[Accurate localization and successful treatment of 23 cases of migrating pharynx and cervical esophageal foreign bodies].

Objective: To explore clinical features, diagnosis, localization, and therapeutic strategy of migratory pharyngeal and cervical esophageal foreign bodies. Methods: A total 23 cases of pharyngeal and cervical esophageal migratory foreign bodies were admitted between January 2015 and December 2021. There were 14 females and 9 males with the age ranged from 35 to 82 (55.0±12.7)years. In all the cases, esophageal CT was taken to confirm the esophageal foreign body. Multiplanar reconstruction (MPR) was performed to locate the foreign body from the horizontal, coronal and sagittal dimensions as well as the corrected reconstructed MPR. According to the location of the foreign body, appropriate surgical method was selected.The symptoms, complications, types of foreign body, positioning, surgical methods, and relevant information were recorded.Data were analyzed using the descriptive method and SPSS 25.0 software. Results: The clinical symptoms of 23 migrating esophageal foreign bodies included pharyngodynia (20/23), foreign body sensation (6/23), hoarsenss (1/23), difficulty in turning neck(1/23), difficulty in opening mouth (1/23), fever (7/23), poor appetite (1/23), and abdominal pain (1/23). The foreign bodies included 19 fish bones, 2 wires, 1 embroidery needle and 1 chicken bone. There were 9 cases (39.1%) of foreign bodies located in extraluminal cervical esophagus, 2 cases (8.7%) of foreign bodies located in the muscular layer of the cervical esophagus and 12 cases (52.2%) of foreign bodies located in pharynx. Twenty-one cases of foreign bodies were removed by cervical lateral incision, in which 11 were removed by cervical lateral incision directly, 10 by the second lateral cervical incision after the foreign bodies were accurately located by MPR and/or corrected MPR, 1 foreign body was removed by incision of the pharyngeal mucosa under suspension laryngoscope, 1 foreign body was removed by tracheoscopy. Compared with patients with intraluminal foreign bodies (n=308) treated in the same period, intake of fishbone [19 (19/23) vs. 133 (82.6% (43.2%, 133/308), OR=7.31] and first visit was more than 24 hours [20(87.0%, 20/23) vs. 77(25.0%, 77/308),OR=17.2] were the significant risk factors of migratory esophageal foreign bodies. Conclusions: MPR and the corrected MPR can accurately locate the migrating pharyngeal and cervical esophageal foreign bodies, by providing more intuitive imaging evidence for doctors, which provide imaging basis for formulation of surgical programs. Foreign bodies in pharyngeal and cervical esophagus need to be treated as soon as possible, otherwise they are easy to migrate, leading lead to serious complications.

Protective effects of L-carnitine on reproductive capacity in rats with diabetes.

To explore the protective effects of L-carnitine on erectile function and reproductive function in rats with diabetes. A total of 60 male diabetes mellitus induced-erectile dysfunction (DMED) rats were randomly divided into three groups, 20 rats in each group. The blank group was fed normally, the control group was fed with 0.9% sodium chloride solution 5 ml/kg/day, and the experimental group was given L-carnitine 300 mg/kg/day. After six weeks, the Corpus cavernosum penis pressure (ICP) and mean arterial pressure (MAP) were measured. The sperm of epididymis were taken to detect the parameters of sperm. After six weeks of treatment, ICP and MAP in the experimental group were significantly higher than those in the control group and blank group (p < 0.05), and sperm density and PR in the experimental group were significantly higher than those in the control group and the blank group (p < 0.05). Superoxide dismutase (SOD) in the experimental group was significantly higher than that in the control group and blank group (p < 0.05). Malondialdehyde (MDA) in the experimental group was significantly lower than that in the control group and blank group (p < 0.05). The follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone in the experimental group were significantly higher than those in the control group and blank group (p < 0.05). We conclude that L-carnitine can significantly improve erectile function and reproductive function in rats with diabetes and it has great potential in the treatment of systemic organ damage in DMED rats.

Does endoplasmic reticulum stress stimulate the apoptosis of granulosa cells in polycystic ovary syndrome?

