[Clinical characteristics of crystal deposits in joints and tendons in patients with gout].

OBJECTIVE: To explore the abnormal manifestations and clinical features of patients with gout according to the location of crystal deposits: in articulars or in tendons. METHODS: A total of 105 patients with gout who were continuously treated in the Department of Rheumatology and Immunology of Peking University People's Hospital from June 2019 to December 2019 were selected and their knees, ankles, toes and painful joints and tendons were examined by high-frequency ultrasound. Then we grouped them according to the presence or absence of sodium urate crystals and the location of the crystals, collected their clinical data, and analyzed the clinical characteristics. RESULTS: Among the 105 patients, 25 patients had no crystal deposits in the joints or tendons (as the non-crystal group), 43 patients had intra-articular crystals (as the joint group), and 37 patients had intra-tendon crystals with or without intra-articular crystals (as the tendon group). Among them, the most involved part of sodium urate crystals deposited in the joints was the metatarsophalangeal joint (29 cases, 67.4%), followed by knee joints (10 cases, 23.2%), ankle joints (9 cases, 20.9%). The most involved part of sodium urate crystals deposited in the tendon was the quadriceps tendon (16 cases, 43.2%), followed by the Achilles tendon (13 cases, 35.1%), the patellar tendon (12 cases, 32.4%), and the three heads of brachii tendons (5 cases, 13.5%). The three groups were compared using multi-sample analysis of variance/multi-sample rank sum test. Age, age of first increase in uric acid (UA), serum glucose (Glu) level and C reactive protein (CRP) were all significantly different. After multiple comparisons, compared with the non-crystal group, age, the age of first increase in uric acid, and CRP were significantly higher in the tendon group. There was no significant difference between the non-crystal group and the joint group. There was no significant difference between the tendon group and the joint group. CONCLUSION: In patients with gout, it is common for ultrasound to find crystals deposited in joints or tendons. The most commonly affected parts include the metatarsophalangeal joint, knee joint, ankle joint, quadriceps tendon, Achilles tendon, patellar tendon, and triceps tendon. There were significant differences among the three groups in age, age of first increase in uric acid, CRP and blood glucose, and the proportion of urinary calculi in patients with crystal deposits was significantly higher than those without crystal deposits.

[Clinical characteristics and risk factors of cardiovascular disease in patients with elderly-onset rheumatoid arthritis: A large cross-sectional clinical study].

OBJECTIVE: To investigate the clinical characteristics of patients with elderly-onset rheumatoid arthritis (EORA), and the risk factors of EORA complicated with cardiovascular disease (CVD). METHODS: A cross-sectional study was conducted in Peking University People's Hospital from July 2009 to December 2014 and 1 116 patients were recruited. The patients' characteristics and CVD, including ischemic heart disease, cerebral and peripheral vascular disease, were recorded. The patients were divided into EORA group (n=212) and younger-onset rheumatoid arthritis (YORA) group (n=904) according to the age of onset ≥60 years and < 60 years. Then, the differences between the groups were analyzed by Student's t test, Mann-Whitney U test or χ2test, and risk influencing CVD were analyzed using Logistic regression. RESULTS: There was no significant difference in the disease activity between the EORA and YORA groups. The proportion of male, pulmonary interstitial disease (ILD), and numbers of deformity joint count (DJC) were significantly higher in the EORA group compared with the YORA group [32.1% vs. 18.5%, χ2=19.11, P < 0.001; 23.6% vs. 13.6%, χ2=16.50, P < 0.001; 6 (2, 12) vs. 3 (2, 7), Z=-3.60, P < 0.001], while the prevalence of Sjögren's syndrome was lower than that of the YORA group (13.5% vs. 5.2%, χ2=11.29, P=0.001). Moreover, there were lower prevalences in the patients treated with disease-modifying antirheumatic drugs (DMARDs) in EORA group (35.4%) than in YORA group (26.7%) (χ2=6.43, P=0.011), especially in methotrexate (MTX), hydroxychloroquine (HCQ) and sulfasalazine (SSZ). In addition, the patients with EORA had a higher prevalence of CVD (27.8%) than the YORA group (11.6%, χ2=40.46, P < 0.001), accompanied with higher prevalence of smoking, hypertension, and hyperlipidemia. Multivariate Logistic regression analysis showed that elder age (OR=1.10, 95%CI: 1.00-1.20), DJC (OR=3.17, 95%CI: 1.04-9.68), rheumatoid nodules (OR=3.56, 95%CI: 1.03-12.23), hypertension (OR=2.37, 95%CI: 1.09-5.13) and hyperlipidemia (OR=8.85, 95%CI: 2.50-31.27) were independent risk factors, while HCQ (OR=0.22, 95%CI: 0.07-0.70) and MTX (OR=0.32, 95%CI: 0.14-0.73) were protective factors of EORA complicated with CVD. CONCLUSION: Compared with YORA, patients with EORA have higher ratio of male, ILD and DJC, which may be attributed to inappropriate therapies. EORA is more likely to be complicated with CVD than YORA. Elder age, DJC, rheumatoid nodules, hypertension, and hyperlipidemia are independent risk factors, while HCQ and MTX are protective factors of EORA complicated with CVD.

