Development of international guidelines by Tuina practitioners for specific acupoints of paediatrics Tuina (2022 version).

OBJECTIVE: To establish a standardized framework encompassing the precise locations, manipulations, functions and indications of specific acupoints in the field of paediatric Tuina. METHODS: The development of consensus involved three distinct stages. Initially, a list of paediatric Tuina specific acupoints was compiled based on an extensive literature review, which was subsequently supplemented through expert interviews. In the second stage, the Delphi method was employed to assess the significance of acupoint locations, manipulations, functions, and indications. In situations where the questionnaire survey failed to yield agreement or when the experts held reservations, the nominal group approach was utilized during the expert consensus meeting. The final version of the technical standardized material was ultimately determined during an expert consensus conference. After undergoing external peer review and evaluation, the completed draft was prepared for public dissemination RESULTS: The comprehensive list identified a total of 66 specific acupoints. The location and manipulation questionnaire consisted of 156 items based on the literature database, while the function and indication questionnaire contained 116 items. Two rounds of Delphi surveys were conducted for the location and manipulation category, and another two rounds of Delphi surveys were conducted for the function and indication category. During the experts consensus meeting The panel of experts conducted in-depth discussions on 61 questions, resulting in the formulation of technical guidelines for the locations, manipulations, functions, and indications of 64 paediatric Tuina acupoints. Subsequently, the research team compiled and edited the draft of the technical guidelines for acupoints of paediatric Tuina, which was finalized after external review and feedback. CONCLUSION: This study successfully established the recognized technical standards for practitioners of paediatric Tuina, thereby standardizing clinical practices and providing a foundation setting the framework for future research. The guidelines offer theoretical insights and recommendations for conducting clinical studies comparing different acupoint sites, as well as modifying or enhancing treatment regimens.

miR-146a aggravates cognitive impairment and Alzheimer disease-like pathology by triggering oxidative stress through MAPK signaling.

INTRODUCTION: Mir-146a-5p has been widely recognized as a critical regulatory element in the immune response. However, recent studies have shown that miR-146a-5p may also be involved in the development of Alzheimer disease (AD). Regrettably, the related mechanisms are poorly understood. Here, we investigated the effects of miR-146a in mice models and SH-SY5Y cells treated with amyloid ß (Aß)1-42. METHODS: To create a model of AD, SH-SY5Y cells were treated with Aß1-42 and mice received intracerebroventricular injections of Aß1-42. Then, the transcriptional levels of miR-146a were estimated by real-time PCR. We transiently transfected the miR-146a-5p mimic/inhibitor into cells and mice to study the role of miR-146a. The role of signaling pathways including p38 and reactive oxygen species (ROS) was studied by using specific inhibitors. Aß and amyloid-beta precursor protein (APP)levels were measured by immunoblotting. Furthermore, Aß expression was analyzed by immunofluorescence and histochemical examinations. RESULTS: Aß1-42-stimulated SH-SY5Y cells displayed increased transcriptional levels of miR-146a and APP. Moreover, the p38 MAPK signaling pathway and ROS production were activated upon stimulation with a miR-146a-5p mimic. However, treatment with a miR-146a-5p inhibitor decreased the levels of APP, ROS, and p-p38 MAPK. A similar phenomenon was also observed in the animals treated with Aß1-42, in which miR-146a upregulation increased the expression of Aß, p-p38, and ROS, while the inhibition of miR-146a had the opposite effect. This suggests that miR-146a increases Aß deposition and ROS accumulation via the p-p38 signaling pathway. CONCLUSIONS: Our research demonstrates that miR-146a-5pa increases Aß deposition by triggering oxidative stress through activation of MAPK signaling.

miR-146a aggravates cognitive impairment and Alzheimer disease-like pathology by triggering oxidative stress through MAPK signalingmi / R-146a empeora el deterioro cognitivo y la patología tipo Alzheimer al promover el estrés oxidativo a través de la vía de señalización MAPK