The aim of this study was to determine whether endoplasmic reticulum (ER) stress is involved in the apoptosis of granulosa cells in patients with polycystic ovary syndrome (PCOS). A total of 116 patients undergoing in vitro fertilization/intracytoplasmic sperm injection cycles at the Binzhou Medical University Hospital IVF Center between September 2019 and January 2020 were enrolled in the study. Apoptosis of the granulosa cells in each patient was analyzed using flow cytometry, and progesterone and estrogen levels in the cell-culture fluid were measured by enzyme-linked immunosorbent assay. The expressions of X-box-binding protein 1 (XBP1(s)), activating transcription factor 6 (ATF6), C/EBP homologous protein (CHOP), B-cell CLL/lymphoma 2 protein (Bcl-2), and Bcl-2 associated X protein (Bax) at the gene or protein level were analyzed using quantitative real-time polymerase chain reaction and Western blotting, respectively. In patients with PCOS, body mass index and basal serum concentrations of luteinizing hormone, testosterone (T), and anti-Mullerian hormone significantly increased (P < 0.05). The number of oocytes retrieed and the rate of clinical pregnancy after the first frozen embryo transfer also significantly increased (P < 0.05). Although apoptosis rate in the granulosa cells significantly increased in patients with PCOS, progesterone (P) and estrogen (E2) levels in the cell-culture fluid significantly decreased (P < 0.05). The apoptosis of granulosa cells was also found to affect blastocyst formation. Furthermore, the messenger ribonucleic acid (mRNA) expressions of XBP1(s), ATF6, CHOP, and Bax significantly increased in patients with PCOS, while that of Bcl-2 significantly decreased (P < 0.05). The protein expressions of CHOP and Bax significantly increased in patients with PCOS, while that of Bcl-2 significantly decreased (P < 0.05). After treatment of the granulosa cells with tauroursodeoxycholic acid, apoptosis rate and mRNA or protein expressions of XBP1(s), CHOP, and Bax significantly decreased, while the expression of Bcl-2 and levels of progesterone and estrogen significantly increased (P < 0.05). We conclude that ER stress could induce the apoptosis of granulosa cells in patients with PCOS. Cell apoptosis may decrease the number of blastocysts formed.

Repair of spinal cord injury in rats by umbilical cord mesenchymal stem cells through P38MAPK signaling pathway.

OBJECTIVE: To explore the repair of spinal cord injury (SCI) in rats by umbilical cord mesenchymal stem cells (UCMSCs) through the p38mitogen-activated protein kinase (MAPK) signaling pathway. MATERIALS AND METHODS: A total of 45 healthy adult male Sprague-Dawley rats weighing 180-220 g and aged 6-8 weeks old were randomly divided into group A (SCI model + transplantation of UCMSCs, n=15), group B (sham operation), and group C (SCI model + injection of an equal dose of DMEM, n=15) using a random number table. The morphology of spinal cord tissues was observed via hematoxylin-eosin (HE) staining, and the protein expression of phosphorylated p38 (p-p38) in spinal cord tissues, the expression of glial fibrillary acidic protein (GFAP) in the injury region, and the spinal cord neuronal apoptosis were detected via Western blotting, immunofluorescence labeling and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, respectively. RESULTS: In group B, there was no significant damage to the structure of spinal cord tissues. In group C, the spinal cord tissues had a disordered structure and significant fragmentation, the damage to grey matter was the greatest. Also, almost all of the grey matter was destroyed and dissolved, with a large number of scars and cavitation, and it was hard to distinguish the gray matter and white matter. In group A, the spinal cord tissues had a clear structure, there were smaller necrotic cavitation regions in the grey-white matter, and the number of cavitation significantly declined compared with that in group C. The results of immunofluorescence assay revealed that the expression of GFAP in spinal cord tissues was the lowest in group B, while it was remarkably decreased in group A compared with that in group C (p<0.05), suggesting that injecting UCMSCs via the caudal vein can prominently reduce the expression of GFAP in spinal cord tissues. Moreover, the spinal cord neuronal apoptosis rate was (4.21±0.19), (0.72±0.21) and (4.57±0.31), respectively, in group A, group B, and group C. It can be seen that the spinal cord neuronal apoptosis rate significantly declined in group A due to the treatment with UCMSCs. Also, the significant difference compared with that in group C, while it was significantly increased in group A compared with that in group B, but lower than group C (p<0.05). According to the results of Western blotting, the protein expression of p-p38 in spinal cord tissues was remarkably decreased in group B compared with that in group A and group C (p<0.05), while it was also markedly decreased in group A compared with that in group C (p<0.05), indicating that injecting UCMSCs via the caudal vein can significantly lower the protein expression of p-p38 in spinal cord tissues. CONCLUSIONS: UCMSCs promote the recovery of neurological function, inhibit the p38 MAPK pathway activated after SCI, and reduce the spinal cord neuronal apoptosis in SCI rats.

