Objective To detect the expression of CK19 in sentinel lymph node for breast cancer and to evaluate its clinical significance.Methods SLNs of 30 patients with breast cancer were successfully detected with methylene blue,both SLN and ALN were examined by hematoxylin and eosin staining ( HK staining). The expression level of CK19 in SLNs were assessed by RT-PCR and Western Blot for mRNA and Protein respectively. Results Detection positive rate of SLN melastases was 26. 67% (8/30) by HE staining,and 1 case of false negative. Through RT-PCR,12 cases,including all the positive cases detected by routine pathological examination,were detected to be positive in the expression of CK19mRNA, with a positive rate of 40. 0% ( 12/30) , and a positive rate of 36. 67% (11/30) for CK19 was observed by Western Blot There was a significant difference between RT-PCR or Western Blot methods and routine pathological examination ( P < 0.05 ) for the detection efficiency of SI.Ns melastases. Conclusions CK19rnRNA may be a potential tumor marker for detecting micrometastasis in SLNs of breast cancer. The combined utilization of locating SLNs and detecting CK19 mRNA expression by RT-PCR instead of histopathological examination can greatly enhance the detection efficiency of SLN micrometastasis of breast cancer.

Objective To observe the effects of live combined bifidobacterium,lactobacillus and enterococcus powder on immunoglobulin E (IgE) and interleukin-17 (IL-17) in atopic children with bronchiolitis.Methods Sixty cases of atopic children with bronchiolitis were randomly divided into the therapy group (30 cases) and the control group (30 cases).Twenty-five healthy children were enrolled as the healthy control group.Both the therapy group and the control group were given traditional therapy.The therapy group received live combined bifidobacterium,lactobacillus and enterococcus powder for 2 months.The change of IgE and IL-17 levels were observed during the acutestage,remission stage and after receiving live combined bifidobacterium,lactobacillus and enterococcus powder for 2months.Results (1) The levels of IgE and IL-17 of therapy group[(132.36 ±9.50) μg/L and (77.76 ±7.95)μg/L] during acute stage were markedly higher than those in the healthy control group [(52.80 ±4.92) μg/L and (46.92 ±4.79) μg/L] (all P ＜0.001).The levels of IgE and IL-17 of control group [(128.83 ± 8.06) μg/L and (76.61 ±6.18) μg/L] during remission stage were markedly higher than those in the healthy control group [(52.80 ±4.92) μg/L and (46.92 ± 4.79) μg/L] (all P ＜ 0.001).(2) The levels of IgE of therapy group (56.67 ± 9.20)μg/L after receiving live combined bifidobacterium,lactobacillus and enterococcus powder for 2 months were markedly lower than those in the control group (70.50 ± 11.38) μg/L (P ＜ 0.001).The levels of IL-17 of therapy group [(49.63 ± 6.35) μg/L] at the time after receiving live combined bifidobacterium,lactobacillus and enterococcus powder for 2 months were markedly lower than these in the control group (54.77 ± 6.33) μg/L (P =0.003).Conclusion Receiving live combined bifidobacterium,lactobacillus and enterococcus powder for two months can decrease the IgE and IL-17 levels in atopic children with bronchiolitis.