[Clinical characteristics and treatment outcomes of macrophage activation syndrome in adults: A case series of 67 patients].

OBJECTIVE: To described the clinical and laboratory features and outcome of 67 macrophage activation syndrome (MAS). METHODS: A total of 67 MAS patients from three centers from January 2007 to December 2017 were enrolled. Clinical and laboratory features, and response to therapy were analyzed. Predictive factors for remission and survival were explored. RESULTS: We identified a mean age of (36.1±16.3) years at diagnosis of MAS and a median connective tissue disease (CTD) duration of 8 months prior to MAS development. Among 67 MAS patients identified, underlying diseases included adult-onset Still's disease (AOSD) in 56.7% and systemic lupus erythematosus (SLE) in 30.0%. Fever and splenomegaly were found in 100.0% and 82.1% of the patients, respectively. Ferritinemia and elevation of serum soluble interleukin-2 receptor was seen in 100.0% and 93.2% of the patients. Serum levels of alanine aminotransferase, D-dimer, ferritin and C reactive protein were significantly higher in MAS associated with the AOSD patients than in MAS associated with the SLE patients. A significant decrease of erythrocyte sedimentation rate was found in MAS associated with AOSD, as compared with MAS associated with SLE. The most commonly used therapy was corticosteroids, which were initially administered in 100.0% of the patients. Intravenous immunoglobulin (IVIG) was administered in 91.0%, cyclosporine A in 64.2%, and etoposide in 46.3% of the patients, respectively. The induction therapy yielded a complete remission (CR) at the end of week 8 in 47.8% of the MAS patients. The overall mortality rate at the end of week 16 was 22.4%. The median serum levels of gamma-glutamyltransferase, alkaline phosphatase, total bilirubin and direct bilirubin were significantly lower in the patients who achieved complete remission at the end of week 8 than in those who did not, and splenomegaly was significantly less frequent (71.9% vs.91.4%, P=0.037). Both the mean age at diagnosis of MAS and the mean age at diagnosis of underlying CTD of the deceased patients were elder than those of the survived population (P=0.014 and P=0.017, respectively). The platelet count was significantly less in the deceased population as compared with the living population (P=0.018). No addition of cyclosporine A (P=0.004) was identified as risk factors associated with death in Logistic regression analysis. CONCLUSION: MAS secondary to connective tissue disease is most common with AOSD and SLE. In terms of laboratory findings, there were considerable differences between the patients with underlying SLE and those with AOSD. Advanced age and low platelet counts are significant predictive factors for death, while treatment with cyclosporine may reduce the risk.

[Study of bone mineral density and serum bone turnover markers in newly diagnosed systemic lupus erythematosus patients].