Introduction: Mir-146a-5p has been widely recognized as a critical regulatory element in the immune response. However, recent studies have shown that miR-146a-5p may also be involved in the development of Alzheimer disease (AD). Regrettably, the related mechanisms are poorly understood. Here, we investigated the effects of miR-146a in mice models and SH-SY5Y cells treated with amyloid β (Aβ)1–42. Methods: To create a model of AD, SH-SY5Y cells were treated with Aβ1–42 and mice received intracerebroventricular injections of Aβ1–42. Then, the transcriptional levels of miR-146a were estimated by real-time PCR. We transiently transfected the miR-146a-5p mimic/inhibitor into cells and mice to study the role of miR-146a. The role of signaling pathways including p38 and reactive oxygen species (ROS) was studied by using specific inhibitors. Aβ and amyloid-beta precursor protein (APP)levels were measured by immunoblotting. Furthermore, Aβ expression was analyzed by immunofluorescence and histochemical examinations. Results: Aβ1–42-stimulated SH-SY5Y cells displayed increased transcriptional levels of miR-146a and APP. Moreover, the p38 MAPK signaling pathway and ROS production were activated upon stimulation with a miR-146a-5p mimic. However, treatment with a miR-146a-5p inhibitor decreased the levels of APP, ROS, and p-p38 MAPK. A similar phenomenon was also observed in the animals treated with Aβ1–42, in which miR-146a upregulation increased the expression of Aβ, p-p38, and ROS, while the inhibition of miR-146a had the opposite effect. This suggests that miR-146a increases Aβ deposition and ROS accumulation via the p-p38 signaling pathway. Conclusions: Our research demonstrates that miR-146a-5pa increases Aβ deposition by triggering oxidative stress through activation of MAPK signaling.(AU)

Introducción: miR-146a-5p es un elemento regulador clave en la respuesta inmune. Sin embargo, estudios recientes sugieren que miR-146a-5p también está involucrado en el desarrollo de la enfermedad de Alzheimer (EA), aunque aún no se conoce con exactitud el mecanismo por el que esto sucede. Analizamos los efectos de miR-146a en un modelo animal y en células SH-SY5Y expuestas a β-amiloide (Aβ)1-42. Métodos: Tratamos células SH-SY5Y con Aβ1-42 e inyectamos Aβ1-42 en los ventrículos cerebrales de ratones para generar un modelo celular y otro animal de EA. Estimamos los niveles transcripcionales de miR-146a mediante PCR en tiempo real. Al mismo tiempo, transfectamos temporalmente las células y los ratones con imitador/inhibidor de miR-146a-5p para evaluar el papel de miR-146a. Estudiamos el papel de algunas vías de señalización, como la de p38, y los niveles de especies reactivas de oxígeno (ERO) con inhibidores específicos. Los niveles de Aβ y de proteína precursora amiloidea (APP) se determinaron con inmunoblot. También se analizó la expresión de Aβ mediante inmunofluorescencia y análisis histoquímico. Resultados: Las células SH-SY5Y expuestas a Aβ1-42 mostraron altos niveles transcripcionales de miR-146a y APP. La vía de señalización p-38 MAPK y la producción de EROs se activaron al utilizar un imitador de miR-146a-5p. Sin embargo, el bloqueo de miR-146a-5p con un inhibidor redujo los niveles de APP, EROs y p-p38 MAPK. Se observó un fenómeno similar en los ratones tratados con Aβ1-42: la sobrerregulación de miR-146a aumentó la expresión de Aβ, p-p38 y EROs, mientras que la inhibición de miR-146a tuvo el efecto contrario. Esto sugiere que miR-146a está involucrado en el aumento de acumulación de Aβ y de producción de EROs por medio de la vía de señalización p-p38. Conclusiones: Nuestro estudio muestra que miR146a-5p aumenta la acumulación de Aβ al promover el estrés oxidativo a través de la activación de la vía de señalización MAPK.(AU)

[Impact of neoadjuvant immunotherapy on pulmonary function and perioperative outcomes in patients with resectable non-small cell lung cancer].

