Objective: To investigate the correlation between CLOCK and BMAL1 genes and MEN2 medullary thyroid carcinoma (MTC). Methods: Thirteen cases with MEN2 MTC and thirteen cases with non-MEN2 MTC were selected who were treated in the Yantai Yuhuangding Hospital between January 2013 and September 2021. Clinical indicators such as blood calcitonin level, tumor diameter and metastatic lymph node of patients were collected. The expression differences of CLOCK and BMAL1 between MEN2 MTC and para-carcinoma tissue as well as between MEN2 MTC and non-MEN2 MTC were detected by immunohistochemistry and qPCR. The correlation between lymph node metastasis and CLOCK or BMAL1 expression was analyzed. Protein-protein interaction (PPI) network analysis combined with qPCR and correlation analysis was used to explore the expression regulation relationship between RET and circadian clock genes. The rhythm disorder of MEN2 cells was verified by lipopolysaccharide cell stimulation experiment after dexamethasone rhythm synchronization. Results: MEN2 MTC exhibited typical RET gene mutation. The mean blood calcitonin level, the tumor diameter and the number of metastatic lymph nodes of patients with MEN2 MTC were higher than those of patients with non-MEN2 MTC (t value was 2.76, 2.53, 2.26, all P<0.05). Immunohistochemical results showed that the expression levels of CLOCK and BMAL1 in MEN2 MTC were higher than those in non-MEN2 MTC, while negatively expressed in para-cancerous thyroid follicle. qPCR displayed that the expression of CLOCK gene in cancer tissues was higher than that in non-MEN2 MTC and para-cancerous tissues (t value was 2.68 and 2.86, all P<0.05); the expression of BMAL1 gene in MEN2 MTC was higher than that in non-MEN2 MTC and para-cancerous tissues (t value was 2.21 and 2.35, all P<0.05). Correlation analysis showed that the expression levels of CLOCK and BMAL1 genes were positively correlated with the number of lymph node metastases in patients with MEN2 MTC (r=0.65, P<0.001; r=0.52, P=0.005). PPI network analysis indicated that the expression of CLOCK gene was positively correlated with the abnormal expression of RET gene (r=0.96, P<0.001). With lipopolysaccharide to stimulate cultured cells in vitro after dexamethasone rhythm synchronization, the expressions of CLOCK and BMAL1 in MEN2 MTC cells (0.47±0.22 and 2.60±1.48) at 12 hours of synchronization were significantly lower than those in para-cancerous tissues (1.70±1.62 and 8.23±2.52), the difference was statistically significant(t=5.04, P=0.007; t=3.34, P=0.029). Conclusion: CLOCK and BMAL1 are correlated with the occurrence and development of MEN2 MTC, and may be potential targets for the development of new therapeutic strategies for MEN2 MTC.
ARNTL Transcription Factors/genetics* ; CLOCK Proteins/genetics* ; Calcitonin ; Carcinoma, Neuroendocrine/genetics* ; Dexamethasone ; Humans ; Lipopolysaccharides ; Lymphatic Metastasis ; Multiple Endocrine Neoplasia Type 2a/genetics* ; Thyroid Neoplasms/surgery*

Coronavirus disease-2019 (COVID-19) is highly contagious. In the early stages of the disease, the symptoms of coldness, dampness and depression in the lungs often appear, including fever, fatigue, soreness, dry cough, poor appetite, and white greasy tongue coating. During the development of the disease, the damp toxin epidemic often enters the inner and generates heat, and the damp heat epidemic toxin blocks pleurodiaphragmatic sites, the triple energizer and lungs, with manifestations of chest tightness, shortness of breath, exacerbation after exercise, or high fever without bringing down, poor appetite, bitterness in mouth, fatigue, diarrhea, loose stools, and yellow and thick tongue fur. As the pathogen can go outward or enter more deeply inside to cause death at the moment, it is the crucial time to treat the disease. In this study, Haoqin Qingdantang was used to clear dampness and heat, reconcile Shaoyang channel and recover the triple energizer, detoxify the dampness fever epidemic toxin, and block the toxin inside, with a good efficacy. This prescription focuses on smoothing the Shaoyang gallbladder channel and the triple energizer, and regards the spleen and stomach as the acquired essence. In the prescription, Erchentang reconciles the spleen and stomach, elevates clear Qi and lower turbid Qi. Bupleuri Radix is added to increase its detoxification function, and Paeoniae Rubra Radix is added to circulate the blood and prevent pathogen from the blood. When the condition improves, Sweet Wormwood Herb and talc are often withdrawn, and Codonopsis is added to nourish the spleen and stomach, to strengthening vital qi to eliminate pathogenic factor. Based on the cases of COVID-19 treated in Wuhan Jinyintan Hospital, this paper summarized some experience of applying Haoqin Qingdantang.