OBJECTIVE: To investigate the changes of bone mineral density (BMD) and serum bone turnover factor in newly diagnosed systemic lupus erythematous (SLE) patients. METHODS: Eighty newly diagnosed SLE patients and 80 age and gender matched healthy controls were enrolled. None of the SLE patients had ever received glucocorticoid, immunosuppressive agents or vitamin D. BMD was measured at radius,lumbar spine and hip by dual X ray absorptiometry (DXA). Bone turnover markers including serum levels of tartrate-resistant acid phosphatase 5b (TRAP5b),bone alkaline phosphatase (BAP) and 25-hydroxy vitamin D3 (25-OH-VD3) were measured by enzyme-linked immunosorbent assay (ELISA). Logistic regression was employed to analyze the risk factors associated with decreased BMD. RESULTS: Mean age of the SLE patients was (32.8±12.4) years, and 85% were female, none of whom were post-menopausal. BMD was significantly reduced in all the measured sites, compared with the healthy controls. Sixteen (20%) of the patients were osteopenic in at least one site measured locations. The serum levels of 25-OH-VD3 were markedly reduced in the newly diagnosed SLE patients than those of the normal controls [(46.1+12.3) nmol/L vs. (25.4+11.2) nmol/L, P<0.001)]. The serum levels of 25-OH-VD3 in the SLE patients with nephritis were much lower than those without nephritis (P=0.04). A significant negative correlation was demonstrated between the serum concentration of 25-OH-VD3 and the disease activity scores as measured by SLE disease activity index (SLEDAI) (r=-0.3,P=0.001). The serum TRAP5b concentration was positively correlated with SLEDAI (r=0.435,P=0.003). Age (P=0.058) and SLEDAI (P=0.085) were probably associated with decreased BMD in Logistic regression analysis. CONCLUSION: The study showed reduced BMD in untreated SLE patients. The role of chronic inflammation was of probable importance in bone metabolism.

[Clinical and immunological characteristics of patients with systemic lupus erythematosus in Tibet plateau, China].

OBJECTIVE: To describe the clinical, immunological characteristics and organ involvement of patients with systemic lupus erythematosus (SLE) in Tibet plateau, China. METHODS: We retrospectively investigated 70 patients admitted in the Tibet Autonomous Region People's Hospital between May 2014 and April 2016. In the study, 120 hospitalized patients with SLE from the Department of Rheumatology and Immunology of the Peking University People's Hospital were randomly selected as the control (plain) group. The major organ involvement, clinical and immunological characteristics were compared between the two groups. RESULTS: The female to male ratio of Tibet plateau group was 10.7, while the corresponding ratio of plain group was 11.0. The mean age at disease diagnosis was (32.21±11.40) and (35.38±13.25) years, respectively. the most common initial manifestations of SLE were arthritis (78.6%), alopecia (55.7%) and malar rash (48.6%) in Tibet plateau group, the prevalence of arthritis and alopecia was significantly higher than in plain group (P<0.05). The incidence of neuropsychiatric and kidney involvement was significantly lower in Tibet plateau group compared with plain group (P<0.05). As for the serological manifestations, the positivity of anti-double-stranded DNA (dsDNA) (57.1%), anti-Smith (Sm) antibody (55.7%), anti-Sjögren syndrome A (SSA) antibody (72.3%), anti-Sjögren syndrome B (SSB) antibody (41.4%) and anti-u1-ribosenuclear protein (u1RNP) antibody (45.7%) was significantly higher in Tibet plateau group (P<0.05). While the incidence of low serum complement C3 (61.4%), C4 (38.6%) less frequent in Tibet plateau group. Mean SLE disease activity index (SLEDAI) score was similar in the Tibet plateau group (12.18±5.58) and plain group (12.69±7.28). Moreover, there were 13 (18.6%) SLE patients suffering from tuberculosis and 7 (10%) SLE patients infected with hepatitis B virus in Tibet plateau group. The number of recent-onset SLE patients with lower 25-dihydroxy-vitamin D3 (25-OH-VD3) in Tibet plateau group was fewer than that in the plain group (76.7% vs. 90.0%, P=0.046). Serum 25-OH-VD3 levels in Tibet plateau plateau group were (31.14±18.74) nmol/L, those in plain group were (26.91±14.27) nmol/L, and the difference was not significant. CONCLUSION: The age, gender and SLEDAI scores in Tibet plateau group was similar to those in plain group. But there are significant differences in clinical manifestations, distributions of antibodies and immunological changes between Tibet plateau group and plain group. The patients with lower serum 25-OH-VD3 levels were more in plain group than in Tibet plateau group, while there was no significant difference in the 25-OH-VD3 level between the two groups.

