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Background@#Peste des petits ruminants (PPR), caused by the PPR virus (PPRV), is an acute and fatal contagious disease that mainly infects goats, sheep, and other artiodactyls.Peripheral blood mononuclear cells (PBMCs) are considered the primary innate immune cells. @*Objectives@#PBMCs derived from goats were infected with PPRV and analyzed to detect the relationship between PPRV replication and apoptosis or the inflammatory response. @*Methods@#Quantitative real-time polymerase chain reaction was used to identify PPRV replication and cytokines expression. Flow cytometry was conducted to detect apoptosis and the differentiation of CD4+ and CD8+T cells after PPRV infection. @*Results@#PPRV stimulated the differentiation of CD4+ and CD8+ T cells. In addition, PPRV induced apoptosis in goat PBMCs. Furthermore, apoptosis and the inflammatory response induced by PPRV could be suppressed by Z-VAD-FMK and Z-YVAD-FMK, respectively.Moreover, the virus titer of PPRV was attenuated by inhibiting caspase-1-dependent apoptosis and inflammation. @*Conclusions@#This study showed that apoptosis and the inflammatory response play an essential role in PPR viral replication in vitro, providing a new mechanism related to the cell host response.
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Hereditary epidermolysis bullosa (EB) is a rare mutilating and lethal single-gene genodermatosis, and places a heavy burden on society and families. Cell therapy has become a very promising method for the treatment of EB due to its excellent and stable clinical efficacy. This review summarizes progress in laboratory research and clinical application of stem cell- and somatic cell-based therapies in EB in recent years.
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BACKGROUND@#Some scholars have found that dermal papilla spheroid–derived exosomes could promote the development of hair follicles. However, whether adipose-derived stem cell exosomes (ADSC-Exos) have a similar effect on hair growth has not been determined yet. Thus, the purpose of this article was to detect whether ADSC-Exos could promote hair regeneration. @*METHODS@#Adipose-derived stem cells (ADSCs) were isolated from 6-week-old C57BL/6 mice. Then, ADSC-Exos were isolated from the ADSCs. Western blotting was used to detect specific exosome markers. The particle size and distribution of the exosomes were analyzed by NanoSight dynamic light scattering. A total of 12 nude mice were randomly divided into two groups (n = 6 each): the ADSC-Exos group and the control group. For the control group, a mixture of freshly isolated dermal cells (DCs) and epidermal cells (ECs) was grafted. For the ADSC-Exos group, a mixture of DCs, ECs, and 50 lg/ml of ADSC-Exos was grafted. Gross evaluation of the hair regeneration was carried out 2–3 weeks after the transplantation, and the graft site was harvested for histology at the third week. @*Results@#The existence of exosomes derived from ADSCs was evidenced by CD63, ALX1, and CD9 expression. Two or three weeks after the grafting, the number of regenerated hairs in the ADSC-Exos group was higher than that in the control group (p < 0.001). Histologically, the terminal hairs were remarkable in the ADSC-Exos group, whereas the hair follicles observed in the control group were comparatively immature. The ADSC-Exos group had a higher number of regenerated follicles than the control group (p < 0.001). In addition, we found that the skin tissues in the ADSC-Exos group had higher PDGF and vascular endothelial growth factor expressions and lower transforming growth factor beta 1 levels than those in the control group @*CONCLUSION@#Our results indicated that ADSC-Exos could promote in vivo hair follicle regeneration.
