OBJECTIVETo develop a colon-specific prodrug of Indomethacin microbially triggered, carry out in vitro/in vivo evaluation of drug release, and appraise its inhibitory effect on liver metastasis from colon cancer.

METHODSIndomethacin prodrugs were synthesized and characterized by FTIR and NMR, and dissolution test simulating gastrointestinal tract was employed to screen the colon-specific prodrug. Then, the pharmacokinetic profile of portal vein and peripheral blood in Sprague-Dawley rats was studied. Lastly, the inhibitory effect on liver metastasis from colon cancer in nude mice was observed.

RESULTSThe chemical structure characterized by FTIR and NMR demonstrated that six kinds of indomethacin-block-amylose with different drug loading (IDM-AM-1-6) were synthesized, among which IDM-AM-3 was degraded 1.3%, 9.3% and 95.3%, respectively, in simulated gastric fluid for 4 h, small intestine for 6 h, and colon for 36 h. The pharmacokinetic test of IDM-AM-3 showed that absorption was delayed significantly (P < 0.01), peak time [(11.35 + or - 2.45) h], elimination half-life [(16.74 + or - 4.04) h] and mean residence time [(22.27 + or - 0.52) h] were significantly prolonged (P < 0.01), as well as peak serum concentrations [(9.69 + or - 2.40) mg/L] and AUC(0-t) [(236.7 + or - 13.1) mg x L(-1) x h] were decreased markedly (P < 0.01) as compared with those of IDM regarding to portal vein. Additionally, its AUC(0-t) in peripheral blood was remarkably lower than that in Portal vein (P < 0.01). The tumor suppression observation showed that it could remarkably reduce the number of liver metastases in contrast to IDM (P < 0.05).

CONCLUSIONColon-specific IDM-AM-3 possesses advantage of sustained release in portal vein providing some experimental basis for colon-specific delivery system applied to sustained release in the portal vein.

Amylose ; administration & dosage ; chemical synthesis ; pharmacokinetics ; therapeutic use ; Animals ; Colon ; metabolism ; Colonic Neoplasms ; pathology ; Delayed-Action Preparations ; Drug Delivery Systems ; HT29 Cells ; Humans ; Indomethacin ; administration & dosage ; chemical synthesis ; pharmacokinetics ; therapeutic use ; Liver Neoplasms ; prevention & control ; secondary ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Prodrugs ; administration & dosage ; chemical synthesis ; pharmacokinetics ; therapeutic use ; Random Allocation ; Rats ; Rats, Sprague-Dawley

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; China ; Disasters ; statistics & numerical data ; Earthquakes ; Female ; Fractures, Open ; psychology ; surgery ; Humans ; Leg Injuries ; psychology ; surgery ; Male ; Middle Aged

To evaluate the significance of FCM in minimal residual disease (MRD) detection, the immunophenotyping and leukemia-associated immunophenotypes (LAIP) of leukemia cells from 273 adult and 142 childhood patients with B lineage acute lymphoblastic leukemia (B-ALL) were detected by four to six antibody combinations of 4-color CD45/SSC gating multiparametric flow cytometry (FCM). The results showed that the B-ALL patients could be classified into 4 subtypes based on different expression CD34 and CD10: subtype I (CD34(+)/CD10(-)), subtype II (CD34(+)/CD10(+)), subtype III (CD34(-)/CD10(+)), subtype IV (CD34(-)/CD10(-)). The LAIP was observed in 100% and 92% patients of subtype I and subtype II, respectively, whereas only 79.2% in subtype III. The incidence of LAIP in total B-ALL cases was 90% by using the antibodies detected in this investigation. There was no significantce different for incidence of LAIP between adult and pediatric patients. LAIP was observed in 77.6% of patients by labeling only CD34/CD10/CD19/CD45 4-color antibody combination. It is concluded that in 90% of childhood and adult B-ALL patients LAIP can be found, which suits MRD detection by multiparameter flow cytometry.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antigens, CD34 ; analysis ; B-Lymphocytes ; immunology ; Burkitt Lymphoma ; classification ; immunology ; pathology ; Cell Lineage ; Female ; Flow Cytometry ; methods ; Humans ; Immunophenotyping ; Male ; Middle Aged ; Neoplasm, Residual ; diagnosis ; Neprilysin ; analysis ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; classification ; immunology ; pathology

OBJECTIVETo compare the leukemia-associated immunophenotypes (LAIP) in patients with B lineage acute lymphoblastic leukemia (B-ALL) at diagnosis and relapse, and investigate its implications for minimal residual disease (MRD) detection.