[Increased serum C-C chemokine ligand 19 levels correlated with B cell abnormalities in systemic lupus erythematosus].

OBJECTIVE: To detect the levels of serum C-C chemokine ligand 19 (CCL19) in patients with systemic lupus erythematosus (SLE) and to evaluate the correlation between CCL19 expression and clinical features and laboratory parameters, trying to reveal the possible role of CCL19 in the pathogenesis of systemic lupus erythematosus. METHODS: The levels of serum CCL19 were measured by enzyme linked immunosorbent assay (ELISA) in 90 patients with SLE and 30 healthy controls. These SLE patients included 75 patients who received treatment with glucocorticoids and disease-modifying anti-rheumatic drug (DMARD) and 15 patients without therapy. The frequencies of peripheral blood B cells and the B cell subsets were assessed in the patients with SLE by flow cytometry. The correlation between the clinical data, laboratory parameters, B cell subset frequencies and serum CCL19 levels were analyzed. Indepen-dent samples t test, paired t test, Pearson and Spearman correlation were used for statistical analyses. RESULTS: The levels of CCL19 were markedly higher in the SLE patients without therapy and the patients with therapy than in the health controls[(596.25±409.19) ng/L and (422.90±395.84) ng/L vs. (157.79±125.23) ng/L, all P<0.001]. Serum CCL19 levels in the SLE patients without therapy were higher than the SLE patients who accepted glucocorticoids and DMARD treatment (P<0.05). The levels of serum CCL19 were positively correlated with anti-double stranded deoxyribonucleic acid (dsDNA), anti-nucleosome antibody (AnuA), IgA, IgG and IgM (r=0.38, P=0.007; r=0.332, P=0.029; r=0.519, P=0.007; r=0.461, P=0.018, respectively). Serum CCL19 levels in the SLE patients with photosensitivity, arthritis and secondary Sjögren's syndrome were higher than the SLE patients without photosensitivity, arthritis and secondary Sjögren's syndrome, respectively [(562.25±399.12) ng/L, (565.6±435.24) ng/L and (694.9±531.02) ng/L vs. (394.7±281.42) ng/L, (385.90±325.33) ng/L and (424.8±305.46) ng/L, all P<0.05]. The levels of serum CCL19 were positively correlated with the percentage of CD27-B cells and CD27-IgD-double-negative memory B cells (r=0.519, P=0.007; r=0.461, P=0.018, respectively). However, the levels of serum CCL19 were negatively correlated with the percentage of CD27+ memory B cells and CD27+IgD- switched memory B cells (r=-0.433, P=0.027; r=-0.616, P=0.001, respectively). CONCLUSION: The increased serum CCL19 levels in SLE patients were associated with the production of autoantibodies, and CCL19 might be involved in the pathogenesis of SLE by disturbing the homeostasis of B cell subsets.

[Application study of qualitatively diagnosing prostate cancer using ultrahigh b-value DWI].

Objective: To explore the value of ultrahigh b-value DWI in diagnosis of prostate cancer. Methods: From October 2015 to October 2016, a total of 84 cases from Affiliated Changshu Hospital of Soochow University(39 cases of prostate cancer with a total of 57 lesions, 45 cases of benign prostate hyperplasia) were examined with T(2)WI, high b-value DWI (b=1 000 s/mm(2)) and ultrahigh b-value DWI (b=2 000 s/mm(2)) .Three image sets were rated respectively based on PI-RADS V2 by two radiologists and the scores were compared with biopsy results.The differences of the area under the ROC curve (AUC) among the three groups of each observer were compared by Z test. Results: The difference of AUC between ultrahigh b-value DWI and T(2)WI in the diagnosis of peripheral and transitional zone cancer was statistically significant between the two observers (P=0.009 9, 0.008 2, 0.010 8 and 0.004 5 respectively), and there was no significant difference of AUC between ultrahigh b-value DWI and high b-value DWI in the diagnosis of peripheral and transitional zone cancer.The inter-reader agreement was found to be perfect for all lesions, peripheral zone lesions and transition zone lesions at ultrahigh b-value DWI (kappa values were 0.738, 0.709 and 0.768 respectively). Conclusion: The diagnostic performance of ultrahigh b-value DWI is superior to high b-value DWI and T(2)WI in both peripheral zone and transition zone cancers.

