[Analysis of epidemic characteristics of anthrax in China from 2017 to 2019 and molecular typing of Bacillus anthracis].

Objective: To analyze the epidemiological characteristics of anthrax in China from 2017 to 2019 and molecular typing of Bacillus anthracis isolated from some provinces (autonomous regions). Methods: Surveillance data of anthrax cases reported from 2017 to 2019 in the Infectious Disease Surveillance information System of China Disease Prevention and Control and the Public Health Emergency Reporting and Management Information System were collected, and descriptive epidemiological methods were used to analyze the epidemic characteristics, including the temporal, geographic and demographic distribution of this disease. A total of 47 strains of Bacillus anthracis isolated from 2017 to 2019 were analyzed by canSNP and MLVA15. Results: A total of 951 cases of anthrax were reported from 2017 to 2019, of which 938 were cutaneous anthrax, representing 98.63% of the total number reported. It was mainly distributed in the west and northeast of China, and the three provinces with the highest number of cases were Gansu (215), Sichuan (202) and Qinghai (191). Cases had been reported throughout the year, more cases occurred in the summer and autumn, and August was the month with the most cases,66.35% (211/318), 72.32% (243/336) and 68.01% (202/297) of cases were reported during June to September. The age distribution was mainly between 20 and 59 years old, accounting for more than 80% of all cases. The number of male cases was significantly higher than that of female cases, the ratio of male to female was about 3∶1. The occupations were mainly herdsmen and farmers, accounting for 49.70% to 58.18% and 31.45% to 36.70%, respectively. Public health events occurred every year, and 29 events had been reported from 2017 to 2019. canSNP analysis showed that 37 of the 47 strains belonged to the A.Br.001/002 subgroup and 10 belonged to the A.Br.Ames subgroup. MLVA15 analysis showed that there were 17 genotypes, of which 10 genotypes contained only one strain. Conclusion: Cutaneous anthrax was the predominant clinical type in China from 2017 to 2019.The seasonal, geographic and demographic distribution characteristics were evident.Molecular typing methods such as canSNP and MLVA15 can be used to trace the source of infectious diseases and provide technical support for anthrax prevention and control.

Direct Measurement of the Astrophysical

Fluorine is one of the most interesting elements in nuclear astrophysics, where the ^{19}F(p,α)^{16}O reaction is of crucial importance for Galactic ^{19}F abundances and CNO cycle loss in first generation Population III stars. As a day-one campaign at the Jinping Underground Nuclear Astrophysics experimental facility, we report direct measurements of the essential ^{19}F(p,αγ)^{16}O reaction channel. The γ-ray yields were measured over E_{c.m.}=72.4-344 keV, covering the Gamow window; our energy of 72.4 keV is unprecedentedly low, reported here for the first time. The experiment was performed under the extremely low cosmic-ray-induced background environment of the China JinPing Underground Laboratory, one of the deepest underground laboratories in the world. The present low-energy S factors deviate significantly from previous theoretical predictions, and the uncertainties are significantly reduced. The thermonuclear ^{19}F(p,αγ)^{16}O reaction rate has been determined directly at the relevant astrophysical energies.

Fetal growth at different gestational periods and risk of impaired childhood growth, low childhood weight and obesity: a prospective birth cohort study.

OBJECTIVE: To examine the longitudinal associations of fetal growth with adverse child growth outcomes and to assess whether maternal metabolic factors modify the associations. DESIGN: Prospective cohort study. SETTING: Born in Guangzhou Cohort Study, China. POPULATION: A total of 4818 mother-child pairs. METHODS: Fetal growth was assessed according to estimated fetal weight (EFW) from 22 weeks of gestation until birth and the measurement of the birthweight. Fetal growth Z-scores were computed from random effects in the multilevel linear spline models to represent fetal size in early pregnancy (22 weeks of gestation) and growth in mid-pregnancy (22-27 weeks of gestation), early third trimester (28-36 weeks of gestation) and late third trimester (≥37 weeks of gestation). MAIN OUTCOME MEASURES: Z-scores for childhood stunting, low weight, overweight or obesity, length/height for age (LAZ/HAZ), weight for age (WAZ) and body mass index for age (BMIZ) at the age of 3 years. Adjusted associations were examined using multiple Poisson or linear regression models. RESULTS: Increased Z-scores of fetal size in early pregnancy and growth in mid-pregnancy and early third trimester were associated with a higher risk of childhood overweight or obesity (risk ratios 1.25-1.45). Fetal growth in each period was negatively associated with stunting and low weight, with the strongest associations observed for fetal size in early pregnancy and growth in mid-pregnancy. The results for continuous outcomes (LAZ/HAZ, WAZ and BMIZ) were similar. The associations of fetal growth with overweight or obesity in childhood were stronger among mothers who were underweight and who were overweight or obese than among mothers of normal weight. CONCLUSIONS: Accelerated fetal growth before 37 weeks of gestation is associated with children who are overweight or obese, whereas the critical period for stunting and low weight occurs before 28 weeks of gestation. TWEETABLE ABSTRACT: Fetal growth during different periods is differentially associated with childhood stunting, underweight and overweight or obesity.