METHODSThe immunophenotype of leukemia cells from 410 newly diagnosed and 6 relapsed patients with B-ALL were detected by four to six antibody combination, mainly CD34/CD10/CD45/CD19 of 4-color CD45/SSC gating flow cytometry (FCM).

RESULTSThe proportion of CD45 under-expressed or negative in relapsed patients was much higher than that in newly diagnosed patients, being 69.2% and 37.8% respectively. Immunophenotypic changes occurred in 9 relapsed patients (including 8 hematological relapse and 1 central nerves system relapse) when analyzed by paired samples analysis at diagnosis vs at relapse: 4 cases showed CD45 down-modulation and 2 up-modulation; 4 CD34 down-modulation and 2 CD10 up-modulation, while the expression of CD19 remained no change. MRD was observed in all 7 cases of hematological relapse 2 - 4 months before relapse, and the immunophenotype of MRD cells was the same as that in relapse.

CONCLUSIONA high frequency of immunophenotypic changes occurred at relapse and even in MRD before relapse, however the accuracy of MRD monitoring seemed not affected by the FCM strategy used in this investigation.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antigens, CD19 ; immunology ; Antigens, CD34 ; immunology ; Child ; Child, Preschool ; Female ; Flow Cytometry ; methods ; Humans ; Immunophenotyping ; methods ; Leukocyte Common Antigens ; immunology ; Male ; Middle Aged ; Neoplasm, Residual ; diagnosis ; immunology ; pathology ; Neprilysin ; immunology ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ; diagnosis ; immunology ; pathology ; Recurrence ; Young Adult

OBJECTIVETo evaluate the clinical significance for minimal residual disease (MRD) detection by 4 color flow cytometry in B lineage acute lymphoblastic leukemia (B-ALL).

METHODSMRD was analyzed and followed up by using two panels of 4 color antibodies, mainly CD34/CD10/CD45/CD19, in 671 consecutive bone marrow specimens and 1 cerebrospinal fluid from 98 B-ALL patients. In 26 cases of them the immunophenotyping informations at diagnosis were not available.

RESULTSOf 671 bone marrow samples, 579 were MRD negative with leukemic cells below 0.0001 and 93 were MRD positive with leukemic cells over 0.0001. Of 93 MRD positive samples, leukemic cells below 0.05 were found in 64 bone marrow samples, meanwhile in the other 29 samples leukemic cells were over 0.05. Twenty patients relapsed, 19 were bone marrow relapse and one center nerves system. Fifteen of them were found MRD positive 7 - 17 weeks before relapse including 6 patients having no immunophenotyping data at diagnosis. The percentages of leukemia cells in these 15 patients were all over 0.0001. Two relapsed patients were MRD negative in 3 and 9 months before relapse, respectively. Two relapsed after MRD monitoring stopped. If MRD level was > 0.0001 at the end of induction chemotherapy and 12 weeks of treatment, the rate of relapse was 50% (6/12), while, it was 7.5% (3/40) in MRD negative patients (P = 0.000).

CONCLUSIONRelapses can be predicted by MRD monitoring, if MRD was positive in the early phase of treatment, the risk of relapse was higher. Based on the characteristics of B cells ontogeny, MRD detection can be done independently of immunophenotypic information at diagnosis.