One-step synthesis and luminescence properties of tetragonal double tungstates nanocrystals.

A versatile one-step thermolysis protocol is demonstrated to produce a uniform dispersion of tetragonal double tungstates NaRE(WO4)2 (RE = rare earth) nanocrystals (NCs). Oriented attachment in the [001] direction occurred. Doping with luminescent RE(3+) ions resulted in highly luminescent NCs showing characteristic line emission of the dopant as well as a blue emission assigned to surface adsorbed organic species.

Development of polymorphic SSR markers in the razor clam (Sinonovacula constricta) and cross-species amplification.

Next-generation sequencing provides large-scale sequencing data with relative ease and at a reasonable cost, making it possible to identify a large amount of SSR markers in a timely and cost-effective manner. On the basis of the transcriptome database of Sinonovacula constricta obtained by Illumina/Solexa pyrosequencing, 60 polymorphic SSR markers were developed and characterized in 30 individuals. The number of alleles per polymorphic locus ranged from 2 to 7 with an average of 3.75 alleles. The observed and expected heterozygosities varied from 0.050 to 1.000 and from 0.050 to 0.836, respectively. Nineteen loci significantly deviated from Hardy-Weinberg equilibrium (P < 0.01) after Bonferroni's correction for multiple tests. In addition, interspecific transferability revealed that 20 polymorphic loci in Solen linearis were first characterized in this study. To the best of our knowledge, this is the highest number of SSRs in S. constricta and the first report of cross-species amplification. These novel polymorphic SSR markers will be particularly useful for conservation genetics, evolutionary studies, genetic trait mapping, and marker assisted selection in the species.

Development of novel polymorphic microsatellite markers for the blood clam Tegillarca granosa by pyrosequencing.

Large amounts of expressed sequence tags (ESTs) generated using next-generation sequencing technologies provide a cost-effective and valuable genomic resource for the development of microsatellite markers. In this study, we isolated 115 novel polymorphic microsatellite markers for the blood clam Tegillarca granosa from ESTs in 454 sequencing data. All the loci were characterized in 30 individual clams from a natural population in Xiangshan (Zhejiang Province, China). The number of alleles per locus varied from 2 to 10, with an average of 3.78. The observed and expected heterozygosities ranged from 0 to 1 and from 0.040 to 0.799, respectively. The polymorphic information content (PIC) ranged from 0.038 to 0.825, and 29 highly polymorphic loci (PIC ≥ 0.5) and 42 moderately polymorphic loci (0.25 < PIC < 0.5) were identified. Thirty-eight of the 115 loci deviated significantly from the Hardy-Weinberg equilibrium (P < 0.01) after a Bonferroni correction. A BlastX search revealed that 46 (40%) of the polymorphic loci identified were from transcript regions of known genes. The microsatellite markers developed in the present study will greatly enrich the microsatellite resources of T. granosa, and are available for further population genetic analysis, genetic trait mapping, and molecular-assisted selection.

Microsatellite marker analysis reveals the distinction between the north and south groups of hard clam (Meretrix meretrix) in China.

Meretrix meretrix is one of the important commercial bivalves in China. A total of 198 individual clams were collected from 5 locations characteristic of the clam's 5 main natural habitats in China, that is, Shandong, Jiangsu, Fujian, Guangdong, and Guangxi. Ten polymorphic microsatellite markers were selected to examine the genetic diversity and identify genetic differences between the 5 populations. A total of 183 alleles across 10 loci were detected in the individual clams. The observed heterozygosity and expected heterozygosity ranged from 0.197 to 0.7026 and from 0.6264 to 0.9408, respectively. The genetic diversity within samples was high (8.6-11.2 alleles per locus, observed heterozygosity = 0.25-0.875 and expected heterozygosity = 0.6848-0.9259). Most of the genotype distributions significantly deviated from Hardy-Weinberg equilibrium. Genetic structure analysis showed that the 5 populations could be divided into 2 groups, the north and south groups. Neighbor-joining analysis revealed a clear distinction between the north group (Shandong and Jiangsu) and the south group (Fujian, Guangdong, and Guangxi). Locus MM1031 was used to distinguish between groups. Our results can be used for population identification and crossbreeding of M. meretrix.

