Azole Affinity of Sterol 14α-Demethylase (CYP51) Enzymes from Candida albicans and Homo sapiens

Candida albicans CYP51 (CaCYP51) (Erg11), full-length Homo sapiens CYP51 (HsCYP51), and truncated Δ60HsCYP51 were expressed in Escherichia coli and purified to homogeneity. CaCYP51 and both HsCYP51 enzymes bound lanosterol (K_s, 14 to 18 μM) and catalyzed the 14α-demethylation of lanosterol using Homo sapiens cytochrome P450 reductase and NADPH as redox partners. Both HsCYP51 enzymes bound clotrimazole, itraconazole, and ketoconazole tightly (dissociation constants [K_ds], 42 to 131 nM) but bound fluconazole (K_d, ∼30,500 nM) and voriconazole (K_d, ∼2,300 nM) weakly, whereas CaCYP51 bound all five medical azole drugs tightly (K_ds, 10 to 56 nM). Selectivity for CaCYP51 over HsCYP51 ranged from 2-fold (clotrimazole) to 540-fold (fluconazole) among the medical azoles. In contrast, selectivity for CaCYP51 over Δ60HsCYP51 with agricultural azoles ranged from 3-fold (tebuconazole) to 9-fold (propiconazole). Prothioconazole bound extremely weakly to CaCYP51 and Δ60HsCYP51, producing atypical type I UV-visible difference spectra (K_ds, 6,100 and 910 nM, respectively), indicating that binding was not accomplished through direct coordination with the heme ferric ion. Prothioconazole-desthio (the intracellular derivative of prothioconazole) bound tightly to both CaCYP51 and Δ60HsCYP51 (K_d, ∼40 nM). These differences in binding affinities were reflected in the observed 50% inhibitory concentration (IC₅₀) values, which were 9- to 2,000-fold higher for Δ60HsCYP51 than for CaCYP51, with the exception of tebuconazole, which strongly inhibited both CYP51 enzymes. In contrast, prothioconazole weakly inhibited CaCYP51 (IC₅₀, ∼150 μM) and did not significantly inhibit Δ60HsCYP51.     

Expression,Purification, and Characterization of Aspergillus fumigatus Sterol 14-α Demethylase (CYP51) Isoenzymes A and B

Aspergillus fumigatus sterol 14-α demethylase (CYP51) isoenzymes A (AF51A) and B (AF51B) were expressed in Escherichia coli and purified. The dithionite-reduced CO-P450 complex for AF51A was unstable, rapidly denaturing to inactive P420, in marked contrast to AF51B, where the CO-P450 complex was stable. Type I substrate binding spectra were obtained with purified AF51B using lanosterol (K_s, 8.6 μM) and eburicol (K_s, 22.6 μM). Membrane suspensions of AF51A bound to both lanosterol (K_s, 3.1 μM) and eburicol (K_s, 4.1 μM). The binding of azoles, with the exception of fluconazole, to AF51B was tight, with the K_d (dissociation constant) values for clotrimazole, itraconazole, posaconazole, and voriconazole being 0.21, 0.06, 0.12, and 0.42 μM, respectively, in comparison with a K_d value of 4 μM for fluconazole. Characteristic type II azole binding spectra were obtained with AF51B, whereas an additional trough and a blue-shifted spectral peak were present in AF51A binding spectra for all azoles except clotrimazole. This suggests two distinct azole binding conformations within the heme prosthetic group of AF51A. All five azoles bound relatively weakly to AF51A, with K_d values ranging from 1 μM for itraconazole to 11.9 μM for fluconazole. The azole binding properties of purified AF51A and AF51B suggest an explanation for the intrinsic azole (fluconazole) resistance observed in Aspergillus fumigatus.The sterol pathways of eukaryotes are highly conserved and are part of a larger biosynthetic pathway that includes the formation of dolichols, coenzyme Q, heme A, and isoprenylated proteins. In Saccharomyces cerevisiae, the first step exclusively involved in sterol synthesis is the formation of squalene, with the first sterol intermediate in the pathway being lanosterol, culminating in ergosterol some 15 enzymatic steps later. In fungi, these reactions are governed by individual enzymes, but closer examination of other fungal genome sequences has revealed that there is often duplication (29) and, in some instances, triplicate versions of the same gene (1). We are interested as to why fungi have kept multiple copies of these genes, the roles of the proteins in vivo, and their contribution to both sterol biosynthesis and fungal resistance.14-α Demethylase (CYP51) is an ancestral activity of the cytochrome P450 superfamily, which is also the target of azole antifungals (18). The isolation of CYP51 was initially from Saccharomyces cerevisiae (17), and in the fungal pathogen Aspergillus fumigatus, cytochrome P450 was first observed in 1990 (5, 6). Evidence that alteration of CYP51 activity might contribute to azole resistance first emerged in 1997 (11). In this particular study, we have looked in detail at the biochemical properties of the two CYP51 forms in Aspergillus fumigatus (29) encoded by CYP51A (Afu4g06890) and CYP51B (Afu7g03740). A comparison of the deduced amino acid sequences show 63% identity between them; and both orthologues in A. fumigatus have been shown to act in a compensatory manner in the ergosterol pathway; i.e., neither is essential individually, but a double knockdown is lethal (13). It is postulated that CYP51A may encode the major sterol 14-α demethylase activity required for growth on the basis of accumulation of multiple missense mutations linked to azole resistance (31), with CYP51B either being functionally redundant or having an alternative function under particular growth conditions still to be defined. We expressed both proteins in Escherichia coli to investigate their azole binding properties.     