OBJECTIVETo investigate the value of multiparameter flow cytometry (MFC) and flow cytometric scoring system (FCSS) in the diagnosis and prognostic evaluation of childhood myelodysplastic syndrome (MDS).

METHODSA retrospective analysis was performed for the clinical data of 42 children who were diagnosed with MDS. MFC was performed to investigate the phenotype and proportion of each lineage of bone marrow cells. The correlations of FCSS score with MDS type, International Prognostic Scoring System (IPSS) score, and revised IPSS (IPSS-R) score were analyzed.

RESULTSOf all the 42 children, 20 (48%) had an increase in abnormal marrow blasts, 19 (45%) had a lymphoid/myeloid ratio of >1, 14 (33%) had abnormal cross-lineage expression of lymphoid antigens in myeloid cells, 8 (19%) had abnormal CD13/CD16 differentiation antigens, 5 (12%) had abnormal expression of CD56, 3 (7%) had reduced or increased side scatter of granulocytes, 3 (7%) had reduced expression of CD36 in nucleated red blood cells, 2 (5%) had reduced expression of CD71 in nucleated red blood cells, 1 (2%) had absent expression of CD33 in myeloid cells, 1 (2%) had reduced or absent expression of CD11b in granulocytes, and 1 (2%) had absent expression of CD56 and CD14 in monocytes. There were significant differences in the median overall survival time and event-free survival time among the low-, medium-, and high-risk FCSS groups (P<0.05). Among the low-, medium-, and high-risk FCSS groups, the low-risk FCSS group had the highest 2-year overall survival rate, while there was no significant difference between the medium- and high-risk FCSS groups (P>0.05). The three groups had a 2-year event-free survival rate of 95%, 60%, and 46% respectively (P<0.05). FCSS score was positively correlated with MDS type, IPSS score, and IPSS-R score (P<0.05).

CONCLUSIONSMFC and FCSS help with the diagnosis and prognostic evaluation of childhood MDS.

OBJECTIVETo elucidate the mechanism of Chinese tuina in treating sciatic nerve crush injury, and to detect the levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1), which is thought to play an important role in nerve regeneration.

METHODSThirty-two adult male Sprague-Dawley rats were subjected to sciatic nerve crush injury and 16 rats (sham-operated group) went through a sham operation. Control group was given no treatment while tuina group received tuina therapy since day 7 post-surgery. Tuina treatment was performed once a day and lasted for 20 days. The sciatic functional index was examined every 5 days during the treatment session. The rats' gastrocnemius muscles were evaluated for changes in mass and immunohistochemistry techniques were performed to detect the levels of tPA and PAI-1.

RESULTSTuina therapy improved the motor function of sciatic nerve injured rats (P<0.05), however, it did not increase muscle volume (P<0.05). Tuina downregulated the levels of tPA and PAI-1 (P<0.05).

CONCLUSIONSThe present study implies that tuina treatment could accelerate rehabilitation of peripheral nerve injury.

OBJECTIVETo explore the protective effects of Tibetan medicine Zuo-Mu-A Decoction (, ZMAD) on the blood parameters and myocardium of high altitude polycythemia (HAPC) model rats.

