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【Objective】 To investigate the viability of rapamycin-treated rapamycin-treated dendritic cells (DCs) in intervening transfusion-related acute lung injury (TRALI) after infection. 【Methods】 1)The TRALI mouse model was induced by lipopolysaccharide (LPS) combined with anti-H2Kd antibody. The mice anal temperature and the wet/dry ratio of lung, kidney, spleen and brain tissues were measured. 2) Mouse bone marrow-derived DC cells were induced in vitro and treated with rapamycin (10nM) for 24h. 3) Mice were injected with or without rapamycin or rapamycin-treated DC, then injected with LPS intraperitoneally one hour later, finally injected with anti-H2Kd antibody 24 hours later to induce the onset of TRALI. The death situation of the mice was observed and recorded. The condition of mice after the onset of TRALI was analyzed by mouse body temperature, lung wet-dry ratio, and pleural effusion weight and lung histopathological sections. 【Results】 By comparing the induction effects of anti-H2Kd antibody solutions with different concentrations and volumes, the mouse model induced by 0.1mg/kg LPS combined with 4.5 mg/kg anti-H2Kd antibody (infusion volume of 100μL) was selected as the TRALI mouse model for this study. After the onset of TRALI, the wet/dry ratio of the lungs could be significantly increased and the body temperature could be significantly reduced in the model mice. After the intervention of TRALI mice with DCs treated with rapamycin, the mortality rate was significantly reduced, and the lung tissue lesions of the mice were significantly improved, whose protection effect was better than that of the rapamycin-treated group. Compared with the TRALI incidence group, the weight of pleural effusion in the intervention group was significantly reduced (P<0.05), but there was no significant difference in lung wet/dry ratio and body temperature. 【Conclusion】 The combination of LPS and antibodies can effectively induce a stable and typical TRALI mouse model, suggesting that the presence of infectious inflammation and blood transfusion-related inflammatory substances are the decisive factor for the pathogenesis of TRALI. Meanwhile, DCs treated with rapamycin have a protective effect on post-infection transfusion-related acute lung injury, which is expected to be a potential cell therapy strategy to intervene in the exacerbation of TRALI.
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【Objective】 To supervise the clinical blood use of 19 hospitals, covering a district of Shanghai, during two years, and discover the problems in the process of blood transfusion, so as to put forward suggestions for corrective methods in grades and promote continuous improvement of clinical transfusion management. 【Methods】 A total of 19 hospitals were supervised in terms of hardware facilities, management level, professional and technical level, and blood typing test on the site, according to the Administrative Blood Management Measures for Medical Institutions, Technical Specification for Clinical Transfusion and Shanghai Medical Quality Supervision Score Statistical Table.All data were analyzed. 【Results】 These hospitals can properly perform clinical blood transfusion, but there were obvious differences.Tertiary hospitals were relatively better, yet need to strengthen the management of medical documents.Secondary hospitals remained to be improved, mainly in insufficient construction of Blood Transfusion Department (blood bank), the lack of management and maintenance of key equipment and the lack of standardization in medical documents writing.However, flaws in the supervision were general in private hospitals (most of which were affiliated hospitals), so the management of clinical blood use should be further strengthened. 【Conclusion】 For secondary hospitals or above, routinized writing of medical documents and promoted construction of Blood Transfusion Department (blood bank) should be strengthened.For private hospitals, especially affiliated hospitals, the management of clinical blood use should be further improved, including the examination rules corresponding to the blood use process and strict access and exit mechanism, so as to improve the overall management level of clinical blood use and ensure the safety of clinical blood use.
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The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63+ intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles in situ and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.