Aryl hydrocarbon receptor activation by diesel exhaust particles mediates epithelium-derived cytokines expression in severe allergic asthma.

BACKGROUND: Exposure to environmental pollutants promotes Th2 cell responses. Aryl hydrocarbon receptor (AhR) activation aggravates allergic responses. Epithelium-derived thymic stromal lymphopoietin (TSLP), interleukin (IL)-25, and IL-33 are implicated in the dysregulation of Th2 immune responses in severe allergic asthma. METHODS: Bronchial biopsies of 28 allergic severe asthma and 6 mild asthma subjects from highly polluted areas were analyzed for AhR nuclear translocation (NT), cytokine expression, and gene activation. Cultured primary epithelial cells were stimulated with diesel exhausted particles (DEP) to determine AhR-mediated IL-33, Il-25, and TSLP synthesis and release. RESULTS: Primary bronchial epithelial cells exposed to DEP showed upregulation of IL-33, IL-25, and TSLP. These effects were abolished by knockdown of AhR by siRNA. Increased AhR/ARNT binding to promoters of IL-33, IL-25, and TSLP was found using chromatin immunoprecipitation (ChIP) assay. Allergic severe asthma with high AhR NT had higher bronchial gene and protein expression of IL-33, IL-25, and TSLP. These patients derived clinical benefit from anti-IgE treatment. CONCLUSION: Aryl hydrocarbon receptor activation by DEP mediates upregulation of IL-33, IL-25, and TSLP with Th2 activation, potentially linking environmental pollution and allergic severe asthma.

[Clinical utility study of 21-gene assay in 927 Chinese patients with early breast cancer].

Objective: To investigate the distribution patterns of 21-gene assay and its influencing factors in Chinese patients with early breast cancer. Methods: Nine hundred and twenty-seven early breast cancer patients were retrospectively recruited from January 2009 to December 2015 at Ruijin Hospital, Shanghai Jiaotong University School of Medicine. The 21-gene reverse transcriptase-polymerase chain reaction(RT-PCR) assay were conducted in paraffin-embedded tumor tissues to calculate the Recurrence Score(RS). Immunohistochemistry(IHC) assay was used to measure the expression levels of estrogen receptor(ER), progesterone receptor(PR) and Ki-67. Concordances of RT-PCR and IHC results were assessed. Correlations of RS and classical clinicopathological factors were evaluated, and logistic regression were applied to determine independent predictive factors for RS. Results: The median RS of 927 patients was 23(range: 0~90), and the proportions of patients categorized as having a low, intermediate, or high risk were 26.5%, 47.7% and 25.8%, respectively. The distribution of RS varied significantly according to different tumor grade, T stage, PR status, Ki-67 index and molecular subtypes(P<0.05 for all). Grade, PR status and Ki-67 index were independent predictive factors for RS. ER, PR status and Ki-67 index showed significantly correlation between RT-PCR and IHC assays, and the concordance rates for ER and PR status were 98.7% and 87.8%, respectively. Conclusions: RS significantly correlated with tumor grade, T stage, PR status, Ki-67 index and subtypes. Grade, PR status and Ki-67 index can independently predict RS. Remarkable concordances of ER, PR status and Ki-67 index are found between RT-PCR and IHC assays.

[Analysis of the factors influencing adjuvant chemotherapy decisions for triple negative breast cancer].

