A Potential Therapy Using Engineered Stem Cells Prevented Malignant Melanoma in Cellular and Xenograft Mouse Models / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: In the present study, human neural stem cells (hNSCs) with tumor-tropic behavior were used as drug delivery vehicle to selectively target melanoma. A hNSC line (HB1.F3) was transduced into two types: one expressed only the cytosine deaminase (CD) gene (HB1.F3. CD) and the other expressed both CD and human interferon-β (IFN-β) genes (HB1.F3.CD. IFN-β). MATERIALS AND METHODS: This study verified the tumor-tropic migratory competence of engineered hNSCs on melanoma (A375SM) using a modified Boyden chamber assay in vitro and CM-DiI staining in vivo. The antitumor effect of HB1.F3.CD and HB1.F3.CD.IFN-β on melanoma was also confirmed using an MTT assay in vitro and xenograft mouse models. RESULTS: A secreted form of IFN-β from the HB1.F3.CD.IFN-β cells modified the epithelial-mesenchymal transition (EMT) process and metastasis of melanoma. 5-Fluorouracil treatment also accelerated the expression of the pro-apoptotic protein BAX and decelerated the expression of the anti-apoptotic protein Bcl-xL on melanoma cell line. CONCLUSION: Our results illustrate that engineered hNSCs prevented malignant melanoma cells from proliferating in the presence of the prodrug, and the form that secreted IFN-β intervened in the EMT process and melanoma metastasis. Hence, neural stem cell-directed enzyme/prodrug therapy is a plausible treatment for malignant melanoma.

Distribution of clinical isolates of Candida spp. and antifungal susceptibility of high biofilm-forming Candida isolates

Abstract INTRODUCTION: The increase in the incidence of fungal infections, especially those caused by Candida albicans and other Candida species, necessitates the understanding and treatment of Candida-associated infections. In this study, we aimed to investigate the identification, distribution, and biofilm formation ability of different clinical Candida isolates and evaluate the distribution and antifungal susceptibilities of high biofilm-forming (HBF) Candida isolates. METHODS: For identification, carbohydrate fermentation, carbohydrate assimilation, and ChromAgar tests were used. Biofilm formation was assessed using crystal violet binding assay, while the susceptibility to antifungal agents was determined using ATBTM Fungus 3 test kits. RESULTS: The majority of Candida species were C. parapsilosis (31.3%; 31/99) and C. tropicalis (30.3%; 30/99). C. tropicalis was found to be the most frequently isolated species among all HBF Candida species. HBF Candida isolates were more frequently isolated from vaginal swab (35.7%; 10/28), tracheal aspirate (17.9%; 5/28), and urine (17.9%; 5/28). The majority of tested isolates were resistant to itraconazole and voriconazole, whereas no isolate was deemed resistant to 5-flucytosine. CONCLUSIONS: C. tropicalis displays the highest biofilm formation ability among all the Candida species evaluated, and HBF Candida isolates were more frequently seen in vaginal swab, tracheal aspirate, and urine samples. Our findings revealed that 5-flucytosine is the most efficient antifungal agent against HBF Candida isolates.

Anti-proliferative Effect of Engineered Neural Stem Cells Expressing Cytosine Deaminase and Interferon-β against Lymph Node–Derived Metastatic Colorectal Adenocarcinoma in Cellular and Xenograft Mouse Models / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Genetically engineered stem cells may be advantageous for gene therapy against various human cancers due to their inherent tumor-tropic properties. In this study, genetically engineered human neural stem cells (HB1.F3) expressing Escherichia coli cytosine deaminase (CD) (HB1.F3.CD) and human interferon-β (IFN-β) (HB1.F3.CD.IFN-β) were employed against lymph node–derived metastatic colorectal adenocarcinoma. MATERIALS AND METHODS: CD can convert a prodrug, 5-fluorocytosine (5-FC), to active 5-fluorouracil, which inhibits tumor growth through the inhibition of DNA synthesis,while IFN-β also strongly inhibits tumor growth by inducing the apoptotic process. In reverse transcription polymerase chain reaction analysis, we confirmed that HB1.F3.CD cells expressed the CD gene and HB1.F3.CD.IFN-β cells expressed both CD and IFN-β genes. RESULTS: In results of a modified trans-well migration assay, HB1.F3.CD and HB1.F3.CD.IFN-β cells selectively migrated toward SW-620, human lymph node–derived metastatic colorectal adenocarcinoma cells. The viability of SW-620 cells was significantly reduced when co-cultured with HB1.F3.CD or HB1.F3.CD.IFN-β cells in the presence of 5-FC. In addition, it was found that the tumor-tropic properties of these engineered human neural stem cells (hNSCs) were attributed to chemoattractant molecules including stromal cell-derived factor 1, c-Kit, urokinase receptor, urokinase-type plasminogen activator, and C-C chemokine receptor type 2 secreted by SW-620 cells. In a xenograft mouse model, treatment with hNSC resulted in significantly inhibited growth of the tumor mass without virulent effects on the animals. CONCLUSION: The current results indicate that engineered hNSCs and a prodrug treatment inhibited the growth of SW-620 cells. Therefore, hNSC therapy may be a clinically effective tool for the treatment of lymph node metastatic colorectal cancer.