Objective: To explore the effect of neoadjuvant immunotherapy on pulmonary function and the efficacy in patients with resectable non-small cell lung cancer. Methods: Data of 30 patients with non-small cell lung cancer (NSCLC) who received neoadjuvant immunotherapy before surgery in the Chest Hospital of Shanghai Jiaotong University from March 2018 to September 2021 were retrospectively collect. The efficacy and safety of neoadjuvant immunotherapy in the perioperative period and changes in pulmonary function of patients before and after neoadjuvant treatment were valuated. Results: The patients were all-male with age of (61±8)years old, The major pathological response (MPR) rate of patients receiving neoadjuvant immunotherapy was 43%(13 cases), the pathologic complete response (pCR) rate was 37% (11 cases), disease control rate (DCR) was 97% (29 cases), objective response rate (ORR) was 67% (20 cases). The forced expiratory volume in one second (FEV1) after treatment was (2.59±0.63) L, and the ratio of FEV1 to the predicted value (FEV1%pred) was 85.27%±15.86%, which were significantly higher than those before treatment [(2.48±0.59)L, 81.73%±15.94%, respectively] (P=0.013, 0.022, respectively). Forced vital capacity (FVC) after treatment was (3.59±0.77) L, which was also significantly higher than before [(3.47±0.76) L,P=0.036]; while there were no statistical difference in FEV1/FVC and FVC accounted for the proportion of predicted values (FVC%pred) between before and after treatment (P=0.084, 0.344, respectively). The ratio of carbon monoxide dispersion (DLCO) to the predicted value (DLCO%pred) decreased from 83.61%±13.10% to 78.69%±13.85% after treatment (P=0.023). There was no significant difference in the incidence of postoperative complications between the DLCO%pred decreased group and the non-decreased group (3/18 vs 0/6; P=0.546). Conclusions: Neoadjuvant immunotherapy can increase the rate of MPR and PCR, significantly increase FEV1 and FEV1%pred, but also lead to a decrease in DLCO%pred; neoadjuvant immunotherapy does not increase the incidence of postoperative complications.

miR-146a aggravates cognitive impairment and Alzheimer disease-like pathology by triggering oxidative stress through MAPK signaling.

INTRODUCTION: Mir-146a-5p has been widely recognized as a critical regulatory element in the immune response. However, recent studies have shown that miR-146a-5p may also be involved in the development of Alzheimer disease (AD). Regrettably, the related mechanisms are poorly understood. Here, we investigated the effects of miR-146a in mice models and SH-SY5Y cells treated with amyloid ß (Aß)1-42. METHODS: To create a model of AD, SH-SY5Y cells were treated with Aß1-42 and mice received intracerebroventricular injections of Aß1-42. Then, the transcriptional levels of miR-146a were estimated by real-time PCR. We transiently transfected the miR-146a-5p mimic/inhibitor into cells and mice to study the role of miR-146a. The role of signaling pathways including p38 and reactive oxygen species (ROS) was studied by using specific inhibitors. Aß and amyloid-beta precursor protein (APP)levels were measured by immunoblotting. Furthermore, Aß expression was analyzed by immunofluorescence and histochemical examinations. RESULTS: Aß1-42-stimulated SH-SY5Y cells displayed increased transcriptional levels of miR-146a and APP. Moreover, the p38 MAPK signaling pathway and ROS production were activated upon stimulation with a miR-146a-5p mimic. However, treatment with a miR-146a-5p inhibitor decreased the levels of APP, ROS, and p-p38 MAPK. A similar phenomenon was also observed in the animals treated with Aß1-42, in which miR-146a upregulation increased the expression of Aß, p-p38, and ROS, while the inhibition of miR-146a had the opposite effect. This suggests that miR-146a increases Aß deposition and ROS accumulation via the p-p38 signaling pathway. CONCLUSIONS: Our research demonstrates that miR-146a-5pa increases Aß deposition by triggering oxidative stress through activation of MAPK signaling.