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Objective To investigate the value of magnetic resonance imaging-proton density fat fraction (MRI-PDFF) and FibroScan in the quantitative evaluation of liver fat content in patients with chronic hepatitis B (CHB). Methods A total of 96 patients with CHB who were hospitalized in Department of Hepatology, The Second Clinical Medical College of Guangzhou University of Chinese Medicine, from February 2017 to July 2020 were enrolled, and all patients were diagnosed based on liver pathological examination. MRI-PDFF and FibroScan were performed before surgery. According to the results of liver biopsy, the patients were divided into non-fatty liver disease group with 44 patients, mild fatty liver disease group with 33 patients, and moderate-to-severe fatty liver disease group with 19 patients. A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups, and the least significant difference t -test was used for further comparison between two groups; the Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between multiple groups, and the Mann-Whitney U test was used for further comparison between two groups; Bonferroni correction was also performed. The receiver operating characteristic (ROC) curve was plotted to analyze the area under the ROC curve (AUC) of hepatic fat fraction (HFF) and controllable attenuation parameters (CAP) in the diagnosis of fatty liver disease and obtain their sensitivities, specificities, and optimal cut-off values. The intraclass correlation coefficient was used to investigate the consistency of MRI-PDFF data. Results The moderate-to-severe fatty liver disease group had a significant increase in MRI-PDFF HFF compared with the non-fatty liver disease group and the mild fatty liver disease group (all P < 0.05), and the mild fatty liver disease group had a significant increase in MRI-PDFF HFF compared with the non-fatty liver disease group( P < 0.05). The moderate-to-severe fatty liver disease group had a significant increase in FibroScan CAP compared with the non-fatty liver disease group and the mild fatty liver disease group (all P < 0.05), and the mild fatty liver disease group had a significant increase in FibroScan CAP compared with the non-fatty liver disease group ( P < 0.05). In the diagnosis of mild fatty liver disease, MRI-PDFF HFF had an AUC of 0.901 ( P < 0.001), a sensitivity of 90.9%, and a specificity of 82.7% at the optimal cut-off value of 5.1%, and in the diagnosis of moderate-to-severe fatty liver disease, MRI-PDFF HFF had an AUC of 0.972 ( P < 0.001), a sensitivity of 96.1%, and a specificity of 89.5% at the optimal cut-off value of 9.7%. In the diagnosis of mild fatty liver disease, FibroScan CAP had an AUC of 0.829 ( P < 0.001), a sensitivity of 77.3%, and a specificity of 78.8% at the optimal cut-off value of 258.5 dB/m, and in the diagnosis of moderate-to-severe fatty liver disease, FibroScan CAP had an AUC of 0.830 ( P < 0.001), a sensitivity of 76.6%, and a specificity of 78.9% at the optimal cut-off value of 285.5 dB/m. Conclusion Both MRI-PDFF and FibroScan can objectively evaluate the degree of fatty liver disease in patients with CHB. MRI-PDFF HFF and FibroScan CAP can be used as noninvasive markers for the quantitative analysis of CHB with hepatic steatosis, and MRI-PDFF HFF tends to have higher diagnostic efficiency.
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The purpose of the present study was to investigate the effects of salidroside (Sal) on lung injury in lipopolysaccharide (LPS)-induced endotoxemic in vitro and in vivo. SD rats were randomly divided into five groups: control group, LPS group (15 mg kg-1), LPS plus dexamethasone (2 mg kg-1), and LPS plus Sal groups with different Sal doses (20 mg kg-1, 40 mg kg-1). Wet-to-dry weight (W/D) ratio was performed. Hematoxylin-eosin (HE) staining of lung was performed. Lung level of myeloperoxidase (MPO) was measured. Serum levels of the activities of the anti-oxidant superoxide dismutase (SOD), glutathione peroxidase (GSH-px), glutathione (GSH), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) were measured. Caveolin-1 and TLR/NF-κB pathway proteins were detected by Western blot. In vitro, we evaluated the protective effect of Sal on A549 cell line induced by LPS. The activities of the antioxidant SOD, CAT, GSH and GPX, TNF-α, IL-6, and IL-1ß in cellular supernatant were measured. Caveolin-1 and TLR/NF-κB pathway was examined by Western blot. As a result, Sal significantly attenuated the above indices. In addition, Sal exerts pronounced protective effects in rats subjected to LPS possibly through inhibiting the caveolin-1 and TLR/NF-κB pathway in vivo. Our results indicated that Sal could be a potential therapeutic agent for the treatment of lung injury disease.
