Objective To describe the clinical features of invasive fungal disease in Huashan Hospital,Fudan University from January 2004 to December 2006.Methods The medical data were reviewed retrospectively for the patients with fungal infection, which was confirmed by positive fungal culture or microscopic examination with blood,sterile body fluid,deep tissue,sputum specimen or isolation of Aspergillus spp.and Cryptococcus spp.from bronchoalveolar lavage.The proven and probable cases of invasive fungal disease were included in this analysis.Results A total of 111 patients were diagnosed as invasive fungal dis-ease,including 104 proven cases and 7 probable cases.Sixty-one cases were community-acquired and the other 50 were nosoco-mial.The most common site of infection was bloodstream (51,45.9%),followed by central nervous system (44,39.6%)and respiratory system (14,12.6%).The most common pathogens were Candida spp.(50,45%),Cryptococcus (47,42.3%) and Aspergillus spp. (12, 10.8%). The community-acquired fungal infections were mostly found in central nervous system (44,72.1%),and respiratory system (12, 19.7%),mainly caused by Cryptococcus and Aspergillus. The nosocomial fungal infections occurred primarily in blood-stream (96.0%),mainly due to Candida spp.No underlying disease or risk factor was identified in more than half of the pa-tients with community-acquired infection,while almost all the patients with nosocomial fungal infection had underlying disease and predisposing factors.Indwelling venous catheter was closely associated with nosocomial bloodstream infection.Indwelling venous catheter lasted for more than 1 week in 64.7% of the patients with Candida bloodstream infection.The same fungal strain was isolated from both the cather and blood of the same patient in 11 cases.The overall mortality of these invasive fungal diseases was 14.4% (16/111).The mortality rate was 18.0% (9/50)in the patients with nosocomial invasive fungal infection, and 11.5% (7/61)in the patients with community-acquired invasive fungal infection.Conclusions The most common site of in-vasive fungal infection is bloodstream,followed by central nervous system,and respiratory system.Majority of the fungal patho-gens are Candida spp.,Cryptococcus and Aspergillus spp.The community-acquired invasive fungal disease is primarily meningitis caused by Cryptococcus.The nosocomial invasive fungal disease is mainly bloodstream infection caused by Candida spp.

Objective To investigate the biological effect of anti-leukemic cells induced by eosinophilic granulocyte (EOS) in bone marrow of patients with chronic myelogenous leukemia (CML).Methods The BCR-ABL fusion gene as well as the expression of IL-12 and IL-17 mRNA were performed by RT-PCR.The serum concentrations of cytokine IL-12 and IL-17 were determined by enzyme-linked immuno sorbent assay (ELISA).Immunochemistry staining and cytochemistry staining were used to observe the peroxydase (POX) and human Leukocyte antigen (HLA)-DR expression of EOS in bone marrow.Immunofluorescence staining was used to observe mannose receptor (MR),IL-12,IL-17A and IL-17receptor A (IL-17RA) expression of EOS.The results between the CML patients and the healthy controls were compared.Results Serum levels of IL-12 and IL-17 were higher in the 60 CML patients [(196.33 ±21.79) ng/L and (36.55-±3.01) ng/L] than those in the controls [(96.60 ±4.92) ng/L and (23.74 ±1.36) ng/L].In the 32 patients with activated EOS,the levels of IL-12 and IL-17 were (273.12 ± 17.16)ng/L and (40.11 ± 6.13) ng/L,which were significantly higher than those in the non-activated EOS [(126.16 ± 14.27) ng/L and (28.14 ±5.29) ng/L] (P values ＜0.01).IL-12 and IL-17 mRNA were expressed in activated EOS,while BCR-ABL fusion gene was not found.The amounts of EOS were increased abnormally in the bone marrow and peripheral blood of the CML patients with POX positive staining in the cytoplasm and weakly positive HLA-DR staining.It was observed easily by a microscope that EOS could attack leukemic cells in bone marrow through adhesion,capture and phagocytosis.Activated EOS could express IL-12,IL-17A and MR,which was related with the serum levels of these cytokines.Conclusions Activated EOS in bone marrow of CML patients could express IL-12 and IL-17.Activated EOS could induce coup injury to leukemic cell by releasing POX and expressing IL-12 and IL-17.It can also capture or swallow target cells via the expression of MR on the membrane.EOS may play an important role in the anti-tumor immunologic function in bone marrow of CML patients.