[Mechanism of Fuke Qianjin Capsules in treating intrauterine adhesion in rats through TGF-ß1-PI3K/Akt signaling pathway].

To explore the effect of Fuke Qianjin Capsules on anti-endometrial fibrosis in intrauterine adhesion(IUA) rats through TGF-ß1-PI3 K/Akt signaling pathway. With female SD rats as the object, IUA rat models were established through mechanical injury and infection, and they were randomly divided into normal group, sham operation group, Bujiale group(0.63 mg·kg~(-1)·d~(-1)), and high-dose Fuke Qianjin Capsules group(1.008 g·kg~(-1)·d~(-1)), medium-dose Fuke Qianjin Capsules group(0.504 g·kg~(-1)·d~(-1)), low-dose Fuke Qianjin Capsules group(0.252 g·kg~(-1)·d~(-1)). The rats were sacrificed 21 days after drug administration, and the uterus and liver were removed after blood collection from the abdominal aorta. The morphology of the uterus was observed with the naked eyes; the pathological and morphological changes of the uterine tissue and liver were observed by HE staining; the degree of fibrosis of the uterine tissue was observed by Masson staining; the expressions of TGF-ß1, TNF-α and IL-6 in serum were detected; the expressions of TGF-ß1, PI3 K, Akt, p-Akt protein in uterine tissue were detected by Western blot. The results showed that Fuke Qianjin Capsules could improve the pathological changes of uterine tissues in IUA rats, without damage to liver tissues, and reduce the expressions of TGF-ß1, TNF-α and IL-6 in serum(P<0.01); significantly reduce TGF-ß1, PI3 K, p-Akt protein expression in uterine tissues(P<0.05, P<0.01). It is indicated that Fuke Qianjin Capsules could exert the anti-endometrial fibrosis effect by regulating the TGF-ß1-PI3 K/Akt signal pathway, so as to achieve the effect in treating IUA rats, especially with the best effect in medium-dose Fuke Qianjin Capsules group.

Establishment and Its Application of UPLC-MS/MS Method for 20 Fentanyl-related Substances in Blood.

ABSTRACT: Objective To establish an ultra-high performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） rapid determination method for simultaneous analysis of 20 fentanyl-related substances in blood. Methods With fentanyl-D5 as an internal standard, the blood was extracted by liquid-liquid extraction （LLE）, then separated with an ACQUITY UPLC HSS T3 chromatographic column, and finally 20 fentanyl-related substances were simultaneously analyzed with multiple reaction monitoring （MRM） mode. Results The limits of detection （LOD） of all compounds were 0.02-0.03 ng/mL, and the limits of quantitation （LOQ） were 0.05-0.2 ng/mL. Within the mass concentration range of 0.05-40 ng/mL, 20 fentanyl-related substances had a good linear relationship, and correlation coefficients were larger than 0.99. The accuracy of the method was 87.69%-114.68% and the extraction recovery rate was 85.35%-101.80%, and no significant matrix effect was observed. The established method was successfully applied to the detection of sufentanil in rat blood after sufentanil was injected. Sufentanil could still be detected in blood of rats 10 h after sufentanil injection. Conclusion The established method has the advantages of simple pretreatment, high sensitivity and good selectivity, and can be used for the determination of fentanyl-related substances in forensic toxicology analysis.

Rapid Determination of Cocaine and Its Metabolite Benzoylecgonine in Hair by LC-MS/MS.

OBJECTIVES: To establish a rapid determination method with LC-MS/MS for cocaine and its metabolite benzoylecgonine in hair. METHODS: Deuterated internal standards （cocaine-D3 and benzoylecgonine-D8） were added to the decontaminated hair. After the extraction by ultrasonication with methanol, the compounds were separated by the Restek Allure PFP propyl column, and cocaine and benzoylecgonine were simultaneously analysed in multiple reaction monitoring mode. RESULTS: The cocaine and benzoylecgonine in hair showed a good linearity in the range of mass fraction between 0.02 and 10.00 ng/mg with the limits of detection of 0.01 ng/mg. CONCLUSIONS: The developed method is simple and rapid with a good selectivity, which is suitable for the determination of cocaine and its metabolite benzoylecgonine in hair.

