[Objective] To observe the dynamic changes of intestinal IL-17,occludin,and ZO-1 in mice with postinfectious irritable bowel syndrome (PI-IBS).[Methods] Forty C57BL/6 mice were randomly divided into 5 groups:control group and infection groups (2 weeks,4 weeks,6 weeks,and 8 weeks after trichinella infection).Infection groups were given by gavaging of 400～500 Trichinella spiralis in 0.2 mL of normal saline.The body weight of mice were recorded at week 2,4,6,and 8 after infection.The visceral sensitivity of mice was measured by the abdominal withdrawal reflex (AWR).The stool was collected continuously for 8 hours to calculate the percentage of fecal water content.Pathological changes of gut were observed by HE staining.The expressions of IL-17,occludin,and ZO-1 in ileocecus and colon were detected by immunohistochemistry and Western blotting.[Results] At week 2 after infection,the acute inflammation of the intestinal tract was observed and the body weight of mice were significantly decreased (P=0.000).Until week 8 after infection,the intestinal inflammation and body weight of mice recovered to normal.When the colorectal dilatation capacity was 0.35 and 0.5 mL,the AWR scores in the infection groups were significantly higher than those in the control group (P＜0.01).The percentages of fecal water content in the infection groups were significantly higher than those in the control group (P＜0.05).Compared with the control group,the expressions of IL-17 were significantly decreased in week 2 group (P＜0.05) and increased in week 8 group (P＜0.05).The expressions of occludin and ZO-1 in the infection groups were significantly lower than those in the control group (P＜0.05).[Conclusion] The dynamic changes of IL-17 and the decrease of Tight junction proteins may be one of the mechanisms of visceral hypersensitivity and increased percentages of fecal water content.They may be involved in the development of PI-IBS.

Objective:To evaluate the application of visual feedback coaching method, which is embedded in an optical surface monitoring system, in deep inspiration breath holding during the radiotherapy in left breast cancer patients after breast-conserving surgery.Methods:Thirty patients with left breast cancer, who were scheduled to receive the whole breast radiotherapy after breast-conserving surgery, met the requirements of deep inspiration breath holding after respiratory coaching with the visual feedback coaching module in the optical surface monitoring system. Active breathing control equipment was used to control breath-holding state and CT simulation was performed. During treatment, optical surface monitoring system was used to guide radiotherapy. All patients were randomly divided into two groups. In group A ( n=15), visual feedback respiratory training method was utilized and not employed in group B ( n=15). In group A, the visual feedback coaching bar of the optical surface monitoring system was implemented, while audio interactive method was employed to guide patients to hold their breath. Real-time data of optical body surface monitoring were used to compare the interfraction reproducibility and intrafraction stability of breath holding fraction between two groups. Besides, the number of breath holding and treatment time per fraction were also compared. GraphPad prism 6.0 software was used for data processing and mapping, and SPSS 21.0 software was used for analyzing mean value and normality testing. Results:Compared with the control group, the reproducibility in the experiment group was reduced from 1.5 mm to 0.7 mm, the stability was reduced from 1.1 mm to 0.8 mm, the mean number of breath holding required per fraction was decreased from 4.6 to 2.4, the mean beam-on time per fraction from 336 s to 235 s, and the treatment time per fraction was shortened from 847 s to 602 s (all P<0.05), respectively. Conclusions:The application of visual feedback coaching method can improve the reproducibility and stability of breath holding during radiotherapy for left breast cancer, and it can also effectively reduce the number of breath holding and shorten the treatment time per fraction.

OBJECTIVETo investigate the molecular mechanism of a Chinese hemophilia A patient in whom there was a discrepancy between the clinical bleeding symptoms and laboratory assay of FVIII activity (FVIII: C).