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Glucósidos/farmacología , Lesión Pulmonar/tratamiento farmacológico , Fenoles/farmacología , Células A549 , Animales , Antioxidantes , Caveolina 1/antagonistas & inhibidores , Endotoxemia/inducido químicamente , Humanos , Lipopolisacáridos , Pulmón/patología , FN-kappa B/antagonistas & inhibidores , RatasRESUMEN
Objective@#To evaluate the efficiency of maxillary expansion with clear aligners and to analyze the possible influence factors.@*Methods@#Thiry-one clear aligner cases (Invisalign) with maxillary expansion were enrolled in this study. The three-dimensional (3D) digital models of pre-treatment, planned in Clincheck software and post-treatment were collected. Upper dental arch width, buccal inclination of posterior teeth, and the expansion efficiency (expansion acquired/expansion planned) were measured and calculated. The impact of factors such as planned buccal inclination, planned expansion amount, attachment, and the mode of expansion on the expansion efficiency were analyzed.@*Results@#The increases of upper arch width in canine, 1st and 2nd premolar, 1st and 2nd molar were (2.0±1.3), (2.8±1.5), (3.0±1.4), (1.8±1.0) and 0.5 (0.1, 1.1) mm, with their efficiency of 68%, 70%, 68%, 55% and 29%, respectively. The posterior teeth showed significantly more buccal inclination than the planned position (P<0.05). However, the most buccally inclined tooth detected in 1st molars was only 3.1°±3.9°. When the planned inter-molar width increase was less than 2 mm (n=15), the expansion efficiency of premolars was higher compared with those cases with the planned inter-molar width increase more than 2 mm (n=16, P<0.05). The planned buccal inclination, attachments, and the expansion mode had no significant effect on the expansion efficiency (P>0.05).@*Conclusions@#The expansion of maxillary arch with clear aligners was achieved by the buccal movement of the posterior teeth with limited buccal inclination. The efficiency of expansion declines from 1st premolars to second molars. The planned inter-molar width had a significant influence on the efficiency of premolar expansion.
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<p><b>OBJECTIVE</b>To explore the biological characteristics and the immuno-suppression function of tolerogenic dendritic cells (tDC) induced by tacrolimus.</p><p><b>METHODS</b>Human monocytes derived from peripheral blood were cultured in the cGMP-compliant CellGro DC medium supplemented with GM-CSF and IL-4 to obtain dendritic cells (DCs), and 0.1 μmol/L immunosuppressive drug tacrolimus was added to the culture medium at the third and fifth day to obtain tDCs. The molecular markers of them and the livability were assayed by flow cytometry. Then the tolerance functionality of tDCs induced by many agents and these tDCs modulated allogeneic CD4 T cells was determined via CFSE proliferation assay. And the research also analyzed the biological characters and immunosuppression function of tDCs induced by tacrolimus after storing.</p><p><b>RESULTS</b>tDCs induced by tacrolimus exhibit a typical tolerogenic phenotype, whose level of costimulatory molecules CD80, CD83, CD86 and HLA-DR is (2.95 ± 1.32)%, (2.33 ± 1.60)%, (90.02 ± 7.42)% and (91.80 ± 6.18)%, respectively. It's survival rate was (85.2 ± 4.72)%. And immunosuppressive drugs didn't influence the differentiation of tDCs from monocytes. tDCs induced by immunosuppressive drugs dexamethasone, cyclosporin A and tacrolimus had lower immunogenic than control DCs as CD4+ T proliferation rate of tDCs induced by tacrolimus is 0.42% and could not primed allogeneic CD4+ T cells proliferation. Functional analyses showed that tDCs induced by tacrolimus can more effectively suppressed mature DC-induced T cell proliferation than other tDCs, whose inhibition rate can reach (67.01 ± 19.73)%. Importantly, tDCs induced by tacrolimus had phenotypical and functional stability after storing.</p><p><b>CONCLUSION</b>tDCs induced by tacrolimus with tolerance functionality are a promising cellular therapeutic for immunomodulation.</p>
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Humanos , Proliferación Celular , Células Cultivadas , Células Dendríticas , Alergia e Inmunología , Tolerancia Inmunológica , Prueba de Cultivo Mixto de Linfocitos , Tacrolimus , FarmacologíaRESUMEN