[Determination of Sulfide Ion in Blood from Hydrogen Sulfide Poisoning Cases].

OBJECTIVES: To establish a gas chromatography-mass spectrometry （GC-MS） method for the determination of sulfide ion in blood and apply it to the practical cases. METHODS: The 1, 3, 5-tribromobenzene was selected as an internal standard, and 0.2 mL blood sample was collected and analyzed using GC-MS after α-Bromo-2, 3, 4, 5, 6-pentafluorobenzyl bromide derivatization. RESULTS: The mass concentration of sulfide ion in blood had good linearity in the range of 0.2-40 µg/mL with a limit of detection （LOD） of 0.05 µg/mL. The mass concentration of sulfide ion was less than 0.05 µg/mL in blank blood from different sources such as healthy subjects and dead cases. In 3 sulfide poisoning cases, sulfide ion was detected in the blood samples of 6 victims, and the mass concentration range was 1.02-3.13 µg/mL. CONCLUSIONS: This study establishes a method for investigation of sulfide ion in blood which has been applied successfully to the cases of fatal sulfide poisonings.

The association of functional polymorphisms in the aryl hydrocarbon receptor (AHR) gene with the risk of vitiligo in Han Chinese populations.

BACKGROUND: Vitiligo is an acquired depigmentation disorder resulting from selective destruction of melanocytes. The aryl hydrocarbon receptor (AHR) is vital to the regulation of melanogenesis and melanocyte proliferation and differentiation through modulating the expressions of melanogenesis-related genes. AHR mutations may negatively affect AHR proteins and its target genes. Therefore, we hypothesized that AHR polymorphisms might be involved in vitiligo by impacting the transcriptional activities of related genes as mentioned above. OBJECTIVES: To evaluate the potential association between AHR polymorphisms and vitiligo susceptibility. METHODS: We performed a hospital-based, case-control study of 1000 patients with vitiligo and 1000 vitiligo-free but age- and gender-matched controls. Two single nucleotide polymorphisms of the AHR gene (rs10249788 and rs2066853) were selected and genotyped using a polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: A statistically significantly decreased risk of vitiligo was found to be associated with the TT and CT genotypes of rs10249788 [odds ratio (OR) 0·59, 95% confidence interval (CI) 0·38-0·93; P = 0·028 and OR 0·82, 95% CI 0·68-0·98; P = 0·032, respectively] as well as among subgroups: male, active, nonsegmental vitiligo, and onset age ≤ 20 years. Moreover, subjects with the combined (CT + TT)/GG genotype or T/G haplotype (rs10249788/rs2066853) showed a decreased risk for vitiligo (OR 0·57, 95% CI 0·37-0·87, P = 0·009 and OR 0·78, 95% CI 0·64-0·96, P = 0·033, respectively). CONCLUSIONS: These results suggest that the T allele of rs10249788 located in the promoter of the AHR gene is associated with a protective effect on vitiligo in Han Chinese populations.

Inhibitory effects of rosa roxburghii tratt juice on in vitro oxidative modification of low density lipoprotein and on the macrophage growth and cellular cholesteryl ester accumulation induced by oxidized low density lipoprotein.