METHODSFVIII: C was detected by chromogenic and one-stage methods, and FVIII: Ag by ELISA. The APTT corrected test was used to screen the FVIII inhibitor and PCR amplification to analyze all the exons and flanking sequences of F8 gene of the proband, PCR products were purified and sequenced directly. The corresponding gene sites of family members were detected according to the gene mutation sites. Two B domain deleted human FVIII mutant expression plasmids His99Arg and His99Ala (pRC/RS V - BDhFVIIIcDNA) were constructed and transfected into HEK293T transiently. FVIII: Ag and FVIII: C of the expression products were assayed.

RESULTSThe proband APTT was prolonged, FVIII: Ag was 120% but FVIII: C <1% and no FVIII inhibitor in plasma. The results of anticoagulation and fibrinolytic functions were normal. The cross reacting material positive (CRM+) hemophilia A was diagnosed. Gene analysis revealed a A28828G substitution in exon 3 resulted in a H (His) to R (Arg) missense mutation and the same heterozygous was identified in his mother. In vitro expression of FVIII: Ag and FVIII: C of His99Arg were 180.0% and 5.8% , respectively, while FVIII: Ag and FVIII: C of His99Ala were 45.0% and 20.0% of that of wild type, respectively. His99Arg and His99Ala were diagnosed as CRM+ and CRM- mutations, respectively.

CONCLUSIONBoth the two F VIII mutations could express FVIII protein. However, CRM His99Arg mutant protein has little FVIII procoagulant activity and His99Ala has reduced FVIII function by routine methods.

Adult ; DNA Mutational Analysis ; Factor VIII ; genetics ; Genotype ; Hemophilia A ; etiology ; genetics ; Humans ; Male ; Mutation, Missense

BACKGROUNDIn bone marrow transplant patients, the microenvironment in bone marrow is damaged after chemotherapy or radiotherapy. Subsequent to allogenic hematopoietic stem cell transplantation in patients with clinically successful engraftments, the source of mesenchymal stem cells (MSCs) remains controversial. To further verify the stimulatory effect of the simultaneous transplantation of cells from second donors on engraftment success for hematopoietic stem cell transplantation in support of donor MSCs engraftments, the aim of this study is to monitor the dynamics of the engraftment of bone marrow-derived MSCs in patients after transplantation with mismatched-sex hematopoietic stem and third-party cells.

METHODSIn this study, the hematopoietic stem cells from 32 clinical donors of different sexes that resulted in successful engraftments were selected for transplantation and were classified into three groups for research purposes: group A consisted of 14 cases of transplantation with bone marrow and recruited peripheral hematopoietic stem cell transplantation, group B contained 8 cases of simultaneous re-transfusion of MSCs from the second donor, and group C contained 10 cases of simultaneous re-transfusion of umbilical blood from the second donor. The bone marrow from 32 patients with successful engraftments of hematopoietic transplantation were selected and sub-cultured with MSCs. Flow cytometry (FCM) was used to measure the expression of surface antigens on MSCs. Denaturing high-performance liquid chromatography (DHPLC) in combination with polymerase chain reaction amplification of short tandem repeats (STRPCR) was used to measure the engraftment status of fifth-generation MSCs in patients. Fluorescence in situ hybridization (FISH) revealed the sex origin of the fifth-generation MSCs in 32 patients. Dynamic examinations were performed on patients receiving donor transplantations.

RESULTSThe progenies of fifth-generation MSCs were successfully cultured in 32 cases. The results of FCM demonstrated that the expression levels of CD14+ and CD45+ cells were lower than 0.04% in the fifth-generation MSCs. The analysis using DHPLC and FISH showed similar results. One patient from group B also received a temporary transplantation of MSCs from the donor. The MSCs in the remaining 31 patients all originated from the patients themselves.

CONCLUSIONSAfter transplantation, the MSCs present in patients originated from the host. In patients transplanted with MSCs from a second donor, the phenomenon of temporary chimerization of MSCs was observed.