[Clinical analysis of autoimmune hemolytic anemia after allogeneic hematopoietic stem cell transplantation in thalassemia major].

Objective: To explore the diagnosis, treatment and prognosis of autoimmune hemolytic anemia (AIHA) after allo-HSCT in patients with thalassemia major (TM). Methods: A retrospective analysis of AIHA status after allo-HSCT in 291 TM patients from July 2007 to December 2017 was conducted. Results: Five of the 291 TM patients (1.72%) were diagnosed with post-transplant AIHA. The median time of AIHA was 7 (5-12) months after HSCT. All post-transplant AIHA patients were positive in direct and indirect Coombs test, the main clinical manifestations were dizziness, fatigue, pale complexion, skin and sclera yellow, and soy sauce urine. The incidence of AIHA was higher after unrelated donor transplantation (6.36%, 4/63) compared with that of sibling donor transplantation (0.43%, 1/228). One patient who received only prednison was dead. Four patients who received rituximab combined with prednisolone were alive, Coombs test in two of them were negative. Conclusions: AIHA after allo-HSCT developed in 1.72% patients with TM. Monitoring of Coombs test was important for diagnosis of post-transplant AIHA. The incidence of post-transplant AIHA was higher in unrelated donors compared with that of sibling donors transplantation. Treatment of rituximab combined glucocorticoid was effective strategy for post-transplant AIHA.

[Retrospective clinical analyses of otogenic intracranial infections].

Objective: To investigate the clinical characteristics and treatment options for otogenic intracranial infections. Method: Clinical records of all the patients of otogenic intracranial infections admitted to the First Affiliated Hospital of Zhejiang University from 2008 to 2016 were retrospectively analyzed. Their clinical presentations, radiological findings, culture results, and medical and surgical therapy modalities, as well as treatment outcomes were studied. Results: Sixteen cases were identified. The majority of the otogenic patients had a history of cholesteatoma, other rare events included congenital cerebrospinal fluid otorhinorrhea, Gorham-Stout disease and after radiation therapy for nasopharyngeal carcinoma. Meningitis, cerebral venous thrombosis and brain abscess were the primary intracranial infection. Eight patients had received modified radical mastoidectomy at least one time. Results of routine culture for cerebrospinal and pus samples had high negative rate. All patients received initial empirical broadspectrum intravenous antibiotics therapy. Four cases of brain abscesses were drained or excised at the same time for otologic surgery. The mortality rate was 6.25% (1 case). Conclusions: Cholesteatoma is still the most commonly primary disease of otogenic intracranial complications. Diagnosis and treatment of otogenic intracranial infections require multidisciplinary cooperation. Surgical intervention for primary ear lesions and intracranial abscess is still the main option in the treatment of otogenic intracranial infections.

[Long non-coding RNA RAB11B-AS1 prevents osteosarcoma proliferation via its sense gene RAB11B].