Gastric rupture following nasopharyngeal catheter oxygen delivery-a report of two cases.

Iatrogenic gastric distension and subsequent rupture following nasal or nasopharyngeal catheter oxygen delivery is a rare but life-threatening condition that requires urgent laparotomy. We report two cases recently encountered at our institution. Both patients exhibited symptoms of abdominal pain and distension following oxygen delivery involving a nasopharyngeal catheter during procedural sedation. Oxygen flow rates were 4 l/minute in both cases. The diagnosis was made by urgent imaging. Both patients survived following laparotomy and repair of gastric rupture. Seventeen cases have been reported previously in the literature. We recommend avoidance of nasal or nasopharyngeal catheters and the use of alternative oxygen delivery methods such as nasal prongs and face masks.

How to make a gonad: cellular mechanisms governing formation of the testes and ovaries.

Sex determination of the gonad is an extraordinary process by which a single organ anlage is directed to form one of two different structures, a testis or an ovary. Morphogenesis of these two organs utilizes many common cellular events; differences in the timing and execution of these events must combine to generate sexually dimorphic structures. In this chapter, we review recent research on the cellular processes of gonad morphogenesis, focusing on data from mouse models. We highlight the shared cellular mechanisms in testis and ovary morphogenesis and examine the differences that enable formation of the two organs responsible for the perpetuation of all sexually reproducing species.

InGaN self-assembled quantum dots grown by metal-organic chemical vapour deposition with growth interruption.

Self-assembled InGaN quantum dots (QDs) were grown by metal-organic chemical vapour deposition with growth interruption at low V/III ratio and low growth temperature on sapphire substrates. The effects of the interruption time on the morphological and optical properties of InGaN QDs were studied. The results show that the growth interruption can modify the dimension and distribution of InGaN QDs, and cause the QD emission wavelength to blue shift with increasing interruption time. A density of InGaN QDs of about 4.5 × 10(10) cm(-2) with an average lateral size of 11.5 nm and an average height of 1.6 nm can be obtained by using a growth interruption time of 60 s.

Chicken granulosa cells show differential expression of epidermal growth factor (EGF) and luteinizing hormone (LH) receptor messenger RNA and differential responsiveness to EGF and LH dependent upon location of granulosa cells to the germinal disc.

Granulosa cells in the chicken follicle exhibit different phenotypes according to their location relative to the germinal disc (GD). Granulosa cells proximal to the GD (referred to as proximal granulosa cells) are more proliferative, whereas granulosa cells distal to the GD (referred to as distal granulosa cells) are more differentiated. We have shown that epidermal growth factor (EGF) derived from the GD stimulated proliferation of granulosa cells proximal to the GD, whereas extraovarian LH promoted differentiation. We tested the hypothesis that phenotypic differences of granulosa cells are the result of differential responsiveness of granulosa cells to EGF and LH. We found that both granulosa and theca layers of chicken preovulatory follicles expressed mRNA for EGF receptor (EGFr) by polymerase chain reaction (PCR) analysis. However, only the granulosa layer showed differential expression of EGFr and LH receptor (LHr) mRNA. Competitive reverse transcription-PCR revealed that proximal granulosa cells expressed more EGFr mRNA but less LHr mRNA than distal granulosa cells. In addition, proximal granulosa cells proliferated more in response to EGF than their distal counterparts. We further demonstrated that EGF decreased LHr mRNA expression by granulosa cells in a dose-dependent manner, whereas EGF and LH had no effect on EGFr mRNA expression except at one dose of LH (15 ng/ml) that stimulated EGFr mRNA expression. Our findings suggest that EGF derived from the GD influences the phenotypes of granulosa cells. Granulosa cells proximal to the GD exhibit a proliferative phenotype possibly because they are exposed to and are more responsive to GD-derived EGF. Furthermore, GD-derived EGF decreases LHr mRNA expression by proximal granulosa cells and therefore results in less differentiated granulosa cell phenotype. In contrast, granulosa cells distal to the GD are not under the influence of EGF and exhibit a more differentiated phenotype.