METHODSForty male Wistar rats were randomly divided into 4 groups by a random number table, including the normal, model, Rhodiola rosea L. (RRL) and ZMAD groups (10 in each group). Every group was raised in Lhasa to create a HAPC model except the normal group. After modeling, rats in the RRL and the ZMAD groups were administered intragastrically with RRL (20 mL/kg) and ZMAD (7.5 mL/kg) once a day for 2 months, respectively; for the normal and the model groups, 5 mL of distilled water was administered intragastrically instead of decoction. Then routine blood and hematologic rheology parameters were taken, levels of erythropoietin and 8-hydroxy-2'-deoxyguanosine (8-OHdG) were tested, and ultrastructural change in the left ventricular myocardium was observed using transmission electron microscopy.

RESULTSCompared with the model group, ZMAD significantly reduced the red blood cell count, hemoglobin levels, whole blood viscosity at low/middle shear rates, plasma viscosity, erythrocyte electrophoretic time, erythropoietin and 8-OHdG levels, and also increased the erythrocyte deformation index (P<0.05). There was no difference in all results between the RRL and the ZMAD groups. The cardiac muscle fibers were well-protected, mitochondrial matrix swelled mildly and ultrastructure changes were less prominent in the ZMAD group compared with the model group.

CONCLUSIONZMAD has significant protective effects on the blood parameters against HAPC, and also has the beneficial effect in protecting against myocardial injury.

OBJECTIVETo evaluate the clinical characteristics and risk factors of clonal evolution after immunosuppressive therapy (IST) in children with severe/very severe aplastic anemia (SAA/VSAA).

METHODSThe clinical data of 231 children with newly-diagnosed SAA/VSAA who received IST were retrospectively studied. The incidence and risk factors of clonal evolution after IST were analyzed.

RESULTSThe 5-year overall survival rate of the 231 patients was 82.7%. Except for 18 cases of early deaths, 213 patients were evaluated for IST efficacy. Among the 231 patients, cytogenetic abnormalities for at least two chromosome metaphase were detectable in 14 (7.4%) patients, and PNH clones were detectable in either peripheral red blood cells or neutrophils for 95 patients. Among the 213 patients evaluated for IST efficacy, 15 patients experienced clonal evolution after IST. Five patients had PNH and trisomy 8 which were defined as favorable progressions, and ten patients experienced monosomy 7 and MDS/AML as unfavorable progressions. The 5-year accumulative incidence of favorable and unfavorable progression were (2.2±2.2)% and (4.8±3.3)%, respectively. Until the last follow-up, 100% (5/5) of patients with favorable progressions and 50% (5/10) of patients with unfavorable progressions survived. WBC>3.5×10/L, CD3T cell percentage>80%, dosage of antithymocyte globulin >3.0 mg/(kg·d) and no response to IST were related to unfavorable progressions by univariate analysis. Cox multivariate analysis revealed that an increased CD3T cell percentage (>80%) and no response to IST were independent risk factors for unfavorable progressions.

CONCLUSIONSThe children with SAA/VSAA who have an increased CD3T cell percentage at diagnosis or have no response to IST are in high risks of unfavorable progressions.

Adolescent ; Anemia, Aplastic ; drug therapy ; genetics ; immunology ; mortality ; Child ; Child, Preschool ; Chromosome Aberrations ; Clonal Evolution ; Female ; Humans ; Immunosuppressive Agents ; therapeutic use ; Infant ; Male ; Proportional Hazards Models ; Retrospective Studies

OBJECTIVETo observe the anti-virus effects of andrographolide (AD) on the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) signaling pathway when immunological cells were infected with H1N1.

METHODSLeukomonocyte was obtained from umbilical cord blood by Ficoll density gradient centrifugation, and immunological cells were harvested after cytokines stimulation. Virus infected cell model was established by H1N1 co-cultured with normal human bronchial epithelial cell line (16HBE). The optimal concentration of AD was defined by methyl-thiazolyl-tetrazolium (MTT) assay. After the virus infected cell model was established, AD was added into the medium as a treatment intervention. After 24-h co-culture, cell supernatant was collected for interferon gamma (IFN-γ) and interleukin-4 (IL-4) enzyme-linked immunosorbent assay (ELISA) detection while immunological cells for real-time polymerase chain reaction (RT-PCR).