Adolescent ; Adult ; Cells, Cultured ; Chromatography, High Pressure Liquid ; Female ; Flow Cytometry ; Hematopoietic Stem Cell Transplantation ; Humans ; Leukocyte Common Antigens ; metabolism ; Lipopolysaccharide Receptors ; metabolism ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Middle Aged ; Young Adult

OBJECTIVETo study the efficacy and safety of immunotherapy and imatinib mesylate used in early post allogeneic hematopoietic stem cell transplantation (HSCT) for intervention.

METHODSSixty-four chronic myelogenous leukemia (CML) patients received HSCT were analyzed retrospectively based on bcr-abl kinetics post HSCT. Patients were divided into three groups, imatinib group (n = 13), immunotherapy group (n = 20)and combining both group (n = 31). The primary endpoint is molecular response, the second endpoint is side effect related to intervention.

RESULTSThere was no difference among the three groups in converting to bcr-abl negativity (86.0%, 90.0% and 83.9%, respectively, P = 0.126), 4 years cumulative relapse incidence (32.3%, 0% and 16.1%, respectively, P = 0.130) and 4 years OS (90.0%, 89.7%, 83.0%, respectively, P = 0.696). There was a trend of more relapse in Imatinib group than in immunotherapy group (P = 0.052). There were more hematological toxicity in imatinib and combining groups than that in immunotherapy group (30.8%, 38.7%, and 5.0%, respectively, P = 0.001). There was significant difference in the incidence of GVHD among the three groups (P = 0.000), being 95.0%, 0% and 67.7% in immunotherapy, imatinib and combining groups, respectively. Intervention related death occurred in 2 cases, both with discontinuation of CsA, graft failure in another patient with CsA withdrawal. No intervention related death occurred in the other two groups.

CONCLUSIONSAll three regimens can give a quick and durable molecular remission in most of the patients, but side effects are different. The choice of regimen should be balanced with toxicity individually. CsA withdrawal is not the best choice for very early intervention, the long-term effect for patients received imatinib alone without GVHD is needed to be studied.

Fusion Proteins, bcr-abl ; Graft vs Host Disease ; Hematopoietic Stem Cell Transplantation ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; drug therapy ; Recurrence

BACKGROUNDRecent studies have suggested that susceptibility to chronic obstructive pulmonary disease (COPD) might be related to the length polymorphism of (GT)(n) repeat in the 5'-flanking region of heme oxygenase-1 (HOX-1) gene. However, there has been no research about the relationship between the polymorphism of HOX-1 gene and severity of COPD.

METHODSThe polymorphism of HOX-1 gene in 452 patients with COPD from Han population in Southwest China was analysed by fragment analysis. The frequencies of the HOX-1 genotype were compared with the stage of COPD of each patient.

RESULTSThe HOX-1 genotypes were classified into two groups: group I were individuals with class L allele (the number of GT = 32 repeats), and group II were those without class L allele (the number of GT < 32 repeats). The genotypic frequency of the HOX-1 group I was significantly higher than group II in the very severe COPD patients (36.8% vs 22.4%, P < 0.01, OR = 2.0, 95% CI 1.3 - 3.1), while the genotypic frequency of the HOX-1 group II was lower in the mild COPD (16.0% vs 26.0%, P = 0.02, OR = 0.5, 95% CI 0.3 - 0.9). However, in moderate and severe stages COPD, there were similar genotypic frequencies between HOX-1 group I and group II.

CONCLUSIONSGenetic polymorphism in HOX-1 is associated with the severity of COPD in Southwest China. COPD patients with class L allele may be susceptible to develop very severe COPD. Conversely, the COPD patients without class L allele may be more easily stabilized on mild COPD.

Adult ; Aged ; Female ; Forced Expiratory Volume ; Genotype ; Heme Oxygenase-1 ; genetics ; Humans ; Male ; Middle Aged ; Polymorphism, Genetic ; Pulmonary Disease, Chronic Obstructive ; enzymology ; genetics ; physiopathology

OBJECTIVETo analyze phenotype and genotype of three Chinese pedigrees with von Willebrand disease (vWD), and explore the molecular mechanism.