Objective: To analyze adjuvant chemotherapy decisions for triple negative breast cancer (TNBC), and explore the influencing factors in the multidisciplinary treatment (MDT) modality. Methods: A retrospective analysis was performed. The cases with invasive TNBC who underwent surgery and MDT discussion for adjuvant treatment in Ruijin Hospital, from April 2013 to June 2015, were recruited. The patients' clinicopathological characteristics were analyzed and adjuvant treatment suggestions from MDT were obtained. Here the chemotherapy decision alteration was defined as a disagreement in chemotherapy or not, or inconsistence in regimens between the attending doctor and the multidisciplinary team. Results: A total of 194 patients aged ≤70 years old were enrolled in the multidisciplinary discussion, and 187 patients (96.4%) were suggested to receive chemotherapy. When compared the opinions of the attending doctor to suggestions of the multidisciplinary team, we found that the percentage of chemotherapy decision alteration reached 22.7% (39/172), of which 94.9% (37/39) were inconsistence in chemotherapy regimens. There were 119 patients who were recommended to receive epirubicin plus cyclophosphamide (EC) followed by docetaxel (T) or weekly paclitaxel (wP) regimens. Before the announcement of results for the E1199 trial, EC-T accounted for 62.5% (55/88), and EC-wP accounted for 37.5% (33/88) for this group of patients. After that, the proportion of EC-T was decreased to 22.6% (7/31) and proportion of EC-wP increased to 77.4%(24/31) (P<0.001). In addition, a total of 20 patients were suggested to receive platinum based chemotherapy. The proportions were 9.3% in cases with invasive ductal carcinoma, and 33.3% in cases with metaplastic carcinoma, respectively (P=0.016). Conclusions: The adjuvant chemotherapy decision for TNBC patients is altered in 22.7% of the patients after MDT discussion. After the announcement of SABCS E1199 results, more patients are suggested to receive EC followed by weekly paclitaxel. There is a lack of detailed evidence for platinum based adjuvant chemotherapy for TNBC, and more patients with metaplastic carcinoma receive platinum based adjuvant chemotherapy.

[Effect of 21-gene recurrence score on chemotherapy decisions for patients with estrogen receptor-positive, epidermal growth factor receptor 2-negative and lymph node-negative early stage-breast cancer].

Objective: To investigate the effect of 21-gene recurrence score on adjuvant chemotherapy decisions for patients with estrogen receptor (ER)-positive, epidermal growth factor receptor 2 (HER-2)-negative and lymph node (LN)-negative early stage-breast cancer. Methods: One hundred and forty-eight patients with ER+ , HER-2- and LN- early stage breast cancer were recruited in the Ruijin hospital, Shanghai Jiao Tong University School of Medicine. The 21-gene recurrence score (RS)assay was performed and systemic therapeutic decisions were made before and after knowing the RS results under multidisciplinary discussion. The effects of RS assay and the other influential factors on adjuvant chemotherapy decision were further analyzed. Results: After knowing the RS results, treatment decisions were changed in 26 out of 148 patients(17.6%). Among them, 9 out of 26 patients were not recommended for chemotherapy; 16 of 26 had treatment recommendation changed to chemotherapy, and chemotherapy regimen was changed in the last one patient. Multivariate analysis showed that RS, age and histological grade were independent factors of decision-making for adjuvant chemotherapy. Conclusion: Our results suggest that 21-gene recurrence score significantly influences decision making for adjuvant chemotherapy in patients with ER+ , HER-2- and LN- early stage breast cancer.

[Surgical treatment and prognosis of ductal carcinoma in situ: 526 cases analysis].

Objective: To evaluate the choice of surgical treatment of ductal carcinoma in situ (DCIS) and its impact on long-term outcomes. Methods: A retrospective analysis of the clinicopathological features and treatment protocol of DCIS patients who underwent surgical treatment in Comprehensive Breast Health Center, Ruijin Hospital, Shanghai Jiaotong University School of Medicine from January 2009 to August 2016 was done. The factors which could affect surgical treatment were analyzed by χ(2) test and Logistic regression. Survival analysis were performed between different surgical approaches. Kaplan-Meier survival curves and Log-rank tests demonstrated the distribution of disease free survival and overall survival. Results: A total of 526 patients were enrolled in this study, 405 cases (77.0%) underwent mastectomy, 121 cases (23.0%) underwent breast-conserving surgery, of which 88 cases received radiotherapy after breast-conserving surgery. It was shown by univariate and multivariate analysis that age>50 years (OR=0.631, 95% CI: 0.413 to 0.965, P=0.034), first symptom of nipple discharge (OR=0.316, 95% CI: 0.120 to 0.834, P=0.020), excision biopsy (OR=1.831, 95% CI: 1.182 to 2.835, P=0.007) and tumor size >3 cm (OR=0.422, 95% CI: 0.206 to 0.864, P=0.018) were significantly correlated with choice of surgical treatment for breast lesions. Axillary lymph node dissection was performed for 118 cases (22.4%), with sentinel lymph node biopsy for 327 cases (62.2%), and none for 81 cases (15.4%). There was significant statistical difference in the choice of axillary lymph node management in patients of different age (χ(2)=8.124, P=0.017), biopsy type (χ(2)=35.567, P=0.000), breast operation type (χ(2)=149.118, P=0.000) and tumor size (χ(2)=13.394, P=0.010). The 5-year disease free survival rates was 95.7%, 89.6% and 100%, respectively, for mastectomy group, breast-conserving surgery group and breast-conserving surgery plus radiotherapy group. And the 5-year overall survival rates for three groups were 99.0%, 100% and 100%. The differences were not statistically significant (P=0.427, 0.777). Conclusions: For DCIS patients, age, first symptom and tumor size are independent predictors of breast surgery. The choice of axillary lymph node surgery is influenced by age, biopsy, operation type, and tumor size. Different surgical treatment options has no significant effect on disease-free survival and overall survival in DCIS patients.