Objective:To summarize the clinical characteristics, diagnosis and treatment of caspase recruitment domain-containing protein 9 (CARD9) gene deficiency associated invasive candidiasis, and report a novel mutation in CARD9 gene.Methods:The clinical characteristics, laboratory tests, treatment and the outcome of follow-up in a boy with invasive candidiasis were described. The boy′s main clinical manifestations were central nervous system infection and retroperitoneal mass. Whole-exome sequencing was performed and Sanger sequencing was verified to identify the CARD9 gene mutations in the patient and his parents. A literature search for “CARD9”and “invasive candidiasis”was conducted in PubMed, Wanfang and CNKI databases from their establishment to May 2020.Results:A 10-year-old boy suffered onset symptom of chronic diarrhea, which lasted for two months. The symptom was followed by progressive neurological symptoms such as headache, vomiting, seizures and disorder of consciousness. His unusual medical history was absent. Candida albicans were cultured several times in cerebrospinal fluid and blood, and yeast-like fungi were found in the stool high power field of vision. Cerebral magnetic resonance imaging indicated obstructive hydrocephalus and abdominal CT scan showed retroperitoneal mass and thickening of the intestinal wall. The whole-exome sequencing analyses of blood samples from the boy and his parents were performed. The results showed that there was a homozygous mutation of c.952-12_956delinsAG in the CARD9 gene, which was an unreported pathogenic mutation. This was confirmed by Sanger sequencing. There was no significant relief from intravenous combined antifungal medications. After lateral ventricular drainage surgery and injection of amphotericin B into the lateral ventricle, improvement of clinical symptoms and cerebral spinal fluid abnormalities was observed after nine weeks, and the retroperitoneal mass shrank. At follow-up after four-month oral combined antifungal medications, the child had no complaint except fatigue. However, cerebral spinal fluid analysis showed increased protein level and decreased glucose. Persistent hydrocephalus and periventricular white matter abnormal signals were revealed on the brain magnetic resonance imaging and the smaller retroperitoneal mass than before on the abdominal CT scan. In addition to this case, totally 21 cases with CARD9 gene deficiency associated invasive candidiasis have been reported worldwide, most of which featured central nervous system infections.Conclusions:CARD9 gene deficiency is an autosomal recessive primary immunodeficiency that confers human susceptibility to fungal disease. The associated invasive candidiasis often affects the central nervous system and makes the patient severely ill. Adequate systemic antifungal therapies should be given, and patients with hydrocephalus need surgical treatment. A novel mutation is reported that expands the variant diversity of CARD9 gene. For patients with unexplained invasive candidiasis, including those without a history of previous recurrent infection, genetic testing is recommended for primary immunodeficiency including CARD9 gene deficiency.

BACKGROUND: Experimental evidence suggests that growth factors can promote myocardial angiogenesis, but the effect and mechanism of basic fibroblast growth factor (bFGF) in controlled release delivered via fibrin glue has not been fully recognized.OBJECTIVE: To evaluate the effect of controlled-release bFGF delivered via fibrin glue in the myocardium on the expressions of vascular growth factors and cell proliferation in the local acute myocardial infarct area in canines, and assess the therapeutic effect of this strategy.DESIGN: Completely randomized controlled experiment.SETTING: Department of Cardiology, Beijing Anzhen Hospital, Capital University of Medical Sciences; Department of Cardiology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology; Shanghai Xinxing Blood Product Research Institute.MATERIALS: This experiment was carried out in the Laboratory of Animal Surgery, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, and the Experimental Animal Center of Chinese PLA General Hospital between June 2001 And March 2003.Twelve clean healthy adult mongrel dogs of either sex were selected and randomized into transmyocardial laser revascularization group and bFGF group with 6 in each group.METHODS: With appropriate anesthesia, the chest of the dog was opened and the left anterior descending (LAD) branch of the coronary artery was ligated to establish acute myocardial infarction (AMI) model.The dogs were then randomized into transmyocardial laser revascularization group to receive transmural myocardial penetration 30 minutes after AMI and bFGF group with non-transmural myocardial penetration 30 minutes after AMI and subsequent injection of bFGF-containing fibrin glue into the channel. The expressions of vascular epithelial growth factor (VEGF), transforming growth factor beta 1 (TGFβ1) and proliferating cell nuclear antigen (PCNA) in the loacl ischemic myocardium were examined immunohistochemically (IHC) at postoperative 18 weeks.MAIN OUTCOME MEASURES: Quantitative IHC analysis of VEGF,TGFβ1 and the PCNA expressions in the local ischemic myocardia in transmyocardial laser revascularization group and bFGF group.RESULTS: Five dogs in the transmyocardial laser revascularization group and 6 in the bFGF group survived the operations. Quantitative IHC analysis revealed obviously larger positive area stained for myocardial VEGF,TGFβ1 and PCNA in bFGF group than in transmyocardial laser revascularization group (t=-7.505, -2.690 and -6.895, P ＜ 0.05), and the average absorbance of PCNA staining in bFGF group was greater than that in the transmyocardial laser revascularization group (t= -5.271, P ＜ 0.05).CONCLUSION: Controlled-releasing bFGF delivered in the myocardium can increase local expressions of the vascular growth factors in the ischemic myocardium and enhance cell proliferation, promoting revascularization after AMI.