BACKGROUND: Rosa roxburghii tratt juice (RRTJ) administration has been shown to significantly ameliorate atherosclerotic diseases in cholesterol-fed animals. However, the mechanism for the antiatherogenic effect of RRTJ is not clear. METHODS: We investigated the effects of RRTJ on in vitro oxidative modification of LDL and on LDL-induced macrophage growth and cellular cholesteryl ester (CE) accumulation. The effects of RRTJ on LDL oxidative modification were assessed by relative electrophoretic migration, thiobarbituric acid-reactive substance (TBARS) content, and the formation of conjugated dienes. The inhibition of RRTJ on oxidized LDL (Ox-LDL)-induced murine peritoneal macrophage growth was evaluated by a cell-counting assay and an MTT assay. The effect of RRTJ on Ox-LDL-induced cellular CE accumulation was examined after macrophages were incubated with Ox-LDL in the presence of RRTJ. To clarify the mechanism of the inhibitory effect of RRTJ on Ox-LDL-induced CE accumulation in macrophages, its capacity for cholesterol efflux from macrophage-derived foam cells were examined. RESULTS: We showed that RRTJ significantly reduced LDL oxidative susceptibility. In addition, RRTJ effectively suppressed Ox-LDL-induced macrophage growth and especially Ox-LDL-induced CE accumulation in murine peritoneal macrophages by promoting cellular cholesterol efflux. CONCLUSION: These results indicated that RRTJ exerted its antiatherogenic effects largely due to its ability to inhibit the oxidative modification of LDL and to suppress the formation of foam cells.

Relationship between endogenous estrogen concentrations and serum cholesteryl ester transfer protein concentrations in Chinese women.

BACKGROUND: CETP plays an important role in HDL metabolism and in the reverse cholesterol transport pathway. METHODS: The relationship between the changes of endogenous estrogen and the concentration of cholesteryl ester transfer protein (CETP) in the serum of Chinese women was investigated. Serum concentrations of estradiol (E(2)), follicle-stimulating hormone (FSH), CETP and lipid profile were determined in 196 Chinese women (52 premenopausal with ages ranging from 18 to 40 years, 57 perimenopausal from 41 to 60 years, and 87 postmenopausal from 61 to 81 years). RESULTS: Serum CETP concentration was significantly lower in postmenopausal women compared with those in perimenopausal and premenopausal women (1.39+/-1.06, 2.36+/-1.50 and 2.31+/-1.25 mg/l, respectively, P<0.0001). Even in the women around the menopausal, CETP concentration in postmenopause was significantly lower than that in premenopause (1.93+/-1.33 vs. 3.42+/-1.35 mg/l, P<0.01). In addition, CETP concentration had a highly positive correlation with serum concentration of E(2) (r=0.243, P<0.001), while negative correlation of CETP concentration with serum concentration of FSH was found (r=-0.273, P<0.001). CONCLUSIONS: Estrogen may affect the concentration of CETP.

Serum cholesteryl ester transfer protein concentrations in healthy Chinese subjects and cardio-cerebrovascular disease patients.

Serum cholesteryl ester transfer protein (CETP) concentration was measured in 1128 healthy Chinese subjects using a "sandwich" enzyme immunoassay and was 1.84 +/- 1.55 mg/l (mean +/- S.D.). The frequency distribution of CETP in healthy subjects was markedly skewed towards low concentrations. The CETP concentration in females was significantly higher than that in males (2.40 +/- 1.65 mg/l vs. 1.49 +/- 1.37 mg/l, P < 0.001). There was a weak inverse correlation between the CETP concentration and age (r = -0.19, P < 0.001). The CETP concentrations were significantly higher in 117 myocardial infarction (MI) survivors and 110 stroke patients than that in 335 healthy, age-matched males (1.98 +/- 1.68 173 +/- 1.45, and 1.40 +/- 1.37 mg/l respectively, P < 0.01), while no relation was found between CETP concentration and lipids concentration in MI, stroke and healthy group.

Mutagenesis of the rapamycin producer Streptomyces hygroscopicus FC904.