[Factors related to adjuvant ovarian function suppression in premenopausal breast cancer patients].

OBJECTIVE: To analyze the applied condition of ovary function suppression (OFS) before and after joint analysis of TEXT and SOFT trials and SOFT trial, and to identify the relevant factors of OFS usage. METHODS: The analysis was performed in premenopausal women with hormone receptor (HR) positive breast cancer receiving surgical treatment from Apr 2013 to Oct 2015 in Ruijin Hospital, Shanghai Jiaotong University School of Medicine. Adjuvant treatment strategy was made in the multidisciplinary team (MDT) meetings. We analyzed the applied condition of OFS before and after joint analysis, SOFT trial and its relevant factors. RESULTS: Among 454 patients, 114 (25.1%) patients received OFS. Before the results of joint analysis came out, all the patients (38/38) received OFS together with tamoxifen (TAM); after the results came out, clinicians began to put OFS with exemestane into practice, among 76 patients, 41(53.9%) patients received OFS with exemestane while 35 (46.1%) patients received OFS together with TAM. Before the results of SOFT trial came out, 71 out of 310 (22.9%) patients received OFS while 43 out of 144 (29.9%) patients received OFS after that. No significant difference was found between the proportion of patients receiving OFS before and after the results of SOFT trial came out (P=0.112). Age, histological grade, pN status, Ki-67 status, molecular subtype and acceptance of chemotherapy were correlated with OFS treatment (P<0.05). Age, tumor grade and pN were independent significant predictors of OFS usage. CONCLUSIONS: After the results of joint analysis came out, clinicians began to apply OFS with exemestane to premenopausal women with HR positive breast cancer. There is no significant difference between the proportion of patients receiving OFS before and after SOFT trial. Age, tumor grade and pN status are independent significant predictors of OFS treatment. Patients younger than 40, with histological grade Ⅱ or Ⅲ tumor and with pN1 or pN2 status are prone to receive OFS.

[Homology analysis and historical tracing for inter-continental Burkholderia pseudomallei strains of sequence type 562].

Objective: To understand the homology of sequence type 562 (ST562) strains of Burkholderia pseudomallei which circulated in two separate continents (Asia and Australia) at different times. Methods:SpeⅠrestriction fragments and 4-locus multiple locus variable number tandem repeat analysis (MLVA-4) profiles were extracted from MSHR5858 (ST562 Australia strain) and 350105 (ST562 historical strain of Hainan) genomes respectively by in silico analysis and then compared with the PFGE and MLVA-4 results of five ST562 clinical isolates from Hainan to test their homology. Synteny and homology between MSHR5858 and 350105 genomes were evaluated with bioinformatics methods. Results: Five ST562 clinical strains from Hainan shared same PFGE pattern (similarity>97%) and this pattern coincided to the map of SpeⅠrestriction fragments of Australian strain MSHR5858. The amounts of genomic restriction fragments (SpeⅠ) for MSHR5858 and 350105 were 31 and 34 respectively, with 31 of them matched by each other. Five ST562 clinical strains of Hainan were distinct by MLVA-4 profiles, among which HPPH43 (MLVA-4 profile: 10, 8, 10, 8) was close to Australia strain MSHR5858 (10, 8, 8, 6), containing identical repeat numbers at VNTR loci 2341k and 1788k; while HK003 (11, 8, 15, 7) and HK061 (11, 8, 17, 7) similar to Hainan historical strain 350105 (11, 8, 11, 8), with same repeat numbers at loci 2341k and 1788k also. High-degree synteny and consistency on genomic contents were observed between 350105 and MSHR5858, indicating a similar origin for the 2 strains. Conclusion: All inter-continental and historical ST562 strains of B. pseudomallei had similar genomic characteristics, supporting the assumption that they had a common origin. Also, it is possible that Hainan historical strain 350105 is the ancestor of all circulating ST562 strains.

Partially reduced graphene oxide based FRET on fiber-optic interferometer for biochemical detection.

Fluorescent resonance energy transfer (FRET) with naturally exceptional selectivity is a powerful technique and widely used in chemical and biomedical analysis. However, it is still challenging for conventional FRET to perform as a high sensitivity compact sensor. Here we propose a novel 'FRET on Fiber' concept, in which a partially reduced graphene oxide (prGO) film is deposited on a fiber-optic modal interferometer, acting as both the fluorescent quencher for the FRET and the sensitive cladding for optical phase measurement due to refractive index changes in biochemical detection. The target analytes induced fluorescence recovery with good selectivity and optical phase shift with high sensitivity are measured simultaneously. The functionalized prGO film coated on the fiber-optic interferometer shows high sensitivities for the detections of metal ion, dopamine and single-stranded DNA (ssDNA), with detection limits of 1.2 nM, 1.3 µM and 1 pM, respectively. Such a prGO based 'FRET on fiber' configuration, bridging the FRET and the fiber-optic sensing technology, may serve as a platform for the realization of series of integrated 'FRET on Fiber' sensors for on-line environmental, chemical, and biomedical detection, with excellent compactness, high sensitivity, good selectivity and fast response.