Objective: To detect the expression of lncRNA RAB11B-AS1 in osteosarcoma and investigate its role in osteosarcoma cells proliferation and the responsible mechanisms. Methods: Osteosarcoma and corresponding adjacent normal tissues were collected from 24 patients subjected to operations from October 2015 to October 2017 in the Third Affiliated Hospital of Southern Medical University.RAB11B-AS1 expression was detected in osteosarcoma specimens by quantitative real-time polymerase chain reaction (qRT-PCR). Lentiviral vectors that stably over-expressing RAB11B-AS1 were constructed and transfected into U2OS osteosarcoma cell line.The effect of RAB11B-AS1 on osteosarcoma cell proliferation and apoptosis was investigated by cell counting kit (CCK-8) assay and flow cytometry.U2OS osteosarcoma xenograft model of nude mice was established to observe the effect of RAB11B-AS1 on xenograft growth in mice, and the role of RAB11B-AS1 in proliferation and apoptosis of osteosarcoma cells was investigated by immunohistochemistry and TUNEL staining of osteosarcoma slices.The relationship between RAB11B-AS1 and RAB11B was explored using luciferase reporter assay.The data were compared with t test between the two groups. Results: Expression of RAB11B-AS1 was significantly down-regulated in osteosarcoma (0.010±0.015) versus their paired non-neoplastic tissues (0.022±0.030) (t=2.117, P=0.045). Up-regulation of RAB11B-AS1 resulted in decreased proliferative rate of U2OS cells (F=15.659, P<0.001). The ratios of cells in G0-G1 phase, S phase, G2-M phase were 62.6%±6.3%, 21.4%±2.2%, 16.3%±1.6% respectively in RAB11B-AS1 up-regulated group versus 59.4%±5.9%, 25.9%±2.6%, 15.5%±1.1% respectively in control group, and cell ratio in G0-G1 and S phase were increased significantly by RAB11B-AS1 up-regulation (t=17.124, 17.321, both P<0.05). Apoptosis rate was significantly elevated in RAB11B-AS1 over-expressed cells (12.7%±1.3%) when compared with that in control (10.3%±1.0%)(t=17.321, P=0.003). Mice transplanted with osteosarcoma cells that overexpressed RAB11B-AS1 exhibited lower growth rate of tumor (F=8.798, P=0.009). Mechanistically, RAB11B-AS1 expression correlated negatively with RAB11B expression (r=-0.356, P=0.044). Conclusions: lncRNA RAB11B-AS1 expression is down-regulated significantly in osteosarcoma tissues.RAB11B-AS1 may suppress the progression of osteosarcoma via down-regulating RAB11B.

[A clinical analysis of hepatic veno-occlusive disease after hematopoietic stem cell transplantation].

Objective: To analyze the outcome and the prognostic factors of hepatic veno-occlusive disease (HVOD) after hematopoietic stem cell transplantation (HSCT). Methods: A total of 797 patients receiving HSCT were analyzed retrospectively. The prophylaxis regimen of HVOD in the First Affiliated Hospital of Guangxi Medical University consisted of low molecular weight heparin and lipoprostaglandin E1 (PGE1). Results: Fifty-nine patients (7.4%) developed HVOD at 3-49 days after HSCT (median 12 days). Age younger than 15 years at transplant(HR= 6.47, P<0.001), busulphan conditioning (HR=6.40, P<0.001), thalassemia major (HR=6.35, P<0.001), allogeneic transplantation (HR=7.74, P=0.005) were univariate risk factors for HVOD. Multivariate analyses suggested that thalassemia major and busulphan conditioning were independently correlated with the development of HVOD. Conclusion: Thalassemia major and busulphan conditioning are independent risk factors for HVOD after HSCT.

[Clinical features and prognostic analysis of neuromyelitisoptica spectrum disease with sjogrensyndrome].

Objective: To explore the clinical features and conduct prognostic analysis about visual recovery and relapse of neuromyelitisoptica (NMO) spectrum disease (NMOSD) with sjogren syndrome (SS). Methods: A retrospective and prospective observational study was conducted.Between July 2013 and June 2016, 172 patients with NMOSD (NMOSD-non SS: 116/172, 67.4%; NMOSD-SS: 56/172, 32.6%) were assessed at Beijing Tongren Hospital, Capital Medical University, Beijing, China.The prognostic factors of NMOSD-SS patients were also analyzed. Results: As compared with NMOSD-non SS patients, NMOSD-SS patients had worse visual impairment (percentage of patients with visual acuity less than 0.1, 83.9% vs 69.8%, P<0.05), higher positive rate of SSA (92.9% vs 0.0%, P<0.05), higher proportion of dryness of mouth and eye (66.1% vs 5.2%, P<0.05) as well as higher percentage of reduced visual evoked potential (VEP) amplitude (60.7% vs 43.1%, P<0.05). NMOSD-SS patients had a significantly higher average year recurrent frequency (0.58 vs 0.53) and significantly shorter mean recurrence time (6.7 months vs 12.4 months, P<0.05). The results showed that recurrent eyes, the worst visual acuities of onset less than 0.1 were independent risk factors of visual impairment (visual activity <0.1), according to at least six months' follow-up of all NMOSD-SS patients (OR=6.410 and 9.434, respectively, P<0.05). Meanwhile, immunosuppressive drugs were protective factors of relapse in NMOSD-SS patients (OR=0.107, P<0.05). Conclusions: NMOSD-SS patients have worse visual impairment, and they are more vulnerable to relapse than NMOSD-non SS patients, and the vision is lack of recovery for NMOSD-SS with recurrent eyes or the worst vision of onset less than 0.1.Immunosuppressive drugs can reduce the recurrence of NMOSD-SS relapse.