Rapamycin (RPM) is produced by Streptomyces hygroscopicus FC904 isolated from soil in Fuzhou, China. It is a triene macrolide antibiotic with potential application as an immunosuppressant and drug for human gene therapy. In an attempt to improve rapamycin production, mutation and screening of the parent culture have been carried out. Thousands of survivors were obtained after mutagenesis by NTG (3 mg/ml) and UV (30 W, 15 cm, 30 seconds) of spore suspensions. None showed improved production of RPM. We determined the susceptibility to antibiotics of S. hygroscopicus FC904 by two fold dilutions of antibiotics in oatmeal agar plates. It was found that the strain was resistant to penicillin, erythromycin, RPM, tetracycline and chloramphenicol, but susceptible to mitomycin C (MIC, 10 microg/ml) and aminoglycosides such as gentamicin (MIC, 0.1 microg/ml), kanamycin (MIC, 0.1 microg/ml) and streptomycin (MIC, 0.3 microg/ml). Protoplasts of strain FC904 were prepared after finding the best conditions for their formation. They were treated with gentamicin, erythromycin, mitomycin C and NTG. Surprisingly, gentamicin was especially effective for obtaining higher RPM-producing mutants. Mutant C14 was selected by exposing the protoplasts of the parent strain FC904 to 1 microg/ml of gentamicin at 28 degrees C for 2 hours. A higher RPM-producing mutant (C14-1) was obtained from the protoplasts of mutant C14 treated with gentamicin, and its titer was 60% higher than that of the parent strain FC904 by HPLC analysis. Another improved mutant (C14-2) was obtained from the spores of mutant C 14 treated with 1 microg/ml of gentamicin plus 2 mg/ml of NTG at 28 degrees C for 2 hours. Mutant C14-2 had a titer 124% higher than FC904. The possible mechanism for the effect of gentamicin by using protoplasts or spore suspensions will be discussed, i.e. the possibility of gentamicin being a mutagen or a selective agent.

The role of perisynaptic Schwann cells in development of neuromuscular junctions in the frog (Xenopus laevis).

Fluorescence microscopy was used to study the behavior of perisynaptic Schwann cells (PSCs) in relation to motor nerve terminals and postsynaptic clusters of acetylcholine receptors, during the development of the neuromuscular junction (NMJ) in the frog Xenopus laevis. Pectoral (supracoracoideus) muscles were labeled with monoclonal antibody 2A12 for Schwann cells, the dye FM4-64 for nerve terminals (NTs), alpha-bungarotoxin for acetylcholine receptors (AChRs), and Hoechst 33258 for cellular nuclei, in animals from tadpole stage 57 to fully grown adults. When muscle fibers first appeared in stage 57, NMJs consisted of tightly apposed NTs and AChRs and were only partially covered with PSCs or their processes. Within a few stages, PSCs fully occupied and overgrew the NMJs, extending fine sprouts between a few micrometers and hundreds of micrometers beyond the borders of the junction. Sprouts of PSCs were most abundant during the time when secondary myogenesis, synaptogenesis, and synaptic growth occurred at their highest rates. PSCs were recruited to NMJs during synaptic growth, at rates between 1.3 PSCs/100 microm junctional length early on and 0.4 PSCs/100 microm later. Shortly after metamorphosis, PSC sprouts disappeared and NMJs acquired the adult appearance, in which PSCs, NTs, and AChRs were mostly congruent. The results suggest that, although PSCs may not be required for initial nerve-muscle contacts, PSCs sprouts lead synaptic growth and play a role in the extension and maturation of developing NMJs.

Reciprocal interactions between perisynaptic Schwann cells and regenerating nerve terminals at the frog neuromuscular junction.