Gamma-aminobutyric acid-A and -B receptor antagonists increase luteinizing hormone-releasing hormone neuronal responsiveness to intracerebroventricular norepinephrine in ovariectomized estrogen-treated rats.

These studies determined whether endogenous gamma-aminobutyric acid (GABA) secretion affects LHRH neuronal responsiveness to norepinephrine (NE). The intracerebroventricular (icv) infusion of either bicuculline or phaclofen (GABA-A or GABA-B receptor antagonists, respectively) into ovariectomized (OVX) estrogen-treated rats did not affect basal LH levels (95 +/- 8.5 ng/ml) obtained over the 120 min of this study. When NE was infused icv, it produced a modest rise in plasma LH, which peaked within 15 min (240 +/- 25 ng/ml) and then declined toward baseline over the next 90 min. In contrast, if bicuculline was given icv at about time zero, and NE was infused (icv) 15 min later, plasma LH secretion was markedly increased and reached a peak concentration of 723 +/- 98 ng/ml within 15 min after NE treatment. Similarly, when bicuculline was infused into the medial preoptic area (MPOA), and NE was given 15 min later (icv), a peak LH level of 726 +/- 105 ng/ml was obtained within 15 min. If phaclofen was given icv at about time zero, and NE was infused 15 min later, LH rose dramatically to reach a peak concentration of 844 +/- 126 ng/ml within 15 min; a similar amplified LH response occurred when the GABA-B antagonist was infused into the MPOA and icv NE was given 15 min later. Comparisons of the LH responses obtained over the 120 min of the study suggest that icv infusions of the GABA-B receptor antagonist were more effective in sustaining peak LH secretion than the GABA-A receptor antagonist. In other groups of rats, the MPOA was electrochemically stimulated (ECS), and the effects of icv NE alone or combined with GABA receptor antagonists were evaluated. MPOA ECS alone induced a significant rise in plasma LH, which peaked between 35-45 min and then declined to approach basal levels by 150 min. In a second group, the MPOA was ECS, and at 30 min NE was infused icv. Plasma LH levels in these rats remained significantly elevated for the next 30 min before beginning their decline. In other animals, the MPOA was stimulated, and 15 min later either bicuculline or phaclofen was infused icv. Neither drug affected the patterns or concentrations of LH obtained after MPOA ECS alone. However, when rats received MPOA ECS plus either icv bicuculline or phaclofen, and these treatments were followed 15 min later by icv infusions of NE, LH secretion patterns and levels were altered significantly.(ABSTRACT TRUNCATED AT 400 WORDS)

Morphine amplifies norepinephrine (NE)-induced LH release but blocks NE-stimulated increases in LHRH mRNA levels: comparison of responses obtained in ovariectomized, estrogen-treated normal and androgen-sterilized rats.

In these studies we examined the temporal effects of intracerebroventricular (i.c.v.) infusions of norepinephrine (NE) on plasma LH and on LHRH mRNA levels in the organum vasculosum of the lamina terminalis (OVLT) and in neurons located in the rostral (r), middle (m) and caudal (c) preoptic areas (POA) of ovariectomized, estrogen-treated rats. Thereafter, we compared these responses to those which occur in androgen-sterilized rats (ASR). NE infusions not only increased plasma LH concentrations but within 1 h after NE, LHRH mRNA levels also were increased significantly in the OVLT and rPOA but not in the mPOA or cPOA. By 4 h, these message levels still were elevated in the OVLT and rPOA and they now also were significantly higher than control values in the mPOA and cPOA. While NE also increased LH secretion in ASR, the plasma LH concentrations obtained were markedly blunted compared to control values. Moreover, NE infusions did not alter single cell levels of LHRH mRNA in any region of the rostral hypothalamus. Previously, we have reported that morphine (s.c.) markedly amplifies NE-induced LH release and questioned whether these responses are accompanied by concomitant augmented increases in LHRH mRNA levels. Morphine alone did not affect basal LHRH mRNA or plasma LH levels. However, when rats were pretreated with morphine (-15 min) and NE was infused i.c.v. at 0 time, significant amplification of LH release occurred but, unexpectedly, morphine completely blocked NE-induced increases in LHRH mRNA levels in all of the neurons we examined. Morphine also amplified LH release in ASR but these responses were significantly less than those obtained in control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of morphine on hypothalamic tyrosine hydroxylase mRNA levels in dopaminergic neurons and on preoptic DOPAC levels measured by microdialysis.