In Vitro and In Vivo Studies of the Antiparasitic Activity of Sterol 14α-Demethylase (CYP51) Inhibitor VNI against Drug-Resistant Strains of Trypanosoma cruzi

Chagas disease affects more than 10 million people worldwide, and yet, as it has historically been known as a disease of the poor, it remains highly neglected. Two currently available drugs exhibit severe toxicity and low effectiveness, especially in the chronic phase, while new drug discovery has been halted for years as a result of a lack of interest from pharmaceutical companies. Although attempts to repurpose the antifungal drugs posaconazole and ravuconazole (inhibitors of fungal sterol 14α-demethylase [CYP51]) are finally in progress, development of cheaper and more efficient, preferably Trypanosoma cruzi-specific, chemotherapies would be highly advantageous. We have recently reported that the experimental T. cruzi CYP51 inhibitor VNI cures with 100% survival and 100% parasitological clearance both acute and chronic murine infections with the Tulahuen strain of T. cruzi. In this work, we further explored the potential of VNI by assaying nitro-derivative-resistant T. cruzi strains, Y and Colombiana, in highly stringent protocols of acute infection. The data show high antiparasitic efficacy of VNI and its derivative (VNI/VNF) against both forms of T. cruzi that are relevant for mammalian host infection (bloodstream and amastigotes), with the in vivo potency, at 25 mg/kg twice a day (b.i.d.), similar to that of benznidazole (100 mg/kg/day). Transmission electron microscopy and reverse mutation tests were performed to explore cellular ultrastructural and mutagenic aspects of VNI, respectively. No mutagenic potential could be seen by the Ames test at up to 3.5 μM, and the main ultrastructural damage induced by VNI in T. cruzi was related to Golgi apparatus and endoplasmic reticulum organization, with membrane blebs presenting an autophagic phenotype. Thus, these preliminary studies confirm VNI as a very promising trypanocidal drug candidate for Chagas disease therapy.     

A Nonazole CYP51 Inhibitor Cures Chagas’ Disease in a Mouse Model of Acute Infection