Objective To investigate the effect of CCAAT/enhancer-binding protein homologeus protein (CHOP) in Tan Ⅱ A treated acute promyelocytic leukemia (APL) cells.Methods APL cell differentiation was monitored by morphology and membrane differentiation antigens; expression of CHOP was inhibited by siCHOP; mRNA and protein expression of CHOP in Tan Ⅱ A-intervened APL cells were examined by RT-PCR and Western blot.Results Expression of CHOP was increased in Tan Ⅱ A induced differentiated APL cells (proteins levels 1.933±0.987 vs 0.537±0.110,F =114.852,P < 0.01,mNRA levels 1.587±0.815 vs 0.713±0.090,F =52.256,P < 0.01),combination of CHOP gene silencing with Tan Ⅱ A treatment induced strong APL cell differentiation [(50.767±1.241) % vs (16.167±2.122) %,F =989.431,P < 0.05] and apoptosis [(89.233±5.581) % vs (27.433±2.957) %,F =308.961,P < 0.05].Conclusion CHOP acts as a negative regulator in Tan Ⅱ A induced differentiation.Inhibition of CHOP may be a promising therapeutic strategy.
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A method for qualitative analysis of constituents in Panax notoginseng by UPLC-LTQ-Orbitrap mass spectrometry was established. Based on the high-resolution mass information, MS/MS fragmentation behaviors and chemical components from literatures, 43 compounds were identified or tentatively characterized. New type saponin aglycone, combined with malonyl-substituted and acetyl-substituted saponins were discovered and plausibly identified in this study. This work could be helpful for the quality control and further phytochemical studies of Panax notoginseng, and provided a good example for the analysis of chemical constituents in traditional Chinese medicine.
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BACKGROUND:The brain mechanism of semantic processing is one of the focus problems in cognitive neuroscience.With the research technologies plentiful and diversified, the brain mechanism of semantic processing is gradually distinct.However, at present, the related researches on Chinese semantic processing are not enough. The brain mechanism of semantic processing by Chinese language obstacle should be studied more deeply.OBJECTIVE: To further identify the neuropsychological significance of clinical diagnosis, treatment and rehabilitation by concluding the study fruits on the brain mechanism of semantic processing by native Chinese speaker with dysphonia and analyze the relationship between the related brain mechanism and the local brain system of semantic processes as well as brain anatomical parts.RETRIEVE STRATEGY: The retrieve staffs are the research personnel for this paper. The range of retrieve time focuses since 1984. A computer-based online search was conducted in CNKI for literatures related to basic neuropsychology and its clinical application published between January 1994 and May 2006, in Elsevier for articles between January 1984 and May 2006, in Academic Source Premier and MEDLINE of EBSCOhost for studies between January 1984 and May 2006 with the key words of "semantic processing", and the language was limited to English. Meanwhile, relevant data were searched manually. The number of total retrieved articles was 264, among which 43 enrolled studies were in accordance with the inclusion criteria and excluded articles involving semantic processing or encephalic region but without their relationship. The unpublished articles were only used for references LITERATURE EVALUATION: The literatures are selected from related works, collected analyses, reports from single case or research. Evaluated persons are related research staffs.DATA SYNTHESIS: Processing of Chinese semantic relied primarily on the left superior temporal region, middle temporal gyrus, the inferior gyrus of frontal regions as well as the left middle frontal gyrus, which make up a frontal-temporal network for semantic processing. Lexical-semantic processing is strongly correlated with activation in the posterior portion of left superior temporal region and the middle temporal gyrus, which appear to be responsible for storage and automatic of semantic processing. The anterior temporal region is related to integrate different semantic knowledge. More strategic processes and those that require specific memory resources may be represented in the inferior frontal cortex. In addition, the left middle frontal gyrus is special to Chinese semantic process, and some scholars infer the reason is the unique style of Chinese characters.CONCLUSION: At present, there are many methods to study neuropsychology, brain tissues and its functions, with which a great deal of neuropsychological disorder and mechanism of pathological changes in clinic can be studied and comprehended more distinctly, and all these are greatly helpful to the treatment and rehabilitation.