The perisynaptic Schwann cell (PSC) has gained recent attention with respect to its roles in synaptic function, remodeling, and regeneration at the vertebrate neuromuscular junction (NMJ). Here we test the hypothesis that, following nerve injury, processes extended by PSCs guide regenerating nerve terminals (NTs) in vivo, and that the extension of sprouts by PSCs is triggered by the arrival of regenerating NTs. Frog NMJs were double-stained with a fluorescent dye, FM4-64, for NTs, and fluorescein isothiocyanate (FITC)-tagged peanut agglutinin (PNA) for PSCs. Identified NMJs were imaged in vivo repeatedly for several months after nerve injury. PSCs sprouted profusely beginning 3-4 weeks after nerve transection and, as reinnervation progressed, regenerating NTs closely followed the preceding PSC sprouts, which could extend tens to hundreds of microns beyond the original synaptic site. The pattern of reinnervation was dictated by PSC sprouts, which could form novel routes joining neighboring junctions or develop into new myelinated axonal pathways. In contrast to mammals, profuse PSC sprouting in frog muscles was not seen in response to axotomy alone, and did not occur at chronically denervated NMJs. Instead, sprouting coincided with the arrival of regenerating NTs. Immunofluorescent staining revealed that in muscle undergoing reinnervation 4 weeks after axotomy, 91% of NMJs bore PSC sprouts, compared to only 6% of NMJs in muscle that was chronically denervated for 4 weeks. These results suggest that reciprocal interactions between regenerating NTs and PSCs govern the process of reinnervation at frog NMJs: regenerating NTs induce PSCs to sprout, and PSC sprouts, in turn, lead and guide the elaboration of NTs.

Construction and analysis of cDNA library of Necator americanus third stage larvae.

OBJECTIVE: To obtain the genetic information on Necator americanus and to search for the purpose genes. METHODS: mRNA was isolated from the third stage larvae of Necator americanus maintained in hamsters. Double strand cDNA was synthesized and ligated to lambda ZAPII vector to construct the cDNA library. Expressed sequence tages (ESTs) were obtained by single pass sequencing of randomly isolated cDNA clones from the established library. RESULTS: A cDNA library of N. americanus was successfully constructed with high recombinant efficiency. The titer of unamplified library was 1 x 10(7). The insert size was about 750-3,000 bp. Of 11 ESTs obtained from the library, 7 have a significant homology with certain functional genes. CONCLUSION: A high quality and high representative cDNA library of N. americanus was constructed at the first time and some functional genes were identified from the library by ESTs.

Length of protection by murine vaccination with living infective third-stage hookworm larvae.

OBJECTIVE: To determine the length of protection by murine immunization with living third-stage hookworm larvae (L3) as measured by reduction in worm burden and host serologic antibody responses. METHODS: Outbred male (Kunming strain) mice were immunized subcutaneously with 500 L3 once every 2 weeks for a total of immunization for 3 times, and then challenged orally with 1000 L3 for 1 to 8 weeks after the final immunization. Host protective immunity was determined both by the reduction in worm burden as measured by the number of L3 recovered from murine lungs 48-hour post-challenge, as well as by measurement of circulating antibodies. Histopathological responses were also examined. Non-immunized mice served as negative controls. RESULTS: The protection by L3 immunization declined over time. One or 2 weeks after the final immunization, worm burdens were reduced 72% and 77.5% after challenge respectively. In contrast, only 37% reduction in worm burden was observed when the L3 challenge was delayed by 4 weeks and protection was almost entirely lost when there was an 8 week delay between the time of final immunization and challenge. The reduced level of protection over time partially correlated with diminishing L3-specific antibody responses. Host inflammation in the lungs of immunized mice also diminished. CONCLUSION: The protection afforded by living L3 immunization is maximal for the first two weeks after immunization, but then declines significantly over the ensuing weeks.

Enzyme-linked immunoelectrotransfer blotting analysis of human serologic responses to infective hookworm larval antigen.

OBJECTIVE: To explore the possibility of using specific antigens for immunodiagnosis of hookworm disease in endemic area. METHOD: Infective third-stage larvae of the canine hookworm, Ancylostoma caninum (A. caninum), were prepared as the source of antigen. Enzyme-linked immunoelectrotransfer blotting (EITB) was employed as an immunodiagnostic method. RESULTS: Two immunodominant bands of hookworm antigens (42 kDa and 55 kDa) were recognized by the sera of hookworm-infected patients (serum dilution 1:200; antigen centrifuged at 36,000 r/m for 20 minutes, but not by sera from negative controls. CONCLUSION: The 42 kDa and 55 kDa A. caninum antigens might be the specific antigens that could be used for immunodiagnosis of hookworm disease in endemic area.