Germinal disc-derived epidermal growth factor: a paracrine factor to stimulate proliferation of granulosa cells.

The germinal disc (GD) of the chicken oocyte produces factors that influence proliferation and differentiation of granulosa cells. Granulosa cells proximal to the GD are more proliferative, whereas granulosa cells distal to the GD are more differentiated. Previously, we had found epidermal growth factor (EGF) was present in the GD. In this study, we tested the hypothesis that EGF is the GD-derived paracrine factor that stimulates proliferation of granulosa cells. Northern analysis, reverse transcription-polymerase chain reaction, and radioimmunoassay indicated that the GD and granulosa cells but not theca cells are the sources of EGF in chicken preovulatory follicles. However, only the conditioned medium from the GD region (GDR = GD + overlying granulosa cells) but not the granulosa cell-conditioned medium stimulated proliferation of granulosa cells. Pretreatment of conditioned media with EGF antibody abolished the proliferation-stimulating effect of the GDR-conditioned medium. We conclude that EGF is one of the paracrine factors produced by the GD to stimulate proliferation of granulosa cells. Granulosa cells proximal to the GD express a proliferative phenotype possibly because they are exposed to a greater amount of EGF derived from the GD.

Simultaneous determination of ethylene glycol and glycolic acid in serum by gas chromatography-mass spectrometry.

We describe a gas chromatographic-mass spectrometric (GC-MS) procedure for the simultaneous determination of ethylene glycol (EG) and its major toxic metabolite, glycolic acid (GA), in serum. In this method, serum (50 microL) is treated with 150 microL of glacial acetic acid/acetonitrile (1:10, v/v; contains internal standard, 1,3-propanediol, 15 mg/dL) to precipitate protein. After centrifugation, 10 microL of supernate is treated with 500 microL of 2,2-dimethoxypropane/dimethylformamide (80:20, v/v) to convert water to methanol, and the volume is then reduced to < 100 microL of dimethylformamide (but not to dryness). After formation of tertbutyldimethylsilyl derivatives, analysis is performed by capillary column GC-MS in selected ion mode. The method gives a linear response to 1000 mg/L each EG and GA (16.1 mmol/L and 13.2 mmol/L, respectively) and has a lower limit of detection and a lower limit of quantitation of 10 mg/L each EG and GA (0.16 mmol/L and 0.13 mmol/L, respectively). Total assay imprecision is CV < or = 6.4% (200 and 800 mg/L EG and GA [3.2 and 12.9 mmol/L EG; 2.6 and 10.5 mmol/L GA, respectively]). Absolute recovery from human serum was 91.1% for EG and 77.6% for GA. The procedure is free from any known interference. A complete analysis set (three calibrators, patient serum neat, patient serum diluted 1:5 (v/v), and two controls) may be completed in about 2 h. A preliminary result, based on a single calibrator and patient serum diluted 1:5 (v/v), is complete in about 1 h. The method has been used to aid the diagnosis and management in 34 cases of EG intoxication. Selected cases are briefly reviewed.

Restricted expression of WT1 messenger ribonucleic acid in immature ovarian follicles: uniformity in mammalian and avian species and maintenance during reproductive senescence.

