EGTA (23.1%) and 10 mM EDTA (22.6%) groups incubated for 120-180 min. The average number of blastocyst cells in the 50 mM EGTA group (33.1 cells) was significantly higher (p < 0.05) than that in the 10 mM EDTA group (17.8 cells). Finally, we transferred oocytes from 50 mM EGTA or control groups incubated for 0-60 min into estrous-synchronized recipients. The two recipients of the control oocytes became pregnant and one miscarried two fetuses on day 39. The results suggested that fragmentation of DNA in freeze-dried boar sperm is one of the causes of decreased in vitro developmental ability of injected oocytes to the blastocyst stage. Supplementation with EGTA in a freeze-drying buffer improves this ability.
Subject(s)
Sperm Injections, Intracytoplasmic/veterinary , Sus scrofa/embryology , Animals , Chelating Agents , DNA Fragmentation , Egtazic Acid , Embryonic Development , Female , Freeze Drying/methods , Freeze Drying/veterinary , In Vitro Techniques , Male , Oocytes , Pregnancy , Semen Preservation/methods , Semen Preservation/veterinary , Sperm Head/transplantationABSTRACT
It is generally accepted that cumulus cells support the nuclear maturation of mammalian oocytes. In the present study, we examined relationships between the cytoplasmic glutathione (GSH) content of porcine oocytes, and oocyte nuclear maturation, fertilization or subsequent embryonic development. Cumulus-oocyte complexes (COCs; control group) and oocytes denuded of cumulus cells after collection (DO 0h group) were cultured for 24h with dibutyryl cAMP, eCG and hCG (first culture step) and then for a further 20h without supplements (second culture step; 44h total culture). After the first culture step, some of the COCs were denuded, either completely (DO 24h group) or partly (H-DO 24h group), and then matured by the second culture step. Also, in the second culture step, some DOs were co-cultured with cumulus cells that had been pre-cultured for 24h (DO 24h+CC group). The maturation rates of all the cumulus-removed groups (DO 0h, DO 24h, H-DO 24h and DO 24h+CC groups) were lower (34.3-45.0%) than that of the control group (64.5%; P<0.05). The GSH contents of matured oocytes in the completely denuded groups (DO 0h, DO 24h and DO 24h+CC groups) were lower (4.03-5.26pmol/oocyte) than that of the control group (9.60pmol/oocyte; P<0.05); however, the H-DO 24h group had an intermediate value (7.0pmol/oocyte). The male pronuclear formation rates of completely denuded oocytes were lower (41.4-59.3%) than that of the control group (89.4%; P<0.05), whereas the H-DO 24h group had an intermediate rate (80.0%). The blastocyst formation rates of the completely denuded oocytes were lower (3.0-4.5%) than that of the control group (19.9%; P<0.05), and the H-DO 24h group again had an intermediate rate (11.6%). The GSH content was correlated with the rates of male pronuclear formation (P<0.01) and blastocyst formation (P<0.01), and also with the number of cells per blastocyst (P<0.01). In conclusion, we inferred that GSH synthesized by intact cumulus cells during maturation culture improved oocyte maturation and played an important role in fertilization and embryonic development.
Subject(s)
Embryonic Development/physiology , Fertilization in Vitro/veterinary , Glutathione/physiology , Oocytes/physiology , Swine/physiology , Animals , Cytoplasm/metabolism , Cytoplasm/physiology , Female , Glutathione/metabolism , Male , Oocytes/cytology , Pregnancy , Swine/metabolismABSTRACT
Salivary or secreted carbonic anhydrase (CA), which constitutes a new class of CA, designated CA-VI, was isolated. Swine CA-VI purified from swine saliva by inhibitor-affinity chromatography and ion exchange chromatography had a specific activity of 5,468 units/mg. The molecular weight was 250,000, as determined by gel filtration under non-denaturing conditions, and the subunit molecular weight was found to be 37,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that swine CA-VI consists of 7 subunits. The treatment of the enzyme with endo-N-acetylglucosaminidase F reduced its subunit molecular weight from 37,000 to 35,000 and 32,000. We raised a rabbit antibody against purfied swine CA-VI. Double immunodiffusion showed that anti-swine CA-VI serum reacted with swine CA-VI and swine saliva, but not with hemolysate (containing CA-I and CA-Il) or muscle extracts (containing CA-III). The concentration of CA-VI in swine saliva, measured using single radial immunodiffusion, was 0.027 +/- 0.017 mg/mg total protein.