Role of atopy in chronic rhinosinusitis with nasal polyps: does an atopic condition affect the severity and recurrence of disease?

BACKGROUND: The role of atopy in chronic rhinosinusitis is unclear: it is particularly controversial in chronic rhinosinusitis with nasal polyps. METHODS: A prospective study of 210 patients with chronic rhinosinusitis with nasal polyps was performed. Patient demographics, visual analogue scale scores, Lund-Kennedy endoscopy scores, Lund-Mackay computed tomography scores, serum total immunoglobulin E levels, serum eosinophil cationic protein (ECP) levels and Phadiatop test findings were analysed. RESULTS: There were no significant differences in age, sex, visual analogue scale score, Lund-Mackay computed tomography score, total serum immunoglobulin E level, serum ECP level or Phadiatop test results between patients with primary and recurrent chronic rhinosinusitis with nasal polyps. A total of 99 patients (47 per cent) had positive atopy tests. No significant differences in sex, visual analogue scale score, Lund-Kennedy endoscopy score, Lund-Mackay computed tomography score or recurrence rates were found between atopic and non-atopic patients; however, atopic patients were significantly younger than non-atopic patients. Atopy status did not correlate with disease severity. CONCLUSION: There was no association between atopy status and either disease severity or recurrence in patients with chronic rhinosinusitis with nasal polyps, although atopic patients were younger than non-atopic patients.

Mucosolvan serves to optimize perioperative airway management for NSCLC patients in fast track surgery: a randomized placebo controlled study.

OBJECTIVE: This study is conducted to investigate whether mucosolvan may offer therapy optimization initiatives for airway perioperative management of patients with non-small cell lung cancer (NSCLC) in fast track surgery (FTS). PATIENTS AND METHODS: The patients in FTS were treated with and without aerosol (90 mg/day) of mucosolvan in combination of intravenous administration of the agent (180 mg/day) for 8 consecutive days. Postoperative complications and length of time of hospital stay were examined in the patients treated with and without mucosolvan. RESULTS: Prevalence rate for the overall postoperative complication was significantly decreased in the mucosolvan-treated patients as compared to the untreated ones (p < 0.05). In further analysis, an appearance of postoperative pulmonary complication was reduced in the treated patients as well (p < 0.05). There was no statistical difference in a morbidity rate of postoperative cardiac complications between these two groups. Furthermore, treatment with mucosolvan resulted in significantly decreasing length of time of hospital stay as compared to the untreated patients (p < 0.05), indicating that this agent may facilitate early recovery of the patients in FTS after major surgical approaches. CONCLUSIONS: Mucosolvan optimizes the perioperative airway management for NSCLC patients in FTS through reducing postoperative complications and shortening time of hospital stay.

Magnetism-Driven Ferroelectricity in Double Perovskite Y2NiMnO6.

We report the discovery of multiferroic behavior in double perovskite Y2NiMnO6. X-ray diffraction shows that the material has a centrosymmetric crystal structure of space group P2(1)/n with Ni(2+)/Mn(4+) ordering. This result is further confirmed by aberration-corrected scanning transmission electron microscopy combined with atomic resolution electron energy loss spectroscopy. The appearance of ferroelectric polarization coincides with the magnetic phase transition (∼67 K), which indicates that the ferroelectricity is driven by magnetism, and this is further confirmed by its strong magnetoelectric (ME) effect. We proposed the origin of the ferroelectricity is associated with the combination of Ni(2+)/Mn(4+) charge ordering and the ↑↑↓↓ spin ordering. When compared with other known magnetic multiferroics, Y2NiMnO6 displays several attractive multiferroic properties, including high polarization (∼145 µC/m(2)), a high multiferroic transition temperature (∼67 K), and strong ME coupling (∼21%).

MicroRNA screening and functional study of obliterative bronchiolitis in a rat model simulating lung transplantation.