Objective To investigate the polymorphism profile of cytochrome P_(450)2C19 (CYP2C19) in Chinese patients with invasive fungal infections. Methods Two major single nucleotide polymorphism loci of the CYP2C19 gene (CYP2C19 * 2 and CYP2C19 * 3) were genotyped with PCR and restriction fragment length polymorphism (PCR-RFLP) in 134 patients with invasive fungal infections and 134 healthy volunteers. Allele frequencies and the proportions of metabolizer phenotypes were compared. Results In patients with invasive fungal infections, CYP2C19 * 1, CYP2C19 * 2 and CYP2C19 * 3 alleles showed frequencies of 58.2%, 36.6% and 5.2%. In healthy volunteers, the frequencies of CYP2C19 * 1, CYP2C19 * 2 and CYP2C19 * 3 were 63.4% , 34. 3% and 2. 2%. There was no significant difference in allele frequencies between the two groups. Of the patients with invasive fungal infections, 33. 6% were homozygous extensive metabolizers, 50.0% heterozygous extensive metabolizers and 16.4% poor metabolizers. Of the healthy volunteers, 40.3% were homozygous extensive metabolizers, 48.5% heterozygous extensive metabolizers and 11. 2% poor metabolizers. The proportions of metabolizer phenotypes were similar between the two groups. Conclusions Significant CYP2C19 polymorphism was detected in both groups. Approximately two thirds of the Chinese patients were either heterozygous extensive metabolizers or poor metabolizers. The genetic polymorphism may have important effect on drug metabolism in these patients

Objective To analyze the clinical features of patients with uncommon fungal infections in central nervous system (CNS).Methods Thirty-five patients with uncommon CNS fungal infections who were admitted to Huashan Hospital from 1997 to 2010 were retrospectively reviewed.The pathogens,symptoms and signs.treatments of patients were evaluated.The data were analyzed by rank sum test and Fisher'S exact test.Results Twenty-nine of the 35 patients met the definition criteria of prover CNS fungal infections,while the other 6 had probable diagnosis.Predisposing factors were found in 86% of all patients.The most common pathogens were Aspergillus and Candida species.The symptoms and signs commonly occurred including fever(22 cases),headache(19 cases), cranial neuropathy(12 cases),and meningeal irritation sign(12 cases).High white blood cell count,high protein level,and low glucose level were the main findings of cerebrospinal fluid (CSF) analysis.Patients with cerebral aspergillosis were more frequently accompanied with immunocompromised conditions, and they often got CNS aspergillosis from hematogenous dissemination or direct extension of paranasal sinus infection.Cerebral granuloma and abscess were the common clinical characteristics of CNS aspergillosis.Cerebral candidiasis often arose from neurosurgical surgery or traumatic brain injury,and these patients were usually presented with meningitis.All patients were treated with antifungal drugs and (or) surgical intervention and 77%(27/35) of the patients achieved complete or partial responses. Antifungal agents combined with surgical resection might improve outcome of patients with CNS aspergillosis; while removal or replacement of drainage tubes in combination with antifungal treatment showed satisfactory efficacy in patients with cerebral candidiasis who usually had shunt manipulation. Conclusions The incidence of CNS fungal infection, such as cerebral aspergillosis and candidiasis, is increasing. Early diagnose and therapeutic intervention are crucial for improving outcome.

Objective To study the clinical features and antifungal therapeutic effects in nonacquired immune deficiency syndrome(AIDS)patients with cryptococcal meningitis. Methods One hundred and fifty-four non-AIDS patients with cryptococcal meningitis admitted to Huashan Hospital, Fudan University from 1997 to 2007 were reviewed retrospectively. Clinical characteristics, initial antifungal therapies and outcome of these patients were analyzed. Continuous variables were analyzed using t test and categorical variables were compared by X~2 test or Fisher's exact test. Kaplan-Meier survival curves of different therapies were compared with log-rank test. Results Fifty-one patients (33.12%)had one or more predisposing factors. Headache, fever, meningeal irritation, vomiting and altered mental status were common clinical symptoms and signs during the course of diseases. The positive rates of cerebrospinal fluid(CSF)smear, CSF culture and detection of CSF cryptococcal capsular polysaccharide antigen were 88.44%,78.95%and 100.00%,respectively.Twelve cases were excluded because treatment durations were less than 7 days, including 9 died,2 discharged against medical advice due to illness exacerbation and 1 lost after against medical advice discharge. The remaining 142 patients were evaluated for therapeutic effects. The effective rates in amphotericin B (AmB)group, fluconazole group and AmB plus fluconazole group were 78.3%(36/46),33.3%(8/24)and 76.0%(38/50),respectively. The therapeutic effects in AmB group and AmB plus fluconazole group were superior to fluconazole group(X~2=13.6354,12.5509;P＜0.01).Eleven patients were lost during 1-year follow-up. The attributable and overall mortality in the remaining 143 patients were 19.58% and 28.67%,respectively.The 1-year survival rates in AmB group and AmB plus fluconazole group were significantly higher than that in fluconazole group. Conclusions The mortality of non-AIDS cryptococcal meningitis is still high,which is closely correlated with initial antifungal therapies. AmB alone or combined with flucytosine is related to both higher successful response and higher survival rate, while the efficacy of initial fluconazole alone or combined with flucytosine is poor.