Subject(s)
Carbonic Anhydrases/isolation & purification , Saliva/enzymology , Salivary Proteins and Peptides/isolation & purification , Swine/metabolism , Animals , Carbonic Anhydrases/chemistry , Chromatography, Affinity/veterinary , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel/veterinary , Immunodiffusion/veterinary , Isoenzymes/chemistry , Isoenzymes/isolation & purification , Male , Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase/chemistry , Molecular Weight , Salivary Proteins and Peptides/chemistryABSTRACT
This study reports the development of a reliable method for cryopreservation of rat epididymal spermatozoa and the production of live young by artificial insemination using these cryopreserved spermatozoa. The motility and membrane integrity of rat spermatozoa were investigated after spermatozoa had been subjected to physical stress and frozen with various concentrations of glycerol (0, 3 and 6%) either in the presence or absence of Equex Stem as cryoprotective agents. The ability of cryopreserved spermatozoa to generate normal offspring by intrauterine insemination was also evaluated. Rat spermatozoa that had been centrifuged at 700 g for 5 min showed a significant decrease in motility compared with non-centrifuged spermatozoa. In addition, after centrifugation three times the percentage of membrane-intact spermatozoa decreased to approximately 0%. The percentage of membrane-intact spermatozoa was significantly higher (P < 0.01) in semen samples that had been frozen in medium without glycerol than in samples frozen in medium with 3% glycerol. Although the addition of 0.7% Equex Stem to medium without glycerol or with 3% glycerol did not influence rates of sperm motility after freezing and thawing, the percentage of membrane-intact spermatozoa was improved by the presence of 0.7% Equex (P < 0.05). Therefore, rat spermatozoa were handled gently to avoid physical stress and were frozen in medium containing 23% egg yolk, 8% lactose monohydrate and 0.7% Equex Stem, at pH 7.4 adjusted with 10% Tris(hydroxymethyl)aminomethane solution. Thirteen female rats were inseminated into the oviductal end of both uterine horns with frozen-thawed spermatozoa. Forty-one normal live offspring were obtained from nine of the inseminated females. These results indicate that frozen-thawed rat spermatozoa can generate normal offspring. To our knowledge, this procedure is the first successful production of offspring using spermatozoa cryopreserved in liquid nitrogen.
Subject(s)
Cryopreservation , Epididymis/cytology , Insemination, Artificial , Semen Preservation , Spermatozoa/physiology , Animals , Cell Membrane/ultrastructure , Centrifugation , Cryoprotective Agents , Female , Glycerol , Hot Temperature , Litter Size , Male , Nitrogen , Pregnancy , Pregnancy Outcome , Rats , Sperm Motility , Spermatozoa/ultrastructureABSTRACT
The prevalence of parapoxvirus infection was examined in free-ranging wild ruminants in Japan, Japanese serow (Capricornis crispus) and Japanese deer (Cervus nippon centralis), in 1996-9. We collected a total of 151 serum samples from 101 Japanese serows and 50 Japanese deer and tested for antibodies against parapoxvirus by an enzyme-linked immunosorbent assay and an agar gel immunodiffusion test. Overall seroprevalences among Japanese serows were 5/25 (20.0%) in 1996, 4/14 (28.6%) in 1997, 5/32 (15.6%) in 1998 and 2/30 (6.7%) in 1999, respectively. The seroprevalence increased with age but was not affected by sex. No antibodies were detected from any of 50 serum samples taken from Japanese deer. Our results in this study suggest that parapoxvirus infection is widespread among the population of Japanese serows, however, Japanese deer appear to be still free of the disease.
Subject(s)
Animals, Wild , Antibodies, Viral/blood , Deer , Parapoxvirus/immunology , Poxviridae Infections/veterinary , Ruminants , Age Factors , Animals , Enzyme-Linked Immunosorbent Assay/veterinary , Female , Immunodiffusion/veterinary , Japan/epidemiology , Male , Parapoxvirus/classification , Parapoxvirus/isolation & purification , Poxviridae Infections/epidemiology , Seroepidemiologic StudiesABSTRACT
The objective of this study was to evaluate the developmental ability of early porcine embryos produced in vitro and transferred to recipient gilts. Porcine cumulus-oocyte complexes were matured in modified North Carolina State University-37 solution for 44-46 h (in vitro maturation, IVM). In vitro fertilization (IVF) was performed with frozen-thawed epididymal spermatozoa. Inseminated oocytes were cultured in vitro (IVC) for 0, 24, or 48 h in modified NCSU-37 solution. Embryos were surgically transferred to the oviducts of recipients in which estrus had been synchronized with eCG and hCG. On the 29th day post-IVF, the uteri of some recipients were surgically examined for pregnancy; then pregnant females were hysterectomized in order to examine number and weight of the fetuses. Developmental rates to fetuses for IVM/IVF oocytes cultured for 24 and 48 h were significantly lower (p < 0.05, 1.7% and 2.0%, respectively) than that of IVM/IVF oocytes without IVC (6.7%). However, the weights of fetuses (1.0-1.2 g) did not differ among the experimental groups. The other recipients were examined for pregnancy using an ultrasound pregnancy detector, and pregnant females were allowed to go to term. Healthy piglets were delivered by some recipients to which embryos cultured for 0 or 24 h had been transferred; however, no farrow was obtained from embryos cultured for 48 h before the transfer. The results indicate that the viability of in vitro-produced porcine embryos is decreased by IVC after IVF; however, these embryos have competence to develop to term. An improved IVC system of porcine IVM/IVF oocytes is needed to generate advances in this field.