The aim of this study was to provide the experimental basis for effective prevention and treatment of obliterative bronchiolitis (OB) by studying the changes on the microRNA (miRNA) expression profile after an orthotopic tracheal transplantation (OTT) simulating lung transplantation (LT). The OTT was performed on inbred rats to establish an OB animal model simulating LT, which was confirmed successful through pathological examination after 4 weeks. A miRNA microarray was used to screen for the most significantly differentially expressed miRNA in the OB tissues of donor transplanted trachea and real-time quantitative PCR was then used to validate the reliability of the microarray results. The microarray detection obtained 29 OB-related miRNAs, composed of 15 and 14 significantly up- and down-regulated miRNAs, respectively, among which miR-146a, miR-155, and miR-451, whose function is involved in the immune and inflammatory reactions, were subjected to relative quantitation research. The LT-simulated OTT-induced OB showed significantly differential expressions of multiple miRNAs, among which miR-146a and miR-155 were highly expressed, while miR-451 was lowly expressed, suggesting that these miRNAs may play an important regulatory role in the OB pathological process after LT.

Multidetector CT features of pulmonary focal ground-glass opacity: differences between benign and malignant.

OBJECTIVE: To evaluate different features between benign and malignant pulmonary focal ground-glass opacity (fGGO) on multidetector CT (MDCT). METHODS: 82 pathologically or clinically confirmed fGGOs were retrospectively analysed with regard to demographic data, lesion size and location, attenuation value and MDCT features including shape, margin, interface, internal characteristics and adjacent structure. Differences between benign and malignant fGGOs were analysed using a χ(2) test, Fisher's exact test or Mann-Whitney U-test. Morphological characteristics were analysed by binary logistic regression analysis to estimate the likelihood of malignancy. RESULTS: There were 21 benign and 61 malignant lesions. No statistical differences were found between benign and malignant fGGOs in terms of demographic data, size, location and attenuation value. The frequency of lobulation (p=0.000), spiculation (p=0.008), spine-like process (p=0.004), well-defined but coarse interface (p=0.000), bronchus cut-off (p=0.003), other air-containing space (p=0.000), pleural indentation (p=0.000) and vascular convergence (p=0.006) was significantly higher in malignant fGGOs than that in benign fGGOs. Binary logistic regression analysis showed that lobulation, interface and pleural indentation were important indicators for malignant diagnosis of fGGO, with the corresponding odds ratios of 8.122, 3.139 and 9.076, respectively. In addition, a well-defined but coarse interface was the most important indicator of malignancy among all interface types. With all three important indicators considered, the diagnostic sensitivity, specificity and accuracy were 93.4%, 66.7% and 86.6%, respectively. CONCLUSION: An fGGO with lobulation, a well-defined but coarse interface and pleural indentation gives a greater than average likelihood of being malignant.

Monte Carlo simulation of size, random field and temperature dependences of exchange bias in a core/shell magnetic nanoparticle.

By using Monte Carlo simulation on a ferromagnetic core/antiferromagnetic shell nanoparticle, we investigate in detail the exchange bias of the magnetic hysteresis as a function of both core radius and shell thickness, at low temperature. It is found that the exchange bias is very sensitive to the core radius and a small variation of the radius may lead to a big fluctuation of the bias. In a general tendency the exchange bias is enhanced by increasing shell thickness and decreasing core radius. The intrinsic correlation between the exchange bias and the spin configuration on the core-shell interface is demonstrated. We further investigate the dependence of the exchange bias on temperature and random field inside the nanoparticle, indicating a monotonic decreasing of the bias with the magnitude of random field and temperature, respectively.

Validation of a high-performance liquid chromatography method for the assay of and determination of related organic impurities in nevirapine drug substance.

Nevirapine (Viramune), a dipyridiodiazepinone, is a potent and highly specific nonnucleoside inhibitor of HIV-1 reverse transcriptase. This paper describes the validation of a specific, sensitive, and stability-indicating high-performance liquid chromatography method for the assay and determination of related organic impurities in nevirapine drug substance. This method uses a Supelcosil LC-ABZ column, a mobile phase of 20:80 (v/v) acetonitrile-25mM NH4H2PO4 (pH 5.0), and ultraviolet detection at a wavelength of 220 nm. This method was validated for specificity, linearity, accuracy, repeatability, detection limit, quantitation limit, stability of analyte solutions, robustness, and intermediate precision. Nevirapine is completely separated from all impurities. The method is shown to be linear with coefficients of determination r2 greater than 0.999. Average accuracy is 100.4% with a relative standard deviation of 0.7% for the assay. Accuracy ranges from 100.1 to 102.6% for related organic impurities. Repeatability is good, with relative standard deviations not more than 1.4%. The detection limit and the quantitation limit are determined to be 0.001 and 0.003%, respectively. Relative response factors of known organic impurities are determined, permitting the use of nevirapine at the 0.1% level as an external standard for the quantitation of these impurities. Analyte solutions are shown to be stable for at least 2 days at ambient temperature. The method is validated as robust, and intermediate precision is high. A system suitability test is developed and validated, and requirements are set.