RESULTSThe optimal concentration of AD for anti-virus effect was 250 μg/mL. IL-4 and IFN-γ in the supernatant and mRNA levels in RLRs pathway increased when cells was infected by virus, RIG-I, IFN-β promoter stimulator-1 (IPS-1), interferon regulatory factor (IRF)-7, IRF-3 and nuclear transcription factor κB (NF-κB) mRNA levels increased significantly (P<0.05). When AD was added into co-culture medium, the levels of IL-4 and IFN-γ were lower than those in the non-interference groups and the mRNA expression levels decreased, RIG-I, IPS-1, IRF-7, IRF-3 and NF-κB decreased significantly in each group with significant statistic differences (P<0.05).

CONCLUSIONSThe RLRs mediated viral recognition provided a potential molecular target for acute viral infections and andrographolide could ameliorate H1N1 virus-induced cell mortality. And the antiviral effects might be related to its inhibition of viral-induced activation of the RLRs signaling pathway.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Antiviral Agents ; pharmacology ; Cells, Cultured ; Coculture Techniques ; DEAD Box Protein 58 ; DEAD-box RNA Helicases ; genetics ; metabolism ; Dendritic Cells ; drug effects ; immunology ; virology ; Diterpenes ; pharmacology ; Fetal Blood ; cytology ; Humans ; Influenza A Virus, H1N1 Subtype ; drug effects ; immunology ; Influenza, Human ; drug therapy ; immunology ; virology ; Interferon-beta ; genetics ; metabolism ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Leukocytes, Mononuclear ; drug effects ; immunology ; virology ; Macrophages ; drug effects ; virology ; NF-kappa B ; genetics ; metabolism ; Promoter Regions, Genetic ; drug effects ; immunology ; RNA, Messenger ; metabolism ; Signal Transduction ; drug effects ; genetics ; immunology

OBJECTIVETo study the immunologic function of dendritic cells (DCs) cultured in two kinds of hepatoma cell line's supernatant and the enhancing effects of carboxymethylpachymaran (CMP) on DCs.

METHODSDCs were harvested after stimulation by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 from umbilical cord blood using density-gradient centrifugation method. Cultured supernatant of two hepatoma cell lines (HepG2 and HepG2.2.15) were collected for condition medium (CM) according to a volume ratio of supernatant to incomplete RPMI-1640 medium, which was 3:1. CMP was dissolved in incomplete RPMI-1640 medium. Experimental groups were divided according to the culture medium, either CM or with CMP in it. DCs subsets CD83, CD86, CD1a, and d-related human leukocyte antigens (HLA-DR) were analyzed by flow cytometry. The proliferation ability of allogeneic T cells in mixed lymphocyte reaction (MLR) stimulated by DCs was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. IL-12p70, interferon-γ (IFN-γ), and nuclear factor κB (NF-κB) were detected by enzyme-linked immunosorbent assay analysis.

RESULTSThe proliferation of lymphocytes and secreting level of IL-12 and expression of phenotype of DCs cultured in two kinds of CM were lower than those of normal group (P <0.01). Compared with the normal group, groups treated with CMP showed a higher expression level of DCs subsets, lymphocyte reproductive activity, as well as IL-12 and IFN-γ secretion levels. Groups treated with CMP also demonstrated higher levels of DCs phenotype expression and IL-12 and IFN-γ secretion in supernatant of MLR and higher lymphocyte reproductive activity compared with CM group (P <0.05). Compared with the normal group, the expression level of NF-κB in DCs nuclear was higher in CMP groups but lower in two CM groups (P <0.05). After CMP was added, the NF-κB expression levels of two CM groups were increased compared with levels before CMP was added (P <0.05). However, there was no significant difference between the two CM groups (P >0.05).

CONCLUSIONSTwo kinds of hepatoma cell line's supernatant can inhibit the immunologic function of DCs. This suppressive effect may be related to the inhibition of NF-κB/Rel pathway. CMP may up-regulate the DCs function by activating the NF-κB/Rel pathway.