METHODSBleeding time (BT), activated partial thromboplastin time (APTT), ristocetin-induced platelet aggregation (RIPA), von Willebrand factor (vWF): ristocetin cofactor (RCof) (vWF:RCof), vWF antigen (vWF:Ag), vWF activity (vWF:A) test, vWF collagen binding assay (vWF:CB), vWF and Factor VIII (FVIII) binding assay (vWF:FVIII:B) and multimer analysis were used for phenotype diagnosis. Genomic DNA was extracted from the peripheral blood (PB). All the 52 exons and flanking sequences of the probands' vWF gene were amplified by PCR and analyzed by direct sequencing.

RESULTSAPTT were prolonged in all three probands, while BT were normal excepting for proband 3. Plasma RIPA, vWF:RCo, vWF:Ag, vWF:A and vWF:CB were decreased in different extents. In multimer analysis, proband 3 lost the large and intermediate molecular weight multimers, while proband 1 and 2 were normal. Gene analysis in the three probands revealed three heterozygous missense mutations of 144067 G→A (R2287Q) in exon 39, 110374G→A (R1374H) and 110770C→T (S1506L) in exon 28 and heterozygous polymorphism 110667G→A (D1472H) in exon 28, respectively.

CONCLUSIONThe three heterozygous mutations (R2287Q, R1374H and S1506L) and an heterozygous polymorphism (D1472H) are genetic defects of the hereditary vWD of the three pedigrees respectively. R2287Q is a novel mutation reported for the first time in the literature.

Adult ; Child ; DNA Mutational Analysis ; Female ; Genotype ; Heterozygote ; Humans ; Male ; Pedigree ; Phenotype ; von Willebrand Diseases ; diagnosis ; genetics ; von Willebrand Factor ; genetics ; metabolism

BACKGROUNDAnalysis of changes in recipient and donor hematopoietic cell origin is extremely useful to monitor the effect of hematopoietic stem cell transplantation (HSCT) and sequential adoptive immunotherapy by donor lymphocyte infusions. We developed a sensitive, reliable and rapid real-time PCR method based on sequence polymorphism systems to quantitatively assess the hematopoietic chimerism after HSCT.

METHODSA panel of 29 selected sequence polymorphism (SP) markers was screened by real-time PCR in 101 HSCT patients with leukemia and other hematological diseases. The chimerism kinetics of bone marrow samples of 8 HSCT patients in remission and relapse situations were followed longitudinally.

RESULTSRecipient genotype discrimination was possible in 97.0% (98 of 101) with a mean number of 2.5 (1-7) informative markers per recipient/donor pair. Using serial dilutions of plasmids containing specific SP markers, the linear correlation (r) of 0.99, the slope between -3.2 and -3.7 and the sensitivity of 0.1% were proved reproducible. By this method, it was possible to very accurately detect autologous signals in the range from 0.1% to 30%. The accuracy of the method in the very important range of autologous signals below 5% was extraordinarily high (standard deviation <1.85%), which might significantly improve detection accuracy of changes in autologous signals early in the post-transplantation course of follow-up. The main advantage of the real-time PCR method over short tandem repeat PCR chimerism assays is the absence of PCR competition and plateau biases, with demonstrated greater sensitivity and linearity. Finally, we prospectively analyzed bone marrow samples of 8 patients who received allografts and presented the chimerism kinetics of remission and relapse situations that illustrated the sensitivity level and the promising clinical application of this method.

CONCLUSIONThis SP-based real-time PCR assay provides a rapid, sensitive, and accurate quantitative assessment of mixed chimerism that can be useful in predicting graft rejection and early relapse.

Adolescent ; Adult ; Child ; Female ; Genotype ; Hematopoietic Stem Cell Transplantation ; Humans ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; genetics ; Real-Time Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Transplantation Chimera ; genetics ; Young Adult

BACKGROUNDChimerism analysis is an important tool for the surveillance of post-transplant engraftment. It offers the possibility of identifying impending graft rejection and recurrence of underlying malignant or non-malignant disease. Here we investigated the quantitative chimerism kinetics of 21 relapsed leukemia patients after allogeneic hematopoietic stem cell transplantation (HSCT).