Objective:To study the effect of immunological molecules expressive state upon the telomerase activation ( TA) of bone marrow mononuclear cells ( BMMNC ) in the hemopoietic microenvironment of patients with immune related hematocytopenia ( IRHS) ,and to explore the immunologic mechanism as well as the clinical significance of hematoclasis in marrow of IRHS patients .Methods:①TRAP-PCR-ELISA method was performed to detect the TA of BMMNC in marrow of 366 IRHS patients before and after therapy.②The molecules HLA-DR,anti-hunman IgG,FcγⅡR,mannose receptor ( MR),IL-17A and its receptor ( IL-17AR) were analysed by immunochemistry and immunofluorescence staining .③The flow cytometric ( FCM) was used to analyse the proportion of CD3+CD4+T cells as well as CD3+CD8+T cells ,CD3-CD19+B cells and CD3-CD16/56+NK cells in peripheral blood lymphocyte.60 cases of health examination were selected as the control group , and 30 cases hypoferric anemia patients were selected as disease control.The differences between patient group and control group were analysed with statistic method .Immunochemistry and immunofluo-rescence staining were performed to in situ analyze the activation-characteristics of immunocyte in bone marrow slides of IRHS ,and the dependablity of cellular immunologic injury was also checked.Results: ①The levels of TA was 0.261 7 ±0.021 6 before treatment , higher than the disease control group (0.061 6±0.031 3 ,P<0.01).Among of them HLA-B27+patients were higher than HLA-B27-patients (0.301 3±0.020 6 vs.0.192 3±0.012 9,P<0.05).Serious IRP patients with HLA-B27+IgG+were obviously higher than HLA-B27-IgG+patients (0.401 6±0.017 2 vs.0.221 1±0.011 0,P<0.01).②In marrow of HLA-B27+IgG+patients,both cell immunity and humoral immunity were in disorder in the hemopoietic microenvironment ,and immonocyte in marrow expressed HLA-DR, FcγⅡR,IL-17A,IL-17RA and MR,and Th,Ts,B cell and NK cell in peripheral blood increased in different degree ,inducing the in-flammation of haemocyte and lead to destruction.③Humoral immunity was in the dominant level in morrow;humoral immunity of HLA-B27-IgG+patients,immonocyte expressed FcγⅡR in high level,but IL-17A was seldom expressed,only CD19+B cell was increased slightly ,the antibody dependent cellular cytotoxicity ( ADCC) was the main mode of destruction.After therapy glucocorticoids associated with ciclosporin A ,the TA level of BMMNC decreased to 0 with devitalization.Conclusion: The telomerase activation of bone marrow mononuclear cells in IRHS is related with the immune state of hemopoietic microenvironment and the pathologic lesion degree of hema -topoietic cell in marrow.It is viral infection and immunological activation as well as a variety of inflammatory factors play a part in the immunologic injury that might be an important factor of the enhancement in TA.

Objective To describe the distribution of mannose binding lectin (MBL) genetic polymorphisms in non-acquired immunodeficiency syndrome (AIDS) patients with cryptococcosis in China and to verify the association of MBL polymorphisms with susceptibility to cryptococcosis.Methods The case-controlled genetic association study was conducted and 167 non-AIDS patients with cryptococcosis and 208 healthy controls were recruited. Genome DNA was extracted from the peripheral blood and MBL gene was amplified by polymerase chain reaction (PCR). Six singlenucleotide polymorphisms ( SNP) of MBL gene were sequenced. The association of MBL polymorphisms with susceptibility to cryptococcosis were analyzed. The comparison between patients and controls was performed by chi square test or Fisher's exact test. The differences of MBL plasma concentrations between groups with different MBL genotypes were compared by single factor variance analysis. Results There were no differences between patients and controls in terms of MBL genotype frequencies, haplotypes and genotypes (all P>0. 05). Compared with healthy control, the deficient MBL-producing genotypes were strongly associated with cryptococcal meningitis (16. 5% vs 8. 7%,χ2=4.25, P=0.0392, OR = 2.09), particularly in patients without underlying immunocompromised conditions (21. 4% vs 8. 7%, χ2 =7. 15, P = 0. 0075, OR = 2. 88). Individuals with MBL deficiency genotypes showed significantly higher rates of central nervous system (CNS) cryptococcal infection rather than non-CNS cryptococcosis (16. 5% vs 3. 1%, Fisher's exact test, P = 0. 010, OR = 6. 13).The difference was even more significant in the immunocompetent patients (21. 4% vs 4. 0%, P =0.009, OR= 6. 55). Conclusion MBL deficiency is associated with cryptococcal meningitis and may play a role in CNS Cryptococcus infection.