Chagas’ disease, the leading cause of heart failure in Latin America, is caused by the kinetoplastid protozoan Trypanosoma cruzi. The sterols of T. cruzi resemble those of fungi, both in composition and in biosynthesis. Azole inhibitors of sterol 14α-demethylase (CYP51) successfully treat fungal infections in humans, and efforts to adapt the success of antifungal azoles posaconazole and ravuconazole as second-use agents for Chagas’ disease are under way. However, to address concerns about the use of azoles for Chagas’ disease, including drug resistance and cost, the rational design of nonazole CYP51 inhibitors can provide promising alternative drug chemotypes. We report the curative effect of the nonazole CYP51 inhibitor LP10 in an acute mouse model of T. cruzi infection. Mice treated with an oral dose of 40 mg LP10/kg of body weight twice a day (BID) for 30 days, initiated 24 h postinfection, showed no signs of acute disease and had histologically normal tissues after 6 months. A very stringent test of cure showed that 4/5 mice had negative PCR results for T. cruzi, and parasites were amplified by hemoculture in only two treated mice. These results compare favorably with those reported for posaconazole. Electron microscopy and gas chromatography-mass spectrometry (GC-MS) analysis of sterol composition confirmed that treatment with LP10 blocked the 14α-demethylation step and induced breakdown of parasite cell membranes, culminating in severe ultrastructural and morphological alterations and death of the clinically relevant amastigote stage of the parasite.Chagas’ disease, caused by the kinetoplastid protozoan Trypanosoma cruzi, is the leading cause of heart failure in Latin America. The disease is transmitted naturally by hematophagous reduviid insects (6), but human infection may also occur via other routes, including blood transfusion, congenital infection, breast-feeding, organ transplant from chagasic donors, laboratory accidents, and ingestion of contaminated foods and beverages. The acute phase of infection usually occurs in children, and 5 to 10% of symptomatic patients may die. Following a subclinical “indeterminate” phase, a chronic phase involving heart failure and gastrointestinal tract lesions often ensues (37, 42). The only clinically available drugs for Chagas’ disease are nifurtimox and benznidazole, both of which have been in use for 4 decades. While they have significant efficacy in the acute phase, both drugs suffer from the twin liabilities of serious side effects and low efficacy in the chronic phase. New drugs with improved efficacy and less toxicity are needed (14, 29).The biosynthesis of membrane sterols is one of the metabolic pathways successfully targeted in the treatment of diseases caused by pathogenic fungi (48). Clinically employed antifungal azoles target sterol 14α-demethylase (CYP51), a cytochrome P450 enzyme that catalyzes oxidative removal of the 14α-methyl group of a sterol precursor to result in Δ^14,15-desaturated intermediates in ergosterol biosynthesis (19, 20). Close similarities to fungi in sterol composition and biosynthesis, plus an absolute requirement for specific 24-methyl sterols for cell viability and proliferation, provide a basis for development of chemotherapy targeting the sterol biosynthetic pathway in T. cruzi. The successful application of antifungal drugs to anti-chagasic therapy exploits these similarities (38). In addition to compounds optimized for antifungal therapy, other CYP51 inhibitors with strong anti-T. cruzi activity have been reported (3, 8, 24, 44, 45).Inhibitors of CYP51 are in the pipeline for preclinical and clinical development for treatment of Chagas’ disease (11). Although earlier commercially available inhibitors, like ketoconazole and itraconazole, were not powerful enough to eradicate T. cruzi from infected animals or human patients (28), the recently approved inhibitor posaconazole (Noxafil; Schering-Plough) is capable of inducing parasitological cure in murine models of both acute and chronic Chagas’ disease (18). Posaconazole cured 50 to 100% of animals in the acute phase and 50 to 60% of chronically infected animals (2). Very recently, posaconazole cured an immunosuppressed patient with concomitant Chagas’ disease and systemic lupus erythematosus (34). However, the use of posaconazole as an anti-chagasic agent may be limited by the requirement for simultaneous intake of a fatty meal or a nutritional supplement to enhance absorption, the drug''s high cost, and the need for clinical monitoring during treatment (31). Another complication is the rapid appearance of laboratory-induced resistance to azoles in T. cruzi, which may predict the occurrence of drug resistance in chagasic patients (4). Although no data on the development of posaconazole resistance in patients with Chagas’ disease are available, studies of fungal infections indicate that posaconazole resistance occurs mainly by a mechanism involving mutation of the cyp51 gene (23, 33, 35). Posaconazole appears to be less susceptible to the efflux pumps that confer resistance to some other azoles (7, 25, 35). Mapping mutations in cyp51 genes in clinical posaconazole-resistant isolates on the CYP51-posaconazole structure (9) points to the mouth of the posaconazole binding tunnel as a mutation hot spot. Mutations of G54, P216, and M220 in clinical isolates of Aspergillus fumigatus (10, 12, 13, 23, 27, 32) (corresponding to G49, P210, and F214, respectively, in T. cruzi CYP51 [CYP51_Tc]) and of A61 (46) and P230 (25) in clinical isolates of Candida albicans (I45 and P210, respectively, in CYP51_Tc) map directly to the tunnel mouth, where amino acids interact with the dangling long substituent tail of posaconazole extending into the tunnel (9). Mutations of G54 in A. fumigatus to arginine or tryptophan associate with moderate and high levels of resistance, respectively, and confer cross-resistance between itraconazole and posaconazole (27). Mutations of M220 confer cross-resistance to all azole drugs tested, including itraconazole, voriconazole, ravuconazole, and posaconazole (30, 39), and therefore may interfere with the entry of the drugs. In accordance with this assumption, posaconazole is reported to induce resistance to all azole drugs in Candida parapsilosis in vitro (35). The alarming perspective emerging from antifungal therapy efforts must be taken into consideration when designing antichagasic drugs targeting CYP51_Tc. While antifungal azoles do show promise, the less than 30% sequence identity between fungal and protozoan CYP51 targets suggests that a more direct approach may be a better route toward developing novel potent therapeutic CYP51 inhibitors.Using clues from our previous work on CYP51 from Mycobacterium tuberculosis (CYP51_Mt), we focused on rationally designed nonazole inhibitors of CYP51_Tc. These inhibitors were based on an experimental hit obtained from screening a small-molecule-compound library against CYP51_Mt (36). Analysis of the X-ray structure revealed that the N-[4-pyridyl]-formamide scaffold group (Fig. ​(Fig.1A,1A, highlighted in gray) binds in the CYP51 active site via conserved residues and the heme prosthetic group. Structural characterization confirmed that these interactions in the complexes were preserved between CYP51_Mt and five different compounds (8, 36), suggesting that this scaffold could be used efficiently instead of the azole or triazole groups to target a variety of chemotypes to the CYP51 active site. Based on the similarity of the chemical structures, the expanded-spectrum compound LP10 (Fig. ​(Fig.1B)1B) was selected for its nanomolar binding affinity to CYP51_Tc and its potent efficacy against T. cruzi in mammalian cells (8). As the pyridyl group of LP10 presumably coordinates to the heme iron, the indole substituent may fill the space occupied by the 2,4-difluorophenyl ring of fluconazole or posaconazole in their structurally characterized complexes with CYP51 (9).Open in a separate windowFIG. 1.Screen hit (A) and the expanded-spectrum compound LP10 (B) containing the N-[4-pyridyl]-formamide scaffold (highlighted in gray), which unvaryingly binds in the CYP51 active site. The chiral center is labeled with an asterisk.In the present work, we evaluated the efficacy of LP10 in an animal model of acute Chagas’ disease. The curative effect of LP10 in vivo was comparable to that of the protease inhibitor K777, an antichagasic drug in preclinical development, used as a positive control (15, 16). Electron microscopy and gas chromatography-mass spectrometry (GC-MS) analysis demonstrated that treatment with LP10 disrupted cell membranes in T. cruzi amastigotes and altered sterol composition via accumulation of the C-14-methylated precursors lanosterol and 24-methylene-dihydrolanosterol (eburicol). There was concomitant reduction of 14-desmethylated fecosterol and episterol. LP10-induced alterations are consistent with the inhibition of T. cruzi CYP51.     