Acute Disease ; Adolescent ; Adult ; Aged ; Antigens, CD19 ; immunology ; Antigens, CD34 ; immunology ; Child ; Child, Preschool ; Female ; Flow Cytometry ; methods ; Follow-Up Studies ; Humans ; Immunophenotyping ; Leukocyte Common Antigens ; immunology ; Male ; Middle Aged ; Neoplasm, Residual ; diagnosis ; immunology ; Neprilysin ; immunology ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ; diagnosis ; immunology ; Young Adult

This study was purposed to investigate the effect of adenovirus-mediated transfer of PDCD5 gene on apoptosis of K562 cells induced by etoposide. Recombinant adenovirus PDCD5 (Ad-PDCD5), control vectors Ad-null and Ad-eGFP were constructed by AdMax vector system respectively. After K562 cells were transfected by Ad-PDCD5, Ad-null or Ad-eGFP with different multiplicity of infection (MOI), the expression level of the PDCD5 gene was examined by RQ-RT-PCR assay. The effects of etoposide in combination with Ad-PDCD5 on the proliferation and apoptosis of K562 cells were measured by using MTT assay and flow cytometry with Annexin-V-FITC/PI dual labeling technique, respectively. The results showed that the transfection efficiencies of Ad-eGFP in K562, Jurkat and CEM cells ranged from 60% to 86%. Expression level of PDCD5 gene in K562 cells was evidently increased following transfection with Ad-PDCD5. The Ad-PDCD5 synergistically enhanced the apoptotic percentage of K562 cells induced by VP-16, as compared with that of Ad-null + VP16 and VP-16 alone respectively. It is concluded that Ad-PDCD5 may be a potential agent for enhancing the chemotherapy effect.
Adenoviridae ; genetics ; metabolism ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Etoposide ; pharmacology ; Humans ; K562 Cells ; Neoplasm Proteins ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo evaluate the method and value of detecting bone marrow minimal residual disease (MRD) in B lineage acute lymphoblastic leukemia (B-ALL) by multiparameter flow cytometry (FCM).

METHODSThe FCM immunophenotyping of B-lymphocyte precursors in regenerating stage and MRD was analyzed by using two sets of 4-color antibody panels, including mainly CD34, CD10, CD45, CD19, in 26 regenerating bone marrow samples after chemotherapy and 297 consecutive bone marrow specimens from 50 patients with B-ALL, respectively. The immunophenotype of leukemia cells of B-ALL was also detected by four to six antibodies combination of 4-color CD45/SSC gating FCM. The CD19, CD10, CD34 fluorescence intensity of B-cell precursors in normal and leukemic bone marrow was compared by relatively quantitative method.

RESULTSTwelve patients were MRD positive (MRD(+)) among 50 patients during MRD monitoring, the percentages of residual leukemia cells were 0.06% to 7.73%. Eleven cases displayed CD45, CD34, CD19, CD10 antigens aberrant expression. Five patients were bone marrow relapsed and 4 of them were MRD (+) 7-17 weeks before relapse. The percentages of leukemia cells in all the 4 patients were over 0.1%. Only one patient relapsed with MRD negative. The regenerated B precursors could be divided into 3 stages according to the expression of CD45, CD34, CD19, CD10, CD22, and CD20 antigens. Abnormal expression of CD45, CD34, CD19, and CD10 were detected in 71.8% of 213 patients with B-ALL, the percentage of only aberrant expression of CD38 and myeloid antigen was 8.1%.

CONCLUSIONDetection of MRD by multiparameter flow cytometry is a rapid, quantitative and sensitive approach, and has a higher predictability for relapse.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Child, Preschool ; Female ; Flow Cytometry ; methods ; Humans ; Immunophenotyping ; methods ; Leukemia, B-Cell ; diagnosis ; Male ; Middle Aged ; Neoplasm, Residual ; diagnosis