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Objective To explore the diagnostic value and clinical significance of simultaneous detection of AFP,AFU and TSGF in patients with primary hepatic carcinoma(PHC).Methods Serum AFP,AFU and TSGF were detected simultaneously in 62 patients with PHC and 30 controls.Results The levels of AFU and TSGF in primary hepatic carcinoma were all significantly higher than that of controls(P
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<p><b>OBJECTIVE</b>To explore the relationship between NB4 cell apoptosis induced by tanshinone IIA (TanIIA) and the cell mitochondrial transmembrane potential (DeltaPsim).</p><p><b>METHODS</b>NB4 cells were treated with TanIIA, As(2)O(3), TanIIA plus 1.0 micro g/ml CsA and As(2)O(3) plus 1.0 micro g/ml CsA, respectively. Morphological changes were observed under light microscope and transmission electron microscope. The percentages of sub-G(1) cells and DeltaPsim of cells doublely stained with PI and Rh123 were assayed by flow cytometry.</p><p><b>RESULTS</b>The percentages of sub-G(1) cells after treatment with 1.0 micro g/ml and 2.0 micro g/ml TanIIA had no significant difference but was higher than that of 0.5 micro g/ml. After treatment with TanIIA, NB4 cells appeared the classical apoptotic morphology. The percentages of sub-G(1) cells were increased, while the DeltaPsim reduced (P < 0.01) and there was a linear correlation between them. The increment of sub-G(1) cell percentages and decrement of DeltaPsim induced by TanIIA were partly inhibited by CsA (P < 0.01).</p><p><b>CONCLUSIONS</b>TanIIA can induce NB4 cells apoptosis through opening the mitochondrial permeability transition pore and reducing DeltaPSgr;m, and this effect could be inhibited by CsA.</p>
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Humanos , Antineoplásicos Fitogénicos , Farmacología , Apoptosis , ADN , Metabolismo , Abietanos , Potenciales de la Membrana , Mitocondrias , Fenantrenos , Farmacología , Células Tumorales CultivadasRESUMEN
OBJECTIVE To study commonly encountered pathogenic bacteria leading to lung infection and their drug sensitivity. METHODS In the period of Jan 2003 to Feb 2005,the pathogenic species and drug sensitivity of 233 positive samples isolated from 496 phlegm samples were analyzed.RESULTS Totally 234 strains of pathogenic bacteria were(isolated) from 233 phlegm samples.It had been found that 132 strains of Gram-negative bacilli,(accounted) for(56.4%);57 strains of Gram-positive cocci,accounted for 24.4 %;41 strains of fungi,accounted for 17.5%;and 4 strains of Gram-positive bacilli,accounted for 1.7%.The pathogens of the highest infective rate were:(Pseudomonas) aeruginosa(15.4%),Candida albicans(11.6 %),Enterobacter cloacae(6.0%),Klebsiella(pneumoniae)(5.6%),Escherichia coli(5.1%),Staphylococcus aureus(4.3 %),Streptococus pyogenes((4.3%)),Acinetobacter baumannii(3.6%),and C.tropicalis(3.6 %).Results of drug sensitivity indicated that drugs selected sensitive to Gram-negative bacilli were:imipenem(75.4%),piperacillin/tazobactam(38.9%),(ciprofloxacin)(37.3%),gentamicin(37.3%),and amikacin(36.5%).Drugs sensitive to Gram-positive cocci were:vancomycin(75%),rifampin(55%),and erythromycin(35%).CONCLUSIONS The commonly encountered pathogens of lung infection in our hospital are: P.aeruginosia,C.albicans,E.cloacae,K.pneumoniae,E.coli,S.aureus,and S.pyogenes.Infective rate of Candida is increasing evidently.Antibiotic resistance of bacteria(becomes) severe,drugs applied became less and less.Clinic should(actively) develop phlegm cultivation and drug sensitivity test,applying antibiotics reasonably,reducing production of bacteria resistant to drugs.