Morphine not only suppresses norepinephrine-induced increases in LHRH mRNA levels but, in these same animals, it simultaneously amplifies norepinephrine (NE)-induced LH release. These observations suggest that NE may activate parallel mechanisms which independently increase LHRH mRNA levels and LHRH release and suggest that some of these effects could be mediated indirectly via morphine's action on different components of the hypothalamic dopamine (DA) system. Accordingly, in the present studies we examined the effects of morphine on various components of this dopamine system using as our index of altered DA neuronal activity, the changes which occur in tyrosine hydroxylase (TH) mRNA levels following morphine. As an ancillary index of changes which occur in dopamine neuronal activity, we measured, by microdialysis, the changes which occur in preoptic dihydroxyphenylacetic acid (DOPAC) levels after either subcutaneous injections or following microinfusions of morphine into the zona incerta (ZI). In a final study, we evaluated whether DA when given alone (icv infusion) or prior to icv NE would altered LH release. Single cell levels of TH mRNA in preoptic A15 and periventricular anterior hypothalamic A14 DA neurons were not affected by morphine 1, 5 and 24 h later. In contrast, within 1 h after morphine, TH mRNA levels in ZI A13 neurons were significantly elevated and they remained high at 5 nd 24 h compared to controls. Morphine also resulted in a significant rise in TH mRNA levels in tuberoinfundibular DA neurons (TIDA) (A12) within 1 h and these levels remained high to 5 h. Thereafter, by 24 h, message levels had returned to control values. Morphine also resulted in a rapid rise in plasma prolactin (Prl) with peak values occurring at 20 min and then returning to baseline by 90 min. When morphine was given sc it resulted, within 15 min, in a rapid rise in preoptic DOPAC levels and these levels continued to rise such that they were 217% higher than pretreatment values by 105 min. Preoptic 5-hydroxyindoleacetic acid (5-HIAA) levels also increased by 25-75% after sc morphine. The microinfusion of morphine into ZI also resulted in elevated preoptic DOPAC but not 5-HIAA levels within 15 min. The icv infusion of DA alone had no effect on plasma LH whereas, NE (icv) produced a modest but significant increase in plasma LH. When DA was given 15 min prior to the infusion of NE, neither amplification nor inhibition of NE-induced LH release occurred. From these and other studies we conclude that the morphine-induced suppression of TIDA neuronal activity may allow NE to release greater amounts of LHRH from axon terminals in the median eminence.(ABSTRACT TRUNCATED AT 400 WORDS)

Estrogen inhibits hypothalamic pro-opiomelanocortin gene expression in hypothalamic neuronal cultures.

Many in vitro studies show estrogen regulation of the hypothalamic pro-opiomelanocortin (POMC) system, including a decrease in hypothalamic POMC mRNA after estradiol treatment. Because such in vivo experiments do not allow one to determine whether peripheral, interacting systems or extra-hypothalamic brain regions are involved in this regulation, we sought to establish whether estrogen acts directly in hypothalamus to decrease POMC mRNA. Using an in vitro approach, we studied effects of estradiol (E2) on POMC/cyclophilin mRNA concentrations (RNAse protection assays) in neuronal cultures derived from day 17 fetal rat hypothalamus. Chemically defined medium was deprived of progesterone for 2 days prior to E2 treatment and for the duration of the study. E2 (10(-13)-10(-9) M) dose-dependently decreased POMC mRNA concentrations during a 2-day treatment. Whereas the lowest dose (10(-13) M) of E2 resulted in a statistically significant 44% decrease in POMC mRNA concentrations relative to control cultures, this inhibitory effect was lost because higher doses (10(-11) and 10(-9) M) did not produce statistically significant decrements (22 and 16%, respectively) in POMC mRNA concentrations. Additional time course studies revealed that this decrease in POMC mRNA can be seen as early as 4 h after E2 (10(-13) M) treatment. We conclude that E2 inhibition of POMC mRNA concentrations in hypothalamic neuronal cultures indicates that this inhibition can occur directly in hypothalamus.

Temporal changes in tyrosine hydroxylase mRNA levels in A1, A2 and locus ceruleus neurons following electrical stimulation of A1 noradrenergic neurons.