Azole Antifungal Agents To Treat the Human Pathogens Acanthamoeba castellanii and Acanthamoeba polyphaga through Inhibition of Sterol 14α-Demethylase (CYP51)

In this study, we investigate the amebicidal activities of the pharmaceutical triazole CYP51 inhibitors fluconazole, itraconazole, and voriconazole against Acanthamoeba castellanii and Acanthamoeba polyphaga and assess their potential as therapeutic agents against Acanthamoeba infections in humans. Amebicidal activities of the triazoles were assessed by in vitro minimum inhibition concentration (MIC) determinations using trophozoites of A. castellanii and A. polyphaga. In addition, triazole effectiveness was assessed by ligand binding studies and inhibition of CYP51 activity of purified A. castellanii CYP51 (AcCYP51) that was heterologously expressed in Escherichia coli. Itraconazole and voriconazole bound tightly to AcCYP51 (dissociation constant [K_d] of 10 and 13 nM), whereas fluconazole bound weakly (K_d of 2,137 nM). Both itraconazole and voriconazole were confirmed to be strong inhibitors of AcCYP51 activity (50% inhibitory concentrations [IC₅₀] of 0.23 and 0.39 μM), whereas inhibition by fluconazole was weak (IC₅₀, 30 μM). However, itraconazole was 8- to 16-fold less effective (MIC, 16 mg/liter) at inhibiting A. polyphaga and A. castellanii cell proliferation than voriconazole (MIC, 1 to 2 mg/liter), while fluconazole did not inhibit Acanthamoeba cell division (MIC, >64 mg/liter) in vitro. Voriconazole was an effective inhibitor of trophozoite proliferation for A. castellanii and A. polyphaga; therefore, it should be evaluated in trials versus itraconazole for controlling Acanthamoeba infections.     