Objective To investigate the clinical significance and changes of serum interleukin-4 (IL-4),interferon-γ(IFN-γ) and immunoglobulin E (IgE) levels in infants with spasmodic laryngitis.Methods Serum samples were obtained from 50 children with spasmodic laryngitis and 30 healthy children.The changes of IL-4,IFN-γand IgE levels in serum when hospital admission (acute stage),before discharge (the stage of clinical symptom disappeared) and after 2 months of follow-up were observed.Enzyme-linked immunosorbent assay was used to determine the levels of IL-4 and IFN-γ in serum.Serum IgE level was determined with enzyme-linked fluoroimmuneassay.Results The serum level of IL-4 in patients with spasmodic laryngitis during acute stage were much higher than that in normal control group[(20.65 ±5.87) ng/L vs(8.23 ±2.71) ng/L,t =5.536,P ＜0.05].It was decreased during the stage of clinical symptom disappeared and significantly lower than that during acute stage[(11.07 ± 2.93) ng/L vs (20.65 ± 5.87) ng/L,F =5.258,P ＜ 0.05].The serum level of IFN-γ in patients with spasmodic laryngitis during acute stage were much lower than that in normal control group[(61.38 ±6.36) ng/L vs (108.42 ± 13.56) ng/L,t =4.831,P ＜ 0.05],and it was increased during the stage of clinical symptom disappeared [(95.12 ± 11.63) ng/L] and significantly higher than that during acute stage(F =4.239,P ＜ 0.05).There was no difference between the patients at clinical symptom disappeared stage and normal control group (P ＞ 0.05).Two months after discharge,there was no difference in serum INF-γlevel between the patients [(75.68 ±6.29) ng/L] and that in control group (P ＞ 0.05).The serum level of IgE in patients with spasmodic laryngitis during acute stage were much higher than that in normal control group [(136.63 ± 17.86) ng/L vs (47.16 ± 6.83) ng/L,t =6.685,P ＜ 0.05].It was decreased during the stage of clinical symptom disappeared [(88.07 ± 11.83) μg/L] and significantly lower than that during acute stage(F =5.182,P ＜ 0.05),but higher than that of normal control group (P ＜ 0.05).Two months after discharge [(86.72 ± 7.25) μg/L] it was still higher than that in control group (P ＜ 0.05).Conclusions There is TH1/TH2 imbalance in infants with spasmodic laryngitis.Maybe spasmodic laryngitis is one of the clinical manifestations of atopic diseases.IL-4,IFN-γand IgE may play important roles in immunologic pathogenesis.