We examined the effects of electrical stimulation (ES) of right A1 noradrenergic cells on temporal changes in tyrosine hydroxylase (TH) mRNA levels in A1, A2 and locus ceruleus (LC) neurons by in situ hybridization histochemistry and quantitative image analysis methods. The stimulation parameters used previously have been shown to increase hypothalamic norepinephrine (NE) release. Within 1 h after beginning A1 stimulation, TH mRNA levels were significantly increased and they continued to rise to reach plateau by 6 h. TH message levels at 12 h were not difference from 6 h values. A1-ES did not affect TH mRNA levels in contralateral A1 or in A2 or locus ceruleus neurons. These data suggest that changes in TH mRNA levels may serve as an index of increased A1 neuronal activity in circumstances when increases in hypothalamic NE secretion occur.

Effects of reserpine on tyrosine hydroxylase mRNA levels in locus coeruleus and medullary A1 and A2 neurons analyzed by in situ hybridization histochemistry and quantitative image analysis methods.

These studies examined the effects of reserpine on concentrations of norepinephrine (NE), dopamine (DA) and epinephrine (EPI) and on levels of tyrosine hydroxylase (TH) mRNA in locus coeruleus (LC) and medullary A1 and A2 neurons. Noradrenergic neurons in these regions first were identified by immunocytochemistry and, thereafter, by in situ hybridization histochemistry. Levels of TH mRNA were measured by quantitative image analysis methods. Changes in catecholamine concentrations in micropunches of these brain regions were analyzed by HPLC. Epinephrine was not detected in any of the nuclei examined. Twenty-four hours after reserpine treatment, NE concentrations declined in A1, A2 and LC neurons by 46, 69 and 34% respectively while DA declined only in the region of A2 neurons. This reserpine-induced depletion of NE was accompanied by a 2- to 3-fold increase in TH mRNA levels in LC and A1 neurons but no change in message levels occurred in A2 cells 24 h after reserpine. Forty eight hours later, message levels in A1 and LC neurons did not differ significantly from the elevated 24 h values but TH mRNA levels in A2 neurons now were significantly elevated compared to 24 h values. TH mRNA levels 72 h after reserpine did not differ from 48 h values in A1, A2 and LC neurons. Thus, TH gene expression in A1 neurons increases after reserpine treatment in a manner equivalent to that observed in LC, adrenal medulla and superior cervical ganglia. The reason why it required 48 h for TH mRNA to increase in A2 neurons remains unclear.

Changes in tyrosine hydroxylase mRNA levels in medullary A1 and A2 neurons and locus coeruleus following castration and estrogen replacement in rats.

Temporal changes in tyrosine hydroxylase (TH) mRNA levels in medullary A1 and A2 neurons and locus coeruleus (LC) cells were studied 6, 12 and 24 h following orchidectomy in rats. Brains from intact controls and sham castrated rats also were evaluated at these same time periods. In situ hybridization histochemistry and quantitative image analysis techniques were used to quantitate levels of cytoplasmic TH mRNA. Neither the time of day nor the stress of sham castration affected TH mRNA levels in A1, A2 and LC neurons. In contrast, 6 h following castration, TH mRNA levels in A1 neurons had declined significantly. Thereafter, there was a linear increase in A1 message levels such that, by 24 h, TH mRNA values were significantly higher than those obtained in intact controls. Placement of Silastic estrogen capsules immediately after castration prevented the 6 h decline in A1 message levels. At 12 h, TH mRNA levels in A1 neurons were significantly higher in estrogen-treated rats compared to those of the castrate or intact control groups. By 24 h, message levels in A1 neurons of steroid-treated rats were comparable to the intact control. Neither castration nor estrogen treatment altered TH mRNA levels in A2 neurons. TH mRNA levels in LC neurons increased significantly 6 h after castration and estrogen produced a further significant increase in message levels. Six hours later (12 h), TH mRNA values were still higher than controls but, in the estrogen-treated group, these levels had declined to those observed in the 12 h castrate group.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphine but not Naloxone Enhances Luteinizing Hormone-Releasing Hormone Neuronal Responsiveness to Norepinephrine.