Evaluation of a CYP2C19 genotype panel on the GenMark eSensor® platform and the comparison to the Autogenomics Infiniti™ and Luminex CYP2C19 panels

BackgroundCYP2C19 variants have been demonstrated to play an important role in determining response to clopidogrel and outcomes while on clopidogrel therapy. Predicting patient response through pre-therapeutic genotyping may therefore guide selection of antiplatelet therapy.MethodsCYP2C19 genotypes were determined for 111 samples with the eSensor and compared with the Autogenomics expanded CYP2C19 panel, Luminex reagents, and bi-directional sequencing. Samples were obtained from the University of Chicago, ARUP Laboratories, and the Coriell repositories. Reproducibility studies were performed with 5 DNA samples with known CYP2C19 genotypes.ResultsComplete concordance was observed for all samples and all platforms. DNA concentrations as low as 0.05 ng/μl may be used on the eSensor platform. There was 100% reproducibility observed with 2.5% incidence of invalid tests.ConclusionsThe eSensor CYP2C19 genotyping assay is accurate and compares well with 2 current commercial platforms. With a relatively rapid turn-around time of ~ 4 h and a high rate (97.5%) of valid tests, the eSensor can be translated into clinical use to identify slow and rapid metabolizers of clopidogrel who may benefit from alternate therapy or unconventional dosing of clopidogrel. An observed limitation of the eSensor is a maximum capacity of 24 tests/run.     

Comparison of simultaneous plasma clearance of 99mTc-DTPA and 51Cr-EDTA: can one tracer replace the other?

Abstract

Both ^99mTc-DTPA and ⁵¹Cr-EDTA are widely used to determine glomerular filtration rate (GFR), but few direct comparative studies exist. The shortage of ⁵¹Cr-EDTA makes a direct comparison highly relevant. The aim of the study was to investigate if there is any clinically relevant difference between plasma clearance of ^99mTc-DTPA and ⁵¹Cr-EDTA. Patients ≥18?years of age referred for routine GFR measurement by ⁵¹Cr-EDTA were prospectively enrolled. The two tracers (10 MBq ^99mTc-DTPA (CaNa₃-DTPA) and 2.5 MBq ⁵¹Cr-EDTA) were intravenously injected at time zero. A standard 4-sample technique was applied with samples collected at 180, 200, 220 and 240?min, if the estimated GFR (eGFR) was ≥30?mL/min. A comparison of single-sample GFR based on the 200?min sample was also conducted. Fifty-six patients were enrolled in the study. All patients had an estimated GFR >30?mL/min/1.73 m². No patients suffered from ascites or significant oedema. The mean ⁵¹Cr-EDTA plasma clearance was 82?mL/min (range 16–226). The plasma clearances determined by the two methods were highly correlated (r?=?0.993). The plasma clearance was significantly higher when measured by ^99mTc-DTPA than by ⁵¹Cr-EDTA (p?=?0.01), but the numerical difference was minimal (mean difference 1.4?mL/min; 95% limits of agreement (LOA) –6.6 to 9.4). The difference between the two methods was independent of the level of renal function. Similar results were found for one-sample GFR. No clinically relevant differences were found between the plasma clearance of ^99mTc-DTPA and that of ⁵¹Cr-EDTA. Therefore, ^99mTc-DTPA can replace ⁵¹Cr-EDTA when needed.     

Evaluation of 6β-hydroxycortisol, 6β-hydroxycortisone, and a combination of the two as endogenous probes for inhibition of CYP3A4 in vivo

An endogenous probe for CYP3A activity would be useful for early identification of in vivo cytochrome P450 (CYP) 3A4 inhibitors. The aim of this study was to determine whether formation clearance (CL(f)) of the sum of 6β-hydroxycortisol and 6β-hydroxycortisone is a useful probe of CYP3A4 inhibition in vivo. In human liver microsomes (HLMs), the formation of 6β-hydroxycortisol and 6β-hydroxycortisone was catalyzed by CYP3A4, and itraconazole inhibited these reactions with half maximal inhibitory concentration (IC(50))(,u) values of 3.1 nmol/l and 3.4 nmol/l, respectively. The in vivo IC(50,u) value of itraconazole for the combined CL(f) of 6β-hydroxycortisone and 6β-hydroxycortisol was 1.6 nmol/l. The greater inhibitory potency in vivo is probably due to circulating inhibitory itraconazole metabolites. The maximum in vivo inhibition was 59%, suggesting that f(m,CYP3A4) for cortisol and cortisone 6β-hydroxylation is ~60%. Given the significant decrease in CL(f) of 6β-hydroxycortisone and 6β-hydroxycortisol after 200-mg and 400-mg single doses of itraconazole, this endogenous probe can be used to detect moderate and potent CYP3A4 inhibition in vivo.     