Effects of neural stem cells and 5-fluorocytosine in canine metastatic lung tumor

This is the first case report to describe the tumor regressive effect of systemic human neural stem cell (NSC)/5-fluorocytosine (5-FC) therapy on canine metastatic lung tumor. The therapeutic effects appeared approximately two weeks after 5-FC administration. Thoracic radiographs revealed a reduced number of lung nodules and decreased nodule size. However, there were no significant antitumor effects on primary lesions in abdominal organs. In conclusion, human NSC/5-FC prodrug therapy can secure patient quality of life with the same or more therapeutic effects and fewer side effects than other recommended chemotherapies.

Meta-analysis of flucytosine or fluconazole plus amphotericin B for the treatment of acquired immunodeficiency syndrome-associated cryptococcal meningitis / 中华传染病杂志

Objective To evaluate the survival benefit of amphotericin B (AmB) plus flucytosine or fluconazole for treatment of patients with acquired immunodeficiency syndrome (AIDS)-associated cryptococcal meningitis.Methods The following database were searched from the beginning to October 2013,including Cochrane library,PubMed,OVID,Embase,Wanfang Date,CNKI and Chinese Biomedical Database,and the references of eligible studies were manually screened.Reference lists of relevant articles were screened according to selection and extraction criteria.Meta-analysis was performed using RevMan 5.2.Results Four prospective controlled studies with a total of 399 patients with cryptococcal meningitis were identified,including 386 patients with AIDS-associated cryptococcal meningitis and 13 human immunodeficiency virus (HIV)-negative patients.Two hundred and twentyseven patients were treated with AmB and flucytosine combination therapy,including 217 patients with AIDS-associated cryptococcal meningitis and 10 HIV-negative patients.One hundred and seventy-two patients were treated with AmB and fluconazole combination therapy,including 169 patients with AIDS-associated cryptococcal meningitis and 3 HIV-negative patients.The Meta-analysis revealed that the mortality rate in AmB plus flucytosine combination therapy group was 6.6％ (95％ CI:18.5％-31.6 ％) at two weeks point,which was significantly lower than that in AmB plus fluconazole combination group (19.7％,95％CI:-23.6％-62.9％; OR=0.51,95％CI:0.27-0.93,P＜0.05).But at 10 weeks point,the mortality rate in flucytosine combination group was 12.9％ (95％oo CI:-22.2％-48.0％),which was lower than that in fluconazole combination group (31.4％,95％ CI:-23.1％-85.9 ％).However,there was no statistically significant difference between these two groups at 10 weeks point (OR=0.70,95％CI:0.44-1.13,P=0.15).Conclusion Administration of AmB plus flucytosine at early stage can reduce the mortality rate in patients with AIDS-associated cryptococcal meningitis.