Carcinoma, Hepatocellular ; pathology ; ultrastructure ; Cell Line, Tumor ; Cell Shape ; Dendritic Cells ; drug effects ; immunology ; Glucans ; pharmacology ; Humans ; Immunophenotyping ; Interferon-gamma ; metabolism ; Interleukin-12 ; metabolism ; Liver Neoplasms ; pathology ; ultrastructure ; Lymphocyte Culture Test, Mixed ; Signal Transduction ; drug effects ; Subcellular Fractions ; drug effects ; Transcription Factor RelA ; metabolism

Objective To describe the pathogenic and serological test results of the plague in China from 2001 to 2009, and human and animal plague distribution. Methods Through access to information of the plague surveillance report in China from 2002 to 2010, national plague pathogenic and serological test results and the epidemic situation were analyzed from 2001 to 2009. Results From 2001 to 2009, 2966 strains of Yersinia pestis were isolated in the seven provinces which were Guizhou, Guangxi, Yunnan, Qinghai, Tibet, Gansu and Inner Mongolia. Of these, 1 138 000 animals were detected by bacteriological method, 1998 strains of Yersinia pestis were isolated;379 227 groups of intermediary animals were detected, 927 strains of Yersinia pestis were isolated;41 strains of Yersinia pestis were isolated from human body. Animal serums of 1 169 702 were detected by indirect hemagglutination assay(IHA), of these 3177 animal serums were positive, 168 human serums were positive;53 323 animal samples were detected by reverse indirect hemagglutination assay(RIHA), of these 500 were positive. There were outbreak or epidemic of plague in 9 types of plague foci, 2925 strains of Yersinia pestis were isolated. Two animals and 6 fleas were judged as new reservoir and new vector. There were 23 counties of 6 provinces were judged as plague new natural foci counties. Conclusions The plague epidemic in China is still serious between 2001 and 2009. There are nine types of foci in the active state.

OBJECTIVETo evaluate the significance of a panel of immunohistochemical markers for distinguishing hepatocellular carcinoma (HCC) from intrahepatic cholangiocarcinoma (ICC).

METHODSTen markers including hepatocyte paraffin 1 (Hep Par 1), polyclonal carcinoembryonic antigen (pCEA), CD34, CD10, CD105, multidrug resistance-associated protein-3 (MRP-3), cyclooxygenase-2 (COX-2), mucinous glycoprotein-1 (MUC-1), aquaporin-1 (AQP-1) and CK19 were immunohistochemically stained in the samples from 90 surgically resected HCC and 80 ICC, respectively,and the positive rate of their expression were compared statistically.

RESULTSThe positive expression rates of Hep Par 1, pCEA, CD34, CD10, CD105, MRP-3, COX-2 were 85.6%, 82.2%, 87.8%, 18.9%, 8.9%, 11.1% and 48.9%, respectively, in HCC. While the positive expression rates of MUC-1, AQP-1 and CK19 were 73.8%, 65% and 92.5%, respectively, in ICC.

CONCLUSIONBased on our results, Hep Par 1 and CD34 can be used as the first line markers, and pCEA and COX-2 as the second line makers, for differential diagnosis of hepatocellular carcinoma from intrahepatic cholangiocarcinoma. While MUC-1 and CK19 can be used as the first line markers and AQP-1 as the second one for the differential diagnosis of intrahepatic cholangiocarcinoma from hepatocellular carcinoma.

Bile Duct Neoplasms ; chemistry ; diagnosis ; Bile Ducts, Intrahepatic ; chemistry ; Biomarkers, Tumor ; analysis ; Carcinoma, Hepatocellular ; chemistry ; diagnosis ; Cholangiocarcinoma ; chemistry ; diagnosis ; Diagnosis, Differential ; Female ; Hepatocytes ; chemistry ; pathology ; Humans ; Immunohistochemistry ; Liver Neoplasms ; chemistry ; diagnosis ; Male ; Middle Aged