Genotyping for CYP2C9 and VKORC1 alleles by a novel point of care assay with HyBeacon® probes

BackgroundCoumarin anticoagulants such as warfarin are used to treat and prevent thromboembolic events in patients. The required dosage is difficult to predict and the risk of over or under anticoagulation are dependent on several environmental and clinical factors, such as concurrent medication, diet, age and genotype for polymorphisms in two genes CYP2C9 and VKORC1.MethodsA novel fluorescent PCR genotyping assay using HyBeacon® probes, was developed to enable clinical staff to genotype the CYP2C9*2 and CYP2C9*3 alleles and the VKORC1 G-1639A polymorphism directly from unextracted blood samples. A prototype PCR instrument, Genie 1, suitable for point of care use was developed to carry out the assays. The panel of tests was validated by analysing blood samples from 156 individuals and comparing genotypes with data obtained using DNA samples from the same individuals. The accuracy of genotypes obtained with the Genie 1 was compared against results from well validated real time PCR and PCR-restriction fragment length polymorphism analysis.ResultsIdentical results were obtained for the newly developed HyBeacon® method and the validation method in all cases except for one where no result was obtained for the VKORC1 polymorphism on the Genie instrument. The samples used for validation represented all six possible *2 and *3 allele-related CYP2C9 genotypes and all three VKORC1 G-1639A genotypes.ConclusionsWe observed excellent accuracy for the newly developed method which can determine genotype in less than 2 h.     

PDGFRα and CD51 mark human Nestin+ sphere-forming mesenchymal stem cells capable of hematopoietic progenitor cell expansion