Some axon terminals of hypothalamic opiate neurons directly synapse on luteinizing hormone-releasing hormone (LHRH) neurons. To determine whether such synaptic connections affect LHRH neuronal activity, we have examined the profiles and concentrations of LH released in response to intracerebroventricular (icv) norepinephrine (NE, 45 µg) infusions alone or following medial preoptic area (MPOA) electrochemical stimulation (ECS) in estrogen-treated ovariectomized rats. Similar studies were performed in rats treated with naloxone (5 mg/kg ip) or morphine (20 mg/kg sc) given 15 min prior to MPOA-ECS or 30 min prior to icv NE. Naloxone neither augmented nor suppressed the LH response obtained with NE alone. MPOA-ECS evoked a significant increase in plasma LH. When the preoptic area was stimulated (0 min) and NE was infused at 30 min, a significant amplification of LH release occurred. Prior treatment of rats (-15 min) with naloxone had no effect on LH responses elicited by either preoptic stimulation alone or combined with icv NE. In the second study, morphine was given sc and had no effect on basal plasma LH levels. However, when morphine was given (-15 min) and icv NE infusions were made (30 min), the rise in plasma LH induced by NE was significantly enhanced. Preoptic ECS (0 min) evoked a rise in plasma LH and this response was also enhanced by morphine pretreatment. The major effect on LH release occurred when sc morphine injections (-15 min) were combined with MPOA-ECS (0 min) followed by icv NE (30 min). In these rats, a remarkable and highly significant release of LH occurred which reached peak levels even greater than those observed during spontaneous LH surges (2,392 versus 16 to 1,800 ng/ml). Since morphine has profound effects on the serotonergic system, in the third series of studies, morphine was infused into the dorsal raphe nucleus (DRN) and LH responses to MPOA-ECS or icv NE alone or following combined ECS + NE were examined. DRN morphine did not affect basal LH release but it produced a rapid and highly significant rise in plasma prolactin. When DRN morphine was given (-15 min) and NE was infused icv (30 min), there was marked amplification in LH release compared to those values observed after only NE. However, there were no appreciable differences in LH values obtained after sc versus DRN morphine injections in response to NE. Similarly, the amplification of LH release which occurred following DRN morphine (-15 min) + MPOA-ECS (0 min) was not different from that obtained after sc morphine. In the final group of rats, DRN morphine was given (-15 min), the preoptic area was stimulated (0 min) and NE was infused (30 min). Following this treatment, plasma LH release was also markedly enhanced and did not differ appreciably (except at 60 and 120 min) from the levels of LH released after sc morphine. Prolactin concentrations rose slowly after icv NE to reach peak levels 75 min after treatment. Combinations of morphine + MPOA-ECS without or with NE neither augmented nor suppressed the high prolactin concentrations achieved after only DRN morphine infusions. We conclude from these data that: 1) those opiate neuronal terminals which synapse directly on LHRH neurons do not affect LHRH neuronal responsiveness to either NE, to MPOA-ECS or to combined preoptic stimulation+ NE, and 2) morphine has profound effects on LHRH neuronal responsiveness to both NE, to MPOA-ECS and, in particular, to combined ECS + NE. Since amplification of LH release occurs after treatment of rats with morphine either by sc injections or DRN infusions, the augmented LH and prolactin responses observed are most likely due to the morphine-induced release of serotonin and not to direct morphine effects on LHRH neurons.

Tyrosine Hydroxylase Messenger Ribonucleic Acid Levels Increase in A1 but not Locus Ceruleus Noradrenergic Neurons in Proestrous Rats but not in Diestrous or Androgen-Sterilized Animals.

Norepinephrine (NE) turnovers (an index of secretion) increase in the hypothalamus of proestrous rats concomitant with luteinizing hormone surges, whereas, neither of these events are observed in diestrous nor in androgen-sterilized rats. Increased hypothalamic NE release may occur as a consequence of the withdrawal of local inhibitory γ-aminobutyric acid and opiate controls on specific presynaptic NE terminals and/or as a result of an increase in activity within noradrenergic neurons. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the synthesis of NE and our earlier studies revealed that increases in TH mRNA in A1 and locus ceruleus (LC) neurons can serve as an index of increased activity within these cells. In the present study, we evaluated whether TH message levels change in A1 and LC neurons prior to and during the hours when luteinizing hormone surges and increased NE turnovers are observed. As controls, TH mRNA levels in A1 and LC neurons were evaluated at the same hours of day in diestrous day 2 and in androgen-sterilized rats. In situ hybridization histochemistry and quantitative image analysis methods were used to measure changes in TH mRNA levels. Luteinizing hormone surges in proestrous rats began at 1500 h, peaked between 1600 and 1700 h and declined, thereafter, to 2000 h. In contrast, plasma luteinizing hormone remained basal throughout the day in diestrous and androgen-sterilized rats. While A1 neuronal TH mRNA levels did not differ in the three groups of rats during the morning (0930 to 1030 h), these message levels were significantly elevated in proestrous rats during the afternoon (1645 to 1715 h) and remained high at 2000 to 2030 h. In contrast, no changes in TH mRNA levels were observed in A1 neurons throughout the afternoon in diestrous animals or androgen-sterilized rats. TH mRNA levels in the LC did not differ in the three groups of rats and they remained unchanged throughout the afternoon hours we examined. From these observations we conclude that concomitant with afternoon proestrous luteinizing hormone surges and the accompanying increase in hypothalamic NE secretion, there is an increase in activity within A1 but not LC neurons. These data suggest that the proestrous increase in hypothalamic NE turnover we previously observed is not due solely to withdrawal of local inhibitory controls of presynaptic NE release but it also involves an increase in activity within A1 but not LC neurons.