Objective To evaluate the role of a color jaundice card (6 colors) as a possible screening tool for detecting neonatal hyperbilirubinemia.Methods During February 1,2016 and May 31,2017,neonates were enrolled in the study,with gestational age ≥35 weeks,birth weight ≥2 000 g,postnatal age 3-28 days,who were the outpatients or inpatients of the 9th People's Hospital of Wuxi Affiliated to Soochow University and the People's Hospital of Anyang.In a well-lighted room,the card measurements were performed at the infants' forehead,the cheek and the sternum.The skin was pressed with a finger for 2 seconds and left quickly,and then the card was used to compare with the exposed yellow skin.Within 2 hours after jaundice card measurement,blood was obtained by venipuncture and total serum bilirubin (TSB) levels were measured.The sensitivity,specificity,positive predictive value (PPV),negative predictive value (NPV),positive likelihood ratio (PLR) and negative likelihood ratio (NLR) were calculated at each measurement sites.Results One hundred and thirty-two neonates were enrolled,of whom 68 cases (51.5％) were male and 64 cases(48.5％) were female and 18 cases (13.6％) were preterm and 114 cases (86.4％) were term neonates.Among all neonates,TSB was ＜5.00 mg/dL(1 mg/dL =17.1 μmol/L) in 21 cases (15.9％),5.00-9.99 mg/dL in 26 cases (19.7％),10.00-14.99 mg/dL in 34 cases (25.8％),15.00-19.99 mg/dL in 37 cases (28.0％) and ≥ 20.00 mg/dL in 14 cases (10.6％).The card had the highest cap ability to recognize jaundice at the cheek,slightly lower at the sternum and the worst in the forehead.The cut-off of ≥ 12 on the six-color card at the cheek had a sensitivity of 95.95％,specificity of 74.14％,PPV of 82.56％,NPV of 93.48％,PLR of 3.710 and NLR of 0.055 for identifying neonates with TSB ≥ 12 mg/dL,with sensitivity being 98.08％,specificity 57.50％,PPV 60.00％,NPV 97.87％,PLR 2.308 and NLR 0.033 for TSB≥ 15 mg/dL.The identification rate was as follows:sensitivity of 100.00％,specificity of 46.00％,PPV of 37.21％,NPV of 100.00％ and PLR of 1.852 for predicting TSB ≥ 17 mg/dL.In addition,in the forehead,cheeks and sternum,the sensitivity of the cut-off of ≥ 12 on the card was 100.00％ for identifying neonates with TSB≥20 mg/dL.In the cheeks and the sternum,the cut-off of ≥ 15 on the card was with a sensitivity of 100.00％ for predicting TSB ≥ 20 mg/dL.Conclusion The six-color jaundice card is a potential screening tool for neonatal hyperbilirubinemia,and the cheek is the best measurement site.

Objective To quantify the setup errors for the different anatomical sites of patients who received intensity-modulated radiotherapy (IMRT) with linear accelerator on-board kilovolt fan beam CT(kV-FBCT) as non-isocenter IGRT and megavolt cone beam CT (MV-CBCT) as isocenter IGRT. Methods A retrospective analysis was performedon 70 patients who underwent radiotherapy, kV-FBCT, and/or MV-CBCT scans after each routine setup prior to IMRT. The average displacement (M), systematic error (Σ), and random error (б) at different treatment sites in the left-right, anterior-posterior, and cranial-caudal directions were calculated according to the individual displacements. The formula 2.5Σ+0.7б was used to estimate the PTV margin in respective direction. For each single patient, the root mean square in three directions was used as 3D displacement. Results A total of 1130 displacements were recorded in the 70 patients. The PTV margin was estimated to be 1.9-3.1 mm in head and neck cancer, 2.8-5.1 mm in thoracic cancer, 4.6-5.1 mm in breast cancer, 3.0-5.5 mm in upper abdominal cancer, and 3.5-6.8 mm in pelvic tumor. For the 3D mean displacements, the head and neck, thoracic, breast, upper abdominal, and pelvic cancer were 2.4±1.0, 4.0±1.6, 4.1±2.0, 4.6±2.1, and 4.6±2.1 mm, respectively. The average 3D displacement obtained by kV-FBCT and MV-CBCT were 4.1 and 3.4 mm, respectively (P=0.212). Conclusion The quantitative setup-error data can be obtained using linear accelerator on-board FBCT, and the non-isocenter IGRT induced set-up error cannot be negligible.