Correlation analysis of lower respiratory tract fungal infection in hospital of Zhoushan archipelago area / 中华传染病杂志

Objective To study the microbial strains,risk factors and resistance profiles of lower respiratory tract fungal infection in hospital of Zhoushan archipelago area.Methods A total of 204 patients who were hospitalized for lower respiratory tract infection were retrospectively analyzed from May 2008 to April 2011 in Zhoushan archipelago area,and collected 204 fungal strains isolated from confirmed lower respiratory tract fungal infection cases.Chi-square test and Logistic regression analysis were performed.Results Among the 204 fungal strains isolated from lower respiratory tract specimens,110 (53.8％) strains of Candidaalbicans,32 (15.7％) strains of Candida tropicalis,24 (11.8％) strains of Candida glabrata,12 (5.9％) strains of Candida krusei,14 (6.9％) strains of other Candida,and 12 (5.9％) strains of Aspergillus were detected.Logistic regression analysis showed that chronic obstructive pulmonary disease,bacterial pneumonia,long-term use of broadspectrum antibiotics and corticosteroids,endotracheal intubation or incision,old age,exposure in intensive care unit (ICU),and hospitalization ≥7 days were major risk factors (P=0.000,0.001,0.000,0.000,0.012,0.000,0.000,0.000).The resistance rates of isolated Candida against amphotericin B,5-flucytosine,voriconazole,itraconazole and fluconazole were 0,2.1％,4.2％,14.8％ and 22.9％,respectively.Conclusions Candida albicans is the major pathogen of lower respiratory tract fungal infection in hospital of Zhoushan archipelago area,and Candida is sensitive to amphotericin B,5-flucytosine and voriconazole.

Antifungal Susceptibility to Amphotericin B, Fluconazole, Voriconazole, and Flucytosine in Candida Bloodstream Isolates from 15 Tertiary Hospitals in Korea

The in vitro antifungal susceptibility of 636 Candida bloodstream isolates collected from 15 tertiary hospitals in Korea was determined using the Vitek-2 yeast susceptibility system (bioMerieux, France). Overall susceptibility rates were 98.1%, 95.9%, 99.1%, and 97.3% for amphotericin B, fluconazole, voriconazole, and flucytosine, respectively. The results show that the rates of resistance to 4 antifungal drugs remain low among Candida bloodstream isolates in Korea.

The 10-year clinical analysis of cryptococcal meningitis treated with fluconazole and flucytosine / 中华传染病杂志

ObjectiveTo investigate the therapeutic effects of patients with non-acquired immunodeficiency syndrome ( AIDS )-related cryptococcal meningitis treated with fluconazole and flucytosine.MethodsA retrospective study was conducted including 52 cases of non-AIDS-related cryptococcal meningitis,which were divided into two groups:the amphotericin B combined with flucytosine group (n =32) and the fluconazole combined with flucytosine group (n =20 ). The comparison between groups was done by t test and chi-square test.ResultsThe intracranial pressure of the amphotericin B combined with flucytosine group and the fluconazole combined with flucytosine group were (221.9±76.2) and (213.4±99.2) mm H2O,respectively (t=0.302,P＞0.05) ; the cryptococcus counts in the cerebrospinal fluid (CSF) were (351 ± 1180) and (508 ± 943)/mL,respectively (t=- 0.473,P＞0.05) ; the CSF protein levels were (0.754 ± 0.726) and (0.649 ±0.308) g/L,respectively (t=0.578,P＞0.05) ; the CSF white blood cell (WBC) counts were (16±25) × 106/L and (28±32) × 106/L,respectively (t=-1.348,P＞0.05).The cured rates were 59.38％ (19/32) and 35.00％ (7/20),respectively in the amphotericin B combined with flucytosine group and the fluconazole combined with flucytosine group,and the improved rates were 6.25％(2/32)and 25.00％ (5/20),respectively,the uncured or mortality rates were 34.38％ (11/32) and 40.00％ (8/20),respectively,and the difference were not significant of cured rates (x2 =2.925,P＞0.05) and uncured or mortality rates (x2 =0.168,P＞0.05) between two groups.ConclusionFluconazole combined with flucytosine also has a good effect on the treatment of cryptococcal meningitis.

Treatment of severe chromoblastomycosis with itraconazole and 5-flucytosine association / Tratamento de cromoblastomicose severa com a associação itraconazole e 5-flucitosina

Chromoblastomycosis is a chronic human melanized fungi infection of the subcutaneous tissue caused by traumatic inoculation of a specific group of dematiaceous fungi through the skin, often found in barefooted agricultural workers, in tropical and subtropical climate countries. We report the case of a male patient presenting a slow-growing pruriginous lesion on the limbs for 20 years, mistreated over that time, which was diagnosed and successfully treated as chromoblastomycosis. Besides the prevalence of this disease, treatment is still a clinical challenge.