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Objective:To investigate the influence of psychological intervention on depression and quality of life among perinatal women.Methods:60 perinatal women were selected under the condition of the study.30 cases were chosen randomly from the 60 women as the psychological invention group and another 30 cases as the control group.The interventon group received psychological intervention which included supportive therapy,health education,individual psychotherapy,and family and social supportive therapy;the control group received no invention measure.Psychometrics was used for 30-and 38-week gravida of both groups and continuous psychological intervention was enforced for more than seven weeks for the intervention group.Results:The depressive factor of the psychological group was lower than of the control group(P
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Objective To clone shRNA (short hairpin RNA) recombinant plasmid targeting on COX-2 gene and analyze the nucleic acid sequence of the recombinant plasmid. Methods One pair of 21 bp reverse repeated sequence targeting on COX-2 mRNA spaced by 9 bp nucleotides were synthesized. The complement double strands was formed by annealing and inserted into plasmid pGenesil-1 to generate eukaryotic expression plasmid. The recombinant plasmid was transformed into Jm109 strain, and the recombinant plasmid extracted was identified by restriction enzyme and sequence analysis. Inhibition effects of COX-2 mRNA and protein were detected by RT-PCR and Western blotting respectively. Results The target DNA was directly cloned to vector and the result was correct by sequence analysis. Compared to untransfected group, recombinant expression plasmid pshRNA-COX-2 resulted in reduction of COX-2 mRNA and protein expression to 69.9% and 50.3% respectively. Conclusion The recombinant plasmid targeting on COX-2 gene was successfully constructed, and it inhibited the expression of COX-2 gene significantly.
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Aim To study antiangiogenesis and antitumor of thalidomide.Methods In HUVECs, cell viability was determined by MTT assay;death type was observed by electron microscope; ratio of apoptosis was quantitated by flow cytometry.Angiogenesis was tested in chicken embryo chorioallantoic membrane.Effect of thalidomide on S_(180) was examined in homograft mice and microvascular counts were detected through immunochemical staining method.Results Thalidomide might inhibite the growth of HUVECs with a IC_(50) value of(22.91?1.74) ?mol?L~(-1),cells treated by thalidomide for 48 h displayed morphological characteristics of different stages associated with apoptosis,which were irregular nucleus, condensed chromatin, ballooning endoplasmic reticulum, apoptotic bodies,under electron microscope.Thalidomide might be able to cause apoptosis or necrosis of HUVECs in flow cytometry and raised positive of antiangiogenesis with increasing of dosage in chicken embryo chorioallantoic membrane. Thalidomide as a single agent might not significantly prevent tumor growth but decrease microvascular counts in tumors, however, in combination with cyclophosphamide, thalidomide could decrease dosage of cyclophosphamide and enhance antitumor of cyclophosphamide.Conclusion Thalidomide might hold back angiogenesis,as a single agent, could not significantly prevent S_(180) tumor from growing,but acted synergistically with cyclophosphamide.