ObjectiveTo study the effect and mechanism of Xiangsha Liu Junzitang combined with phlegm-removing and detoxifying traditional Chinese medicine on immune escape in Lewis lung cancer mice. MethodA total of 60 specific-pathogen-free （SPF）-grade C57BL/6J male mice were injected subcutaneously with 0.2 mL of Lewis cell suspension （containing 2×10⁶ cells·mL^-1） in the right mid-axillary line. After 7 days， the mice that had been successfully modeled were randomly divided into six groups： the model group， the cisplatin group， the Xiangsha Liu Junzitang low-， medium-， and high-dose groups， and the combined group， with 10 mice in each group. The Xiangsha Liu Junzitang low-， medium- and high-dose groups were gavaged with 17.88， 35.75， 71.50 g·kg^-1 Xiangsha Liu Junzitang solution once a day， respectively， and the dosage of cisplatin intraperitoneally injected into the mice was converted to 5 mg·kg^-1 twice a week， and the tumour volumes of each group were measured every two days. The intervention lasted for 14 consecutive days. At the end of treatment， the tumour mass of mice in each group was weighed and the tumour inhibition rate was calculated. The morphological characteristics of tumours in each group were observed by hematoxylin-eosin （HE） staining. Fluorescent quantitative real-time polymerase chain reaction （Real-time PCR） assay was used to detect messenger ribonucleic acid （mRNA） contents of the natural killer group 2 member D （NKG2D） receptor， ribonucleic acid export-1 （RAE-1）， and γ interferon （IFN-γ） in the tumour tissues of each group. NKG2D， RAE-1， and IFN-γ mRNA in tumour tissues of each group. Immunohistochemistry （IHC） and Western blot were applied to detect the expressions of RAE-1， NKG2D， and IFN-γ in tumour tissues of each group， and Western blot was used to detect the expressions of interleukin-6 （IL-6）， Janus kinase 2 （JAK2）， p-JAK2， signal transducer and activator of transcription 3 （STAT3）， and p-STAT3 in tumour tissues of each group， as well as the protein levels of NKG2D， and RAE-1 in spleen tissues of each group. ResultCompared with that in the model group， the tumour mass decreased in all dose groups of Xiangsha Liu Junzitang， with no statistically significant difference. The tumour volume was reduced （P<0.05， P <0.01）. The pathological morphology was improved. The mRNA contents of NKG2D， RAE-1 and IFN-γ were increased in the medium-dose group （P<0.05， P<0.01）， and the protein expressions of NKG2D， RAE-1， and IFN-γ in tumour tissues were elevated （P<0.05， P<0.01）， and p-JAK2 and p-STAT3 protein expressions were decreased （P<0.05， P<0.01）. In spleen tissues， the protein expressions of NKG2D and RAE-1 in all dose groups of Xiangsha Liu Junzitang were significantly elevated （P<0.01）. Compared with those in the cisplatin group， NKG2D， RAE-1 and IFN-γ mRNA contents were elevated in the middle-dose group of Xiangsha Liu Junzitang， and the difference was not statistically significant. IHC showed that the protein expressions of NKG2D and IFN-γ in the combined group were significantly elevated （P<0.01）， and Western blot results showed that the protein expressions of RAE-1， NKG2D and IFN-γ were elevated （P<0.05， P<0.01）. p-JAK2 and p-STAT3 protein expressions were decreased in the combined group （P<0.05， P<0.01）. NKG2D and RAE-1 protein expressions were significantly increased in spleen tissues of the medium-dose groups and the combined group （P<0.01）. ConclusionXiangsha Liu Junzitang combined with phlegm-removing and detoxifying traditional Chinese medicine can inhibit the growth of tumours in Lewis lung cancer mice by up-regulating the expressions of RAE-1/NKG2D， promoting the activation of NK cells， and inhibiting immune escape， the mechanism of which may be related to down-regulation of the JAK2/STAT3 pathway.

CRISPR-Associated Protein 9 ; Gene Editing ; CRISPR-Cas Systems/genetics*