WT1 is a zinc finger protein with transcriptional repressor activity on several growth factor and growth factor receptor genes. In the ovary, a potential role for WT1 in the suppression of the development of immature follicles has been demonstrated. Here, gel retardation assays further showed that recombinant WT1 protein interacted with consensus DNA sequences in the inhibin-alpha gene promoter. We investigated the pattern of WT1 expression in a wide variety of species and also over the reproductive life span in rats. In chicken ovaries, Northern blot analysis revealed the presence of WT1 transcript in small healthy white follicles (1-5 mm in diameter) and its absence in small yellow (6-12 mm in diameter) or larger follicles (F1-F5). In pig and monkey ovaries, WT1 expression was limited to granulosa cells of preantral follicles, as shown by in situ hybridization analysis. In rats, Northern blot analyses demonstrated the presence of WT1 transcript in the ovaries of young (3-mo-old) and middle-aged (9-mo-old) rats on the proestrous day, with a decrease in old (12-mo-old) rats in persistent estrus. In situ hybridization analysis further suggested that the decrease in WT1 expression in aging ovaries was associated with fewer immature follicles. Thus, WT1 expression is restricted to immature follicles in diverse avian and mammalian species and over the reproductive life span in rats. These data demonstrated that WT1 is a marker for immature follicles and suggested a potential role of this transcriptional repressor in the slow growth of early follicles.

Destruction of the germinal disc region of an immature preovulatory chicken follicle induces atresia and apoptosis.

The germinal disc region (GDR), which contains the germinal disc and overlying granulosa cells, is essential for completion of maturation of the preovulatory chicken follicle. The current study was conducted to test the hypothesis that destruction of the GDR (GDRX) of an immature preovulatory chicken follicle blocks ovulation, induces apoptosis, and causes atresia. The GDR of immature preovulatory follicles (F2) were destroyed by freezing with dry ice (3 mm in diameter) 48-50 h before ovulation. As a control for the effect of freezing, a nonGDR portion (a portion of the follicular wall opposite to the GDR relative to the follicular stalk) of other F2 follicles were destroyed (nonGDRX). Treatment of F2 follicles by GDRX caused atresia and blocked ovulation of all treated follicles (6 of 6), whereas none of the nonGDRX follicles (0 of 5) underwent atresia. Treatment of follicles by GDRX induced apoptotic DNA fragmentation (laddering) in theca and granulosa layers obtained from the frozen area and in the theca layer obtained from the follicular wall distal to the frozen area. In contrast, apoptosis was only present in theca and granulosa layers in the frozen area of the nonGDRX follicle. Furthermore, the in situ DNA end-labeling technique demonstrated that in the GDRX follicle 24 h after treatment, cells in the theca interna, endothelial cells in blood vessels of the theca externa, and a few granulosa cells underwent apoptosis. These results indicate that destruction of the GDR of an immature preovulatory follicle causes atresia and apoptosis and blocks ovulation. These novel findings suggest that the GDR maintains development of the chicken preovulatory follicle by producing one or more survival factors. Without the GDR, chicken follicles cannot develop further and they eventually die.

Epidermal growth factor in the germinal disc and its potential role in follicular development in the chicken.

The germinal disc region (GDR; germinal disc + overlying granulosa cells) of the hen's ovarian follicle secretes one or more factors that stimulate proliferation of, and decrease progesterone (P4) production by, granulosa cells. Destruction of the GDR results in apoptosis and atresia of the follicle. These data suggested that the GDR produces a growth factor(s) to sustain the development of the follicle. These findings prompted us to investigate two questions: 1) Is epidermal growth factor (EGF) or transforming growth factor alpha (TGFalpha), which binds to the EGF receptor, present in the GDR? 2) Does EGF regulate granulosa cell functions in the hen? Immunocytochemistry revealed that EGF, but not TGFalpha, was present in the germinal disc of the four largest preovulatory follicles of the hen. TGFalpha was found only in the theca interna. To determine whether EGF regulates granulosa cell functions, granulosa layer explants (13 mm in diameter) from the second-largest preovulatory follicle were cultured for 36 h with 0,0.017, or 0.17 microM EGF. Proliferation, apoptosis, and P4 production of granulosa layer explants were then measured by using a colorimetric method for determining viable cell number, gel electrophoresis, and RIA, respectively. EGF regulates several functions of granulosa layer explants by stimulating proliferation, inhibiting apoptosis, and decreasing basal P4 production. These data indicate that EGF is present in the germinal disc and may be one of the factors that regulate follicular development in the hen.