[Determination of docosahexenoic acid in human serum by capillary gas chromatography].

A reliable CGC method was developed for the determination of docosahexenoic acid (DHA) in human serum. The serum sample was acidified by adding two drops of 4.5 mol.L-1 H2SO4, and DHA was extracted from serum using ethyl acetate. The extract was evaporated to dryness under nitrogen stream. To each residue, 1 ml 1.3 mol.L-1 methanolic hydrogen chloride solution was added and the mixture was allowed to stand for 30 min in 60 degrees C water bath. After derivatization, the mixture was extracted with 1 ml of n-hexane. The solvent was evaporated under a stream of nitrogen to dryness and the residue was dissolved in 30 microliters n-hexane and subjected to capillary GC, which was equipped with a fused silica capillary column (26.3 m x 0.25 mm ID) coated with FFAP (free fatty acid phase, 0.1 micron film thickness). Tricosanoic acid was used as an internal standard. The retention time of DHA-M and internal standard was 23.41 min and 20.79 min, respectively. The minimum detection concentration of DHA in serum was 40 ng.ml-1 with a serum volume of 200 microliters and S/N value of 2. A good linear relationship between the peak area ratios and concentrations was found at the DHA concentrations ranging from 25 to 200 micrograms.ml-1. The within-day and between-day precision was 5% and 9%, respectively.

Avian LHRH during embryonic development: measurement by competitive ELISA with a monoclonal antibody.

A mouse monoclonal antibody directed against chicken Gln8-luteinizing hormone-releasing hormone (cLHRH-I) was developed and characterized. This antibody was used for the development of a competitive microtiter plate enzyme-linked immunosorbent assay for avian LHRH. The assay was validated for use with tissue and was used at a working range between 5 pg and 10 ng per sample. Using this procedure, cLHRH-I and II were assayed in whole brain extracts of Japanese Quail embryos. Samples were taken at regular intervals between Day 6 of incubation through Day 1 posthatch. There were 10 samples taken at each age with 2 replicates of the entire sampling regime. Data from males and females were pooled. LHRH concentrations were low, then rose to higher levels (15 pg/mg tissue) between Days 10 through 13 and decreased thereafter. These changes are likely to be correlated with the activation of the hypothalamic-pituitary-gonadal axis. This is particularly apparent in later embryonic development.

Production of monoclonal antibodies against avian LHRH-I.

Production of antibodies against peptides or poorly antigenic proteins by conventional methods often requires either large quantities of the native immunogen or some chemical modification to increase their antigenicity. In this study an in vivo and in vitro immunization protocol has been used to generate monoclonal antibodies against the decapeptide luteinizing hormone-releasing hormone (LHRH). Two injections of 100 micrograms of avian LHRH-I into BALB/c mice were given 7 d apart. Dissociated splenocytes were collected under sterile conditions. They were incubated with 100 micrograms of the immunogen in 75-cm2 tissue culture flasks in thymocyte-conditioned media. After 5 to 8 d exposure to the antigen, splenocytes were fused with SP2/O myeloma cells by polyethylene glycol. The cells were plated into 24 wells and then incubated in hypoxanthine aminopterin and thymidine selective media. After 14 d an initial screening was done by enzyme immunoassay. The positive wells (6/24) were expanded into 96-well plates and rescreened. Selected lines were cloned out 3 times by limiting dilution and the most positive expanded for ascites production. The antibody was affinity purified in a protein A column. The antibody cross-reacted with LHRH-I and II but preferentially to LHRH-I, as shown by competitive assay. A hypothalamic extract from a mature chick showed a higher response than preparations from whole brain explants of 1- to 3-d posthatched chicks, mature quail, and mature mouse.