Objective To investigate the associations between normal serum uric acid (UA) levels and knee symptoms and structural changes in patients with knee osteoarthritis (OA).Methods A total of 205 patients with knee OA were enrolled. According to the inclusion and exclusion criteria, 122 patients with OA were included in the study. The general condition of patients were investingated. The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score was used to assess severity of the knee joint symptoms. The Kellgren-Lawrence (K-L) grading system was used to assess radiological severity of the knee joint. Osirix software was used to assess changes of knee structure in magnetic resonance imaging (MRI). Peroxidase method was used to measure the serum levels of uric acid. Patients with normal range of uric acid were included in the study.Results The patients with lower levels of UA (split at the median level, 263 μmol/L) had a higher proportion of women, lower height, weight and lower proportions of K-L grade≥2. After adjustment for sex, age and body mass index, serum UA was negatively associated with total WOMAC scores (β=-16.15, P=0.018), WOMAC pain (β=-3.15, P=0.037), and WOMAC stiffness (β=-1.65, P=0.025). However, there were no significant associations between serum UA levels and knee joint structure changes.Conclusions In OA patients with normal serum UA, serum UA is negatively associated with the severity of joint symptoms, suggesting that UA within normal range may play a protective role in OA symptoms.

To clone,express and purify the E Protein and EDⅢ of Zika virus in E.coli and prepare two kinds of polyclonal antibodies,the virus was amplified by Vero E6 cell culture.Total RNA was extracted by RT-PCR and reverse transcribed into cDNA.The prokaryotic expression vectors pET32a/E and pET28a/EDⅢ were constructed by cDNA sequence of E and EDⅢ gene.Then,recombinant plasmids were transformed into E.coli BL21 and induced by IPTG,and purified by Ni+ column affinity chromatography.BALB/C mice were immunized with purified recombinant proteins.Antiserum was collected and titer was determined by indirect ELISA.Western blot was used to detect the specificity.Results showed that the recombinant proteins were successfully expressed and purified.The titer of the polyclonal antibodies both reached 1:409 600.Western Blot analysis showed that the polyclonal antibodies could specifically recognize the recombinant proteins.Thus,the specific polyclonal antibody were successfully prepared,laying a foundation for further study on the pathogenesis,detection methods and immune strategies of Zika virus.

BACKGROUNDImmune cells within a tumor microenvironment have shown modulatory effects on tumor angiogenic activity. Renal cell carcinoma (RCC) is a hypervascular tumor that reportedly increases the frequency of regulatory T cells (Tregs) in tumor tissues. This study investigated the correlation between Tregs infiltration and angiogenic status in RCC.

METHODSThirty-six patients with RCC were enrolled in the present study, and twenty age-matched healthy donors were included as the control. Tregs were defined as CD4(+)CD25(high)CD127(low/-) T cells. The frequency of Tregs in peripheral blood and tumor infiltrating lymphocytes (TILs) were determined by flow cytometry. The expression of vascular endothelial growth factor (VEGF) in surgical resection specimens were measured with a commercial enzyme-linked immunosorbent assay (ELISA) kit. Microvessel density (MVD) was calculated on slides stained with CD34 antibody. Spearman's rank correlation was performed to evaluate the correlation between the frequencies of Tregs in TILs and VEGF values, as well as between frequencies of Tregs and MVD determinations.

RESULTSCompared to healthy controls, the frequency of peripheral blood Tregs was significantly increased in patients with RCC (P < 0.05). The percentage of tumor-infiltrating Tregs was higher than that of peripheral blood Tregs in patients with RCC (P < 0.01). In addition, the frequency of tumor-infiltrating Tregs was shown to significantly correlate with the pathological stage (P < 0.05) and nuclear grade (P < 0.01). Importantly, a significant positive correlation was observed between the frequency of tumor-infiltrating Tregs and VEGF protein expression (r = 0.51, P < 0.05), as well as between frequencies of Tregs and MVD score (r = 0.39, P < 0.05).

CONCLUSIONSThese observations suggest that the high pro-angiogenic status of RCC may be associated with the accumulation of Tregs in the local microenvironment. Angiogenesis networks may be connected with immune tolerance units and cooperate with each other to facilitate tumor growth and progression.

Adult ; Aged ; Carcinoma, Renal Cell ; immunology ; metabolism ; Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Female ; Flow Cytometry ; Humans ; Immunohistochemistry ; Kidney Neoplasms ; immunology ; metabolism ; Lymphocytes, Tumor-Infiltrating ; immunology ; Male ; Middle Aged ; Neovascularization, Pathologic ; immunology ; metabolism ; T-Lymphocytes, Regulatory ; immunology