Subject(s)
Embryo Transfer , Embryonic and Fetal Development , Fertilization in Vitro , Oocytes/physiology , Swine/embryology , Animals , Chorionic Gonadotropin/pharmacology , Culture Techniques , Female , Pregnancy , Time FactorsABSTRACT
An unusual case of acinic cell tumour of the maxillary sinus is presented. The patient, a 41-year-old male who had undergone incomplete excision of the tumour in the left parotid region previously, was referred to our department for further treatment. The initial pathological diagnosis was adenocarcinoma of the parotid gland. CT-scan not only revealed tumours in the left pre-auricular and upper neck region, but also an enhanced mass in the left maxillary sinus. Although there were neither nasal symptoms nor destruction of the maxillary bone, aspiration biopsy of the maxillary sinus revealed class V. Total maxillectomy, radiotherapy and systemic chemotherapy were performed just after total parotidectomy and radical neck dissection at the left side. The clinical and histopathological findings are discussed.
Subject(s)
Adenocarcinoma/diagnosis , Carcinoma, Acinar Cell/diagnosis , Maxillary Sinus Neoplasms/diagnosis , Parotid Neoplasms/diagnosis , Adenocarcinoma/surgery , Adult , Carcinoma, Acinar Cell/surgery , Diagnosis, Differential , Humans , Male , Maxillary Sinus Neoplasms/surgery , Neck Dissection , Parotid Gland/surgery , Parotid Neoplasms/surgeryABSTRACT
Quantitative high performance liquid chromatographic (HPLC) methods are described for the determination of reduced, oxidized, and total ubiquinone (UQred, UQox, and total UQ, respectively) in biological material. UQ was extracted into n-hexane from an aqueous homogenate after precipitation of protein by addition of alcohol. The n-hexane extract was evaporated to dryness under a N2 atmosphere at 30 degrees C. In order to determine the total UQ, UQred was converted into the corresponding oxidized form (UQox) with ferric chloride. UQox was separated on a reversed-phase column and detected by its ultraviolet absorption (UV275n m). For the simultaneous assay of UQred and UQox, the n-hexane extract was injected onto a HPLC having a UV detector and an electrochemical detector (ECD) coupled in series. UQox was detected by its UV275n m, and UQred by ECD. The HPLC methods described here were applied satisfactorily to the determination of UQred, UQox, and total UQ in human and animal tissues.
Subject(s)
Chromatography, High Pressure Liquid/methods , Ubiquinone/analysis , Animals , HumansABSTRACT
The synthesis of trans-3,4-bis-(3,4-dihydroxyphenyl)pyrrolidine by the Michael reaction of esterenolate with nitro olefin is described. This compound was expected to be the agonist toward dopamine (DA1) receptor with equipotent affinity and to be more selective than dopamine itself.
Subject(s)
Catechols/chemical synthesis , Dopamine Agents/chemical synthesis , Hemodynamics/drug effects , Pyrrolidines/chemical synthesis , Receptors, Dopamine D1/drug effects , Animals , Catechols/pharmacology , Dogs , Dopamine Agents/pharmacology , Pyrrolidines/pharmacology , Radioligand Assay , Rats , Renal Circulation/drug effectsABSTRACT
Pharmacokinetic parameters of E4101,(-)-(3R,4R)-3-(2-chloro-3-hydroxyphenyl)- 4-(3,4-dihydroxyphenyl)-pyrrolidine hydrochloride, in plasma were determined in conscious beagles. E4101 was extracted from plasma using a Bond Elute C18 cartridge and determined by HPLC connected to a reverse phase ODS column and an electrochemical detector. After an i.v. administration of 1 mg/kg, the plasma level of E4101 decreased according to the three compartment model. The half lives of the alpha, beta and gamma phases were 1.3, 8.1 and 27 min, respectively. The mean residence time (MRT) and total clearance were 14.5 min and 78 ml/min/kg, respectively. When 3 mg/kg of E4101 were administered orally under fasting, E4101 in plasma reached the peak (Cmax: 22.4 ng/ml) 45 min after administration. The bioavailability was 3.6%. When 3 mg/kg of E4101 in combination with 15 mg/kg of ascorbic acid was orally administered in capsule, the area under concentration curve (AUC) was increased 7-fold compared with that in a matched control study. A combination with 15 mg/kg of tartaric acid, citric acid, L-aspartic acid or L-cysteine also increased the AUC, byt they were less effective than ascorbic acid. When 10 mg/kg of E4101 in combination with 25 mg/kg of ascorbic acid as a sustained-release dosage form using oily somisolid matrix orally administered to non-fasted beagles, the duration of effective plasma concentration (2 ng/ml plasma in i.d. administration) was 8.4 +/- 1.0 hr (mean +/- SE, n-4). The Cmax was 16.2 +/- 2.5 ng/ml.