METHODSA panel of 29 selected sequence polymorphism (SP) markers was screened by real-time polymerase chain reaction (RT-PCR) to obtain the informative marker for every leukemia patient. Quantitative chimerism analysis of bone marrow (BM) samples of 21 relapsed patients and 20 patients in stable remission was performed longitudinally. The chimerisms of BM and peripheral blood (PB) samples of 14 patients at relapse were compared.

RESULTSTwenty-one patients experienced leukemia relapse at a median of 135 days (range, 30 - 720 days) after transplantation. High recipient chimerism in BM was found in all patients at relapse, and increased recipient chimerism in BM samples was observed in 90% (19/21) of patients before relapse. With 0.5% recipient DNA as the cut-off, median time between the detection of increased recipient chimerism and relapse was 45 days (range, 0 - 120 days), with 76% of patients showing increased recipient chimerism at least 1 month prior to relapse. Median percentage of recipient DNA in 20 stable remission patients was 0.28%, 0.04%, 0.05%, 0.05%, 0.08%, and 0.05% at 1, 2, 3, 6, 9, and 12 months, respectively, after transplantation. This was concordant with other specific fusion transcripts and fluorescent in situ hybridization examination. The recipient chimerisms in BM were significantly higher than those in PB at relapse (P = 0.001).

CONCLUSIONSThis SP-based RT-PCR assay is a reliable method for chimerism analysis. Chimerism kinetics in BM can be used as a marker of impending leukemia relapse, especially when no other specific marker is available. Based on our findings, we recommend examining not only PB samples but also BM samples in HSCT patients.

Adolescent ; Adult ; Child ; Female ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Leukemia ; genetics ; therapy ; Male ; Middle Aged ; Reverse Transcriptase Polymerase Chain Reaction ; Transplantation Chimera ; genetics ; Transplantation, Homologous ; adverse effects ; Young Adult

Background Chimerism analysis is an important tool for the surveillance of post-transplant engraftment.It offers the possibility of identifying impending graft rejection and recurrence of underlying malignant or non-malignant disease.Here we investigated the quantitative chimerism kinetics of 21 relapsed leukemia patients after allogeneic hematopoietic stem cell transplantation (HSCT).Methods A panel of 29 selected sequence polymorphism (SP) markers was screened by real-time polymerase chain reaction (RT-PCR) to obtain the informative marker for every leukemia patient.Quantitative chimerism analysis of bone marrow (BM) samples of 21 relapsed patients and 20 patients in stable remission was performed longitudinally.The chimerisms of BM and peripheral blood (PB) samples of 14 patients at relapse were compared.Results Twenty-one patients experienced leukemia relapse at a median of 135 days (range,30-720 days) after transplantation.High recipient chimerism in BM was found in all patients at relapse,and increased recipient chimerism in BM samples was observed in 90％ (19/21) of patients before relapse.With 0.5％ recipient DNA as the cut-off,median time between the detection of increased recipient chimerism and relapse was 45 days (range,0-120 days),with 76％ of patients showing increased recipient chimerism at least 1 month prior to relapse.Median percentage of recipient DNA in 20 stable remission patients was 0.28％,0.04％,0.05％,0.05％,0.08％,and 0.05％ at 1,2,3,6,9,and 12 months,respectively,after transplantation.This was concordant with other specific fusion transcripts and fluorescent in situ hybridization examination.The recipient chimerisms in BM were significantly higher than those in PB at relapse (P=0.001).Conclusions This SP-based RT-PCR essay is a reliable method for chimerism analysis.Chimerism kinetics in BM can be used as a marker of impending leukemia relapse,especially when no other specific marker is available.Based on our findings,we recommend examining not only PB samples but also BM samples in HSCT patients.