Cromoblastomicose é uma infecção fúngica crônica do tecido subcutâneo causada pela inoculação traumática de um grupo específico de fungos através da pele, encontrados eventualmente em trabalhadores do campo descalços em países de clima tropical e subtropical. Relatamos aqui o caso de um paciente do sexo masculino com uma lesão dermatológica de crescimento lento e pruriginosa nos membros inferiores por 20 anos, diagnosticada e tratada com sucesso para cromoblastomicose. Apesar da prevalência desta doença em nossa região, o tratamento ainda é um desafio.

Clinical features and antifungal therapeutic effects of 154 patients with cryptococcal meningitis / 中华传染病杂志

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.

In vitro antifungal susceptibility of 80 Cryptococcus neoformans isolates / 中华传染病杂志

Objective To determine in vitro drug susceptibility to five antifungal agents of clinical Cryptococcus neoformans strains isolated from different areas of China in recent ten years. Methods Eighty clinical isolates of Cryptococcus neoformans were isolated from Shanghai, Guangdong, Fujian, Beijing and some other areas of China from 1998 to 2007. The minimal inhibitory concentrations (MIC) of the isolates to five antifungal agents, including amphotericin B, fluconazole, flucytosine, itraconazole and voriconazole, were determined using broth microdilution procedure (document M27-A2) recommended by the Clinical and Laboratory Standards Institute (CLSI). Kruskal-Wallis rank sum test was employed for the statistical analysis. Results The MIC50 of the Cryptococcus neoforrnans isolates tested for amphotericin B, fluconazole, flucytosine, itraconazole and voriconazole were 0.5, 4, 2, 0.25 and ≤0.031 3 mg/L, respectively; and the MIC<,90> of the isolates tested for the above antifungal agents were 1, 8, 4, 0.5 and 0.062 5 mg/L, respectively. Among the tested isolates, 3 (3.8 %) were resistant to flucytosine, 4 (5.0 %) were resistant to itraconazole. All isolates were susceptible to amphotericin B and voriconazole. There was no significant difference in MIC of the strains isolated from any particular years to the five agents (χ2=0.500,2.687,2.211, 2.660,0.677,P>0.05). Conclusions The Cryptococcusneoformans isolates are highly susceptible to the five antifungal agents, while a few strains are resistant to flucytosine or itraconazole. The drug susceptibilities of the strains isolated from particular years are similar.

Analysis of 24 cases of cryptococcal meningitis treated with fluconazole / 中华传染病杂志

Objective To evaluate clinical features,therapeutic effects and outcomes of patients with non-human immunodeficiency virus(HIV)-infected cryptococcal meningitis treated with fluconazole or fluconazole and flucytosine.Methods Twenty-four cases of non-HIV-infected cryptococcal meningitis(fluconazole with or without flucytosine as initial therapy)in Huashan Hospital,Fudan University from 1997 to 2007 were retrospectively reviewed.Clinical manifestations,therapeutic effects and outcomes of the patients were collected.Results Fluconazole was administered with median dosage of 400 mg/d,for a median duration of 20.5 days.After fluconazole initial therapy for 2 weeks,16.7% showed partial response,83.3% showed no response,and the overall response rate was 16.7%.After 10 weeks,33.3% showed partial response,29.2% showed complete response,16.7% showed no response,and the overall response rate was 62.5%.Mortality at week 10 was 20.8%.Twenty-two patients who failed to respond to initial therapy were switched to other antifungal drugs(amphotericin B,amphotericin B colloidal dispersion,itraconazole)or other fluconazole containing combined therapy.Eleven out of the 24 patients died during one-year follow-up,8 of whom died of eryptococcal meningitis,and 3 died of other diseases.Conclusions The initial therapy of fluconazole with or without flucytosine is inefficient,and most of the patients need other antifungal drugs because of initial therapy failure.Therefore,fluconazole might not be appropriate for initial therapy in non-HIV-infected cryptococcal meningitis.