Arsenic compounds are widely used for the therapeutic intervention of multiple diseases.Ancient pharmacologists discovered the medicinal utility of these highly toxic substances,and modern phar-macologists have further recognized the specific active ingredients in human diseases.In particular,Arsenic trioxide(ATO),as a main component,has therapeutic effects on various tumors(including leukemia,hepatocellular carcinoma,lung cancer,etc.).However,its toxicity limits its efficacy,and con-trolling the toxicity has been an important issue.Interestingly,recent evidence has pointed out the pivotal roles of arsenic compounds in phase separation and membraneless organelles formation,which may determine their toxicity and therapeutic efficacy.Here,we summarize the arsenic compounds-regulating phase separation and membraneless organelles formation.We further hypothesize their potential involvement in the therapy and toxicity of arsenic compounds,highlighting potential mecha-nisms underlying the clinical application of arsenic compounds.

OBJECTIVETo investigate the effect of amphotericinB (AmB) on migration and invasion of esophageal carcinoma Eca109 cells exposed to hypoxia and explore the molecular mechanisms.

METHODSRoutinely cultured esophageal carcinoma Eca109 cells were treated with 0, 1.25, 2.5, or 5 µg/ml AmB in hypoxic condition (3% O2, 5% CO2, and 92% N2) for 24 h. The cell migration and invasion were assessed by cell scratch test and Transwell chamber assay, respectively. Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expressions of hypoxia-inducible factor-1α (HIF-1α), matrix metalloproteinase-2 (MMP-2), and E-cadherin in the cells, respectively.

RESULTSCompared with the control cells, the cells treated with different doses of AmB showed attenuated ability of migration and invasion (P<0.05). AmB treatment resulted in significantly lowered mRNA and protein expressions of MMP-2 (P<0.05) and increased expressions of E-cadherin (P<0.05); the protein expression of HIF-1α decreased significantly in cells after AmB treatment (P<0.05) but its mRNA levels showed no significant changes (P>0.05).

CONCLUSIONAmB can suppress the migration and invasion of esophageal carcinoma Eca109 cells in hypoxic microenvironment possibly by regulating the expressions of HIF-1α, MMP-2 and E-cadherin.

Amphotericin B ; pharmacology ; Cadherins ; metabolism ; Cell Hypoxia ; Cell Line, Tumor ; drug effects ; Cell Movement ; drug effects ; Down-Regulation ; Esophageal Neoplasms ; metabolism ; pathology ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; RNA, Messenger

Objective To investigate the effect of amphotericinB (AmB) on migration and invasion of esophageal carcinoma Eca109 cells exposed to hypoxia and explore the molecular mechanisms. Methods Routinely cultured esophageal carcinoma Eca109 cells were treated with 0, 1.25, 2.5, or 5μg/ml AmB in hypoxic condition (3%O2, 5%CO2, and 92%N2) for 24 h. The cell migration and invasion were assessed by cell scratch test and Transwell chamber assay, respectively. Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expressions of hypoxia-inducible factor-1α (HIF-1α), matrix metalloproteinase-2 (MMP-2), and E-cadherin in the cells, respectively. Results Compared with the control cells, the cells treated with different doses of AmB showed attenuated ability of migration and invasion (P<0.05). AmB treatment resulted in significantly lowered mRNA and protein expressions of MMP-2 (P<0.05) and increased expressions of E-cadherin (P<0.05); the protein expression of HIF-1α decreased significantly in cells after AmB treatment (P<0.05) but its mRNA levels showed no significant changes (P>0.05). Conclusion AmB can suppress the migration and invasion of esophageal carcinoma Eca109 cells in hypoxic microenvironment possibly by regulating the expressions of HIF-1α, MMP-2 and E-cadherin.