The intermediate filament protein Nestin labels populations of stem/progenitor cells, including self-renewing mesenchymal stem cells (MSCs), a major constituent of the hematopoietic stem cell (HSC) niche. However, the intracellular location of Nestin prevents its use for prospective live cell isolation. Hence it is important to find surface markers specific for Nestin⁺ cells. In this study, we show that the expression of PDGFRα and CD51 among CD45⁻ Ter119⁻ CD31⁻ mouse bone marrow (BM) stromal cells characterizes a large fraction of Nestin⁺ cells, containing most fibroblastic CFUs, mesenspheres, and self-renewal capacity after transplantation. The PDGFRα⁺ CD51⁺ subset of Nestin⁺ cells is also enriched in major HSC maintenance genes, supporting the notion that niche activity co-segregates with MSC activity. Furthermore, we show that PDGFRα⁺ CD51⁺ cells in the human fetal BM represent a small subset of CD146⁺ cells expressing Nestin and enriched for MSC and HSC niche activities. Importantly, cultured human PDGFRα⁺ CD51⁺ nonadherent mesenspheres can significantly expand multipotent hematopoietic progenitors able to engraft immunodeficient mice. These results thus indicate that the HSC niche is conserved between the murine and human species and suggest that highly purified nonadherent cultures of niche cells may represent a useful novel technology to culture human hematopoietic stem and progenitor cells.Hematopoietic stem cells (HSCs) continuously replenish all blood cell lineages throughout their lifetime. Incipient hematopoiesis is first detected extraembryonically in the yolk sac and later in the aorta–gonad–mesonephros region, from where it moves transiently to the placenta and liver before being stabilized in the fetal BM (Wang and Wagers, 2011). In the adult stage, HSCs reside in a highly complex and dynamic microenvironment of the BM commonly referred to as the HSC niche (Schofield, 1978). The interactions between the niche constituents and HSCs ensure hematopoietic homeostasis by regulating HSC self-renewal, differentiation, and migration and by integrating neural and hormonal signals from the periphery (Méndez-Ferrer et al., 2009, 2010; Mercier et al., 2012). However, HSC maintenance and expansion ex vivo still remains challenging mainly because of our limited knowledge on the in vivo HSC niche constituents and the factors that drive HSC self-renewal.Although the cellular constituents of the HSC niche and their role are still poorly understood, in the last decade, several putative cellular components of the murine HSC niche have been proposed, including osteoblastic, endothelial, adipocytic, and perivascular cells (Calvi et al., 2003; Zhang et al., 2003; Arai et al., 2004; Kiel et al., 2005; Sugiyama et al., 2006; Chan et al., 2009; Naveiras et al., 2009; Méndez-Ferrer et al., 2010; Ding et al., 2012). Multipotent BM mesenchymal stem cells (MSCs) have long been suggested to also provide regulatory signals to hematopoietic progenitors, as mixed cultures derived from the adherent fraction of the BM stroma promote the maintenance of HSCs in vitro (Dexter et al., 1977). Although numerous studies explored the ability of mesenchymal stromal cultures to support the ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs), currently these systems are still insufficient to preserve primitive HSCs with long-term multilineage engraftment capacity (Chou et al., 2010; Broxmeyer, 2011). This limitation may in part be associated with the heterogeneous composition of mesenchymal stromal cell cultures. The prospective identification and functional characterization of purified naive populations of mouse and/or human BM stromal MSCs have been mired by the absence of specific cell surface markers allowing prospective isolation. Several MSC-associated antigens have been proposed (such as CD31⁻ CD34⁻ CD45⁻ CD105⁺ CD90⁺ CD73⁺) in cultured cells (Dominici et al., 2006). Nevertheless, these markers are not homogeneously expressed across cultures, varying with isolation protocols and passage and therefore not necessarily representative of MSCs in vivo (Bianco et al., 2013; Frenette et al., 2013). Very few MSC-associated antigens have been validated using rigorous transplantation assays (Sacchetti et al., 2007; Méndez-Ferrer et al., 2010). In the mouse BM, the expression of the intermediate filament protein Nestin characterizes a rare population of multipotent MSCs in close contact with the vasculature and HSCs. Nestin⁺ stromal cells contain all of the fibroblastic CFU (CFU-F) activity within the mouse BM and the exclusive capacity to form clonal nonadherent spheres in culture. The selective ablation of mouse Nestin⁺ cells (Méndez-Ferrer et al., 2010) or CXCL12-abundant reticular (CAR) cells (Omatsu et al., 2010) led to significant alterations in the BM HSC and progenitor maintenance. Serial transplantation analyses revealed that Nestin⁺ cells are able to self-renew and generate hematopoietic activity in heterotopic bone ossicle assays (Méndez-Ferrer et al., 2010). This potential was also associated with a CD45⁻ Tie2⁻ αV⁺ CD105⁺ CD90⁻ subset from the fetal mouse bone (Chan et al., 2009). In the adult mouse BM, PDGFRα⁺ Sca1⁺ CD45⁻ Ter119⁻ cells were also shown capable to give rise to osteoblasts, reticular cells, and adipocytes in vivo upon transplantation into irradiated mice (Morikawa et al., 2009). However, human BM MSCs are still retrospectively isolated based on plastic adherence (Friedenstein et al., 1970; Pittenger et al., 1999). Human CD45⁻ CD146⁺ self-renewing osteoprogenitors isolated from stromal cultures containing all the human BM CFU-F activity were shown capable of generating a heterotopic BM niche in an s.c. transplantation model (Sacchetti et al., 2007). However, a recent study showed that human CD45⁻ CD271⁺ CD146^−/low BM cells also possess these capacities (Tormin et al., 2011).Because Nestin is an intracellular protein, its identification in nontransgenic mice requires cell permeabilization, which precludes prospective isolation of live cells. In this study, we have evaluated putative cell surface MSC markers to identify a stromal population equivalent to Nestin⁺ cells in the mouse and human BM. Our results show that the combination of PDGFRα and CD51 identify a large subset of perivascular Nestin⁺ cells that is highly enriched in MSC and HSC niche activities in both species. Furthermore, we show that PDGFRα⁺ CD51⁺ stromal cells isolated from human BM can also form self-renewing clonal mesenspheres capable of transferring hematopoietic niche activity in vivo and support the ex vivo maintenance and expansion of human HSPCs in a dose-dependent manner.     

Relation research of the expression and function of VEGF and its receptor

VEGF stimulates the formation of vessle and proliferation of endothelial cells．VEGF and its rece ptor，FLK －１have the function of chemotaxing and division．Now ，it is well known that VEGF is involved in growth and metastasis of a number of experimental tu－mors［１］．Neuroblastoma is characterized by higher malignace，early metastasis，abundant vessels．The mechanism of its high malignace is not clear now．We detected expressio n and distribution of VEGF and its receptor in neruob…     

Efficacy and tolerability of a combination of Lyprinol® and high concentrations of EPA and DHA in inflammatory rheumatoid disorders

This 12-week drug-monitoring study was conducted to evaluate the efficacy of Sanhelios Mussel Lyprinol Lipid Complex on 50 adult men and women with inflammatory rheumatoid arthritis. A total of 34 patients required drug therapy before and during the study. By the end of the study, 21 (62%) patients were able to reduce their dosage and 13 were able to terminate drug therapy. At the end of the treatment period, 38% were regarded symptom free, and the number of patients with severe pain decreased significantly from 60% at baseline to 25% at the completion of the trial. A significant effect was observed for each investigated parameter. The special combination of Lyprinol and omega-3 fatty acids was generally very well tolerated, with only one, nonserious adverse event (mild nausea) reported. This dietary supplement may therefore be considered an effective and well-tolerated component of treatment regimens for inflammatory rheumatoid arthritis.     