A Case of Primary Multiple Intracerebral spergillosis Abscess in Neonate:Treatment with Amphotericin B, Flucytosine and Itraconazole

Fungal infection of the central nervous system tends to occur mostly in immunosuppressed patients. In the pediatric population, it is usually seen in severely immunocompromised patients, particulary in children with chronic granulomatous disease and hematopoietic malignancies. Although aspergillosis is considered one of the most frequent agents of mycotic infection of the brain, it is especially rare in the neonatal period, and overwhelming multisystem infection is usually diagnosed postmortem. Manifestations include meningitis, meningoencephalitis, granulomata formation, brain abscess, vasculitis, aneurysm formation, infarct and intracranial hemorrhage. We present a neonate who had brain abscess diagnosed by MRI, and aspergillus was found at surgical exploration. There are very few reported survivors of neonatal aspergillosis. We reported a case of primary multiple brain abscess caused by aspergillus associated with severe hypernatremic dehydration and prerenal azotemia. The patient was treated with amphotericin B combined with flucytosine and itraconazole.

Clinical analysis of 146 patients with cryptococcal meningitis / 中华传染病杂志

Objectives To investigate the clinical features,prognosis and risk factors of patients with cryptococcal meningitis.Methods Totally 146 patients with cryptococcal meningitis who were hospitalized in Huashan Hospital from January 2000 to December 2006 were enrolled in this study.The clinical data including diagnosis and misdiagnosis,experimental and etiology tests,treat- ments and prognosis from all the patients were analyzed retrospectively.Results Among the 146 patients enrolled in the study,78 patients(53.4%)had concomitant diseases.The misdiagnosis rate of all patients was 72.6%(106/146).The positive rate of cerebrospinal fluid(CSF)India ink smear was 59.6%(87/106),while 43.2%(63/146)cases of cryptococcus neoformans culture in CSF was positive.The positive rate of Latex agglutination test(LAT)was 91.7%(134/146)in CSF among all patients.The treatments were as follows:combination of Amphotericin B(AmpB)or its lipid formula- tions with flucytosine(5-FC)(98 cases),including combination with Fluconazole initally(62 cases), single therapy of Fluconazole(13 cases).Ommaya implanted for lateral cerebral ventricle drainage(53 cases)and AmpB intrathecal injection(53 cases).The average dose of AmpB is 3.06 g.The course of treatment lasted from 12 weeks to 20 months.There were 104 patients(71.2%)cured,27(18.5%) improved,15(10.3%)died and 34(23.3%)relapsed.Conclusions High misdiagnosis rate is common in patients with cryptococcal meningitis.Immunodeficiency is the major risk factor for cryp- tococcal meningitis.CSF LAT is the most sensitive diagnostic test.Early diagnosis,combination of AmpB with 5-FC antifungal therapy and control of acute intracranial hypertension are the keys to im- prove prognosis of cryptococcal meningitis.

Susceptibility Testing of 28 Strains of Yeasts to the Combination of Antifungal Drugs in vitro / 中华皮肤科杂志

Objective To study the interaction between antifungal drugs to yeasts in vitro.Methods The in vitro interaction of antifungal drugs was detected with checkerboard microdilution method based on M27-A of NCCLS for28strains of yeasts.Results There was a significant reduction in the geometric means of MICs by the combinations of antifungal drugs to the yeasts compared with the individual agents(P

The study of cytosine deaminase gene/5 - fluorocytosine in tumor gene therapy / 中国病理生理杂志

Cytosine deaminase (CD) gene is a kind of suicide gene, cytosine deaminase can catalyze the conversion from 5 - fluorocytosine (5 - FC) into 5 - fluorouracil (5 - FU), this combination therapy (CDgene/5 - FC) were widely used in cancer therapy. The results of many researches in different cancer cells have demonstrated that it can inhibit tumor growth significantly. The mechanism includes 5 - FU's direct killing tumor cells and its bystander effect. It can also be used as a potential purging method for autologous hematopoietic cells transplantion after chemotherapy in breast cancer patients. CD/5 - FC combined with other therapies would be an important future research approach.

Susceptibility testing of yeasts to combination of amphotericin B and 5-flucytosine in vitro according to M27-A method / 第二军医大学学报

This article is to study the interaction between amphotericin B and 5-flucytosine to yeasts in vitro. The checkerboard microdilution method was applied to study in vitro interaction according to M27-A method of NCCLS. Twenty-eight strains of yeasts were used for combination study. Compared with the single agents,there were significant reductions in the geometric mean of the minimal inhibitory concentration of the combination(P