Experience of prevention and treatment of deep burn of hand

INTRODUCTIONHowtopreventhanddeformitycausedbyburn,restoreappearanceandworkingabilityhasbeenalwaysthemainproblem,thefollow-ingisthereportaboutcausesofdeformityandexperienceofpre-ventionandtreatment.GENERALDATA2037casesofburntreatedfromJanuary1997toOctober2002amongincluding957casesofburnofhandsandtheincidencewas46.9%amongwhichwere229casesofdeepburnand195pa-tientsreceivedearlysurgicaltreatment.6patients(15fingers)un-derwentfingeramputation.38casesneededplasticsbecauseofscardeformityinlat…     

Manifestations and Causes of psychological and behavioral problems in infant patients and patients of early age

Lage amounts clinical facts demonstrate that many diseases can induce emotional disturbances and psychological problems such as fear,anxiety,restlessness,agitation and irritability in infantile patients and patients of early age.The author would like to discuss briefly several common psychological and behavioral problesms and the corresponding causes of them.     

Manifestations and causes of psychological and behavioral problems in infant patients and patients of early age

Largeamountsclinicalfactsdemonstratethatmanydiseasescaninduceemotionaldisturbancesandpsychologicalproblemssuchasfear,anxiety,restlessness,agitationandirritabilityininfantilepatientsandpatientsofearlyage.Theauthorwouldliketodiscussbrieflyseveralcommonpsychologicalandbehavioralproblemsandthecorrespondingcausesofthem.1CommonpsychologicalandbehavioralproblemsininfantpatientsandpatientsofearlyageCryingandmakingnoise:Cryingandmakingnoiseisasignalinfantsandchildrenusetoconveylargeamountsofinformation…     

Investigation of features of hemostasis sponge of collagen and chitosan compound

AIM：To investigate the hemostasis effect of hemostasis sponge of collagen and chitosan compound invitro and in vivo.METHODS:The hemostasis sponge of collagen and chitosan compound was made by mixing collegen I extracted by enzymelytic methods and crosslinked by glutaric dialdeiyde and then lyophilized.The New zealand rabbit were created with a 1cm&;#215;1cm wound in the center artery of the ear.The center artery of the ear were transected and record the hemostasis time since pressured with hemostasis sponge of collagen and chitosan compound and obsevve its effects.In other experiements,1cm&;#215;1cm bleeding wound were established in the liver of the rabbit.Then pressured with hemostasis sponge of collagen and chitosan compound on the wounds and record the hemostasis time;The four corner of the hemostasis sponge of collagen and chitosan compound were sutured to investigate the absorption situation with the control of gelatin treated with same procedures.RESULTS:The bleeding time using hemostasis sponge of collagen and chitosan compound was 120 seconds compared with 600 seconds of using gelatin,and the hemostasis time using hemostasis sponge of collagen and chitosan compound was 40 weconds compared with 75 seconds of using gelatin.Both the hemostasis sponge of collagen and chitosan compound and gelatin can be degradated within 5 months.CONCLUSION:Hemostasis effect of hemostasis sponge of collagen and chitosan compound is better than that of gelatin and it has an promising future in surgery.     

Diagnosis and treatment of 20 cases of phenylpyruvic oligophrenia

BACKGROUND:Phenylpyruvicoligophrenia(Folling'sdisease)isoneofseldomtreatablegeneticmetabolicdiseasesandearlydiagnosisandtreatmentwerethekeytoavoidirreversibleinjuryofnervesystem.OBJECTIVE:Toexplorethediagnosisandtreatmentofphenylpyruvicoligophrenia.UNIT:DepartmentofOtolaryngology,ChildrenHospitalofKunmingCitySUBJECTS:20casesofFolling'sdiseasewereinvestigatedincluding12males,8femalesaged1.4-12yearsold.Patientswasnormalatbirthandsomeappearedvomiting,skineczema,yellowhairandskin,de…