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Background@#Peste des petits ruminants (PPR) is an infectious disease caused by the peste des petits ruminants virus (PPRV) that mainly produces respiratory symptoms in affected animals, resulting in great losses in the world's agriculture industry every year. Singledomain variable heavy chain (VHH) antibody fragments, also referred to as nanobodies, have high expression yields and other advantages including ease of purification and high solubility. @*Objectives@#The purpose of this study is to obtain a single-domain antibody with good reactivity and high specificity against PPRV. @*Methods@#A VHH cDNA library was established by immunizing camels with PPRV vaccine, and the capacity and diversity of the library were examined. Four PPRV VHHs were selected, and the biological activity and antigen-binding capacity of the four VHHs were identified by western blot, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) analyses. ELISA was used to identify whether the four VHHs were specific for PPRV, and VHH neutralization tests were carried out. ELISA and western blot analyses were used to identify which PPRV protein was targeted by VHH2. @*Results@#The PPRV cDNA library was constructed successfully. The library capacity was greater than 2.0 × 106 cfu/mL, and the inserted fragment size was approximately 400 bp to 2000 bp. The average length of the cDNA library fragment was about 1000 bp, and the recombination rate was approximately 100%. Four single-domain antibody sequences were selected, and proteins expressed in the supernatant were obtained. The four VHHs were shown to have biological activity, close affinity to PPRV, and no cross-reaction with common sheep diseases. All four VHHs had neutralization activity, and VHH2 was specific to the PPRV M protein. @*Conclusions@#The results of this preliminary research of PPRV VHHs showed that four screened VHH antibodies could be useful in future applications. This study provided new materials for inclusion in PPRV research.
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BACKGROUND@#Some scholars have found that dermal papilla spheroid–derived exosomes could promote the development of hair follicles. However, whether adipose-derived stem cell exosomes (ADSC-Exos) have a similar effect on hair growth has not been determined yet. Thus, the purpose of this article was to detect whether ADSC-Exos could promote hair regeneration. @*METHODS@#Adipose-derived stem cells (ADSCs) were isolated from 6-week-old C57BL/6 mice. Then, ADSC-Exos were isolated from the ADSCs. Western blotting was used to detect specific exosome markers. The particle size and distribution of the exosomes were analyzed by NanoSight dynamic light scattering. A total of 12 nude mice were randomly divided into two groups (n = 6 each): the ADSC-Exos group and the control group. For the control group, a mixture of freshly isolated dermal cells (DCs) and epidermal cells (ECs) was grafted. For the ADSC-Exos group, a mixture of DCs, ECs, and 50 lg/ml of ADSC-Exos was grafted. Gross evaluation of the hair regeneration was carried out 2–3 weeks after the transplantation, and the graft site was harvested for histology at the third week. @*Results@#The existence of exosomes derived from ADSCs was evidenced by CD63, ALX1, and CD9 expression. Two or three weeks after the grafting, the number of regenerated hairs in the ADSC-Exos group was higher than that in the control group (p < 0.001). Histologically, the terminal hairs were remarkable in the ADSC-Exos group, whereas the hair follicles observed in the control group were comparatively immature. The ADSC-Exos group had a higher number of regenerated follicles than the control group (p < 0.001). In addition, we found that the skin tissues in the ADSC-Exos group had higher PDGF and vascular endothelial growth factor expressions and lower transforming growth factor beta 1 levels than those in the control group @*CONCLUSION@#Our results indicated that ADSC-Exos could promote in vivo hair follicle regeneration.
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Background@#Peste des petits ruminants (PPR) is an infectious disease caused by the peste des petits ruminants virus (PPRV) that mainly produces respiratory symptoms in affected animals, resulting in great losses in the world's agriculture industry every year. Singledomain variable heavy chain (VHH) antibody fragments, also referred to as nanobodies, have high expression yields and other advantages including ease of purification and high solubility. @*Objectives@#The purpose of this study is to obtain a single-domain antibody with good reactivity and high specificity against PPRV. @*Methods@#A VHH cDNA library was established by immunizing camels with PPRV vaccine, and the capacity and diversity of the library were examined. Four PPRV VHHs were selected, and the biological activity and antigen-binding capacity of the four VHHs were identified by western blot, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) analyses. ELISA was used to identify whether the four VHHs were specific for PPRV, and VHH neutralization tests were carried out. ELISA and western blot analyses were used to identify which PPRV protein was targeted by VHH2. @*Results@#The PPRV cDNA library was constructed successfully. The library capacity was greater than 2.0 × 106 cfu/mL, and the inserted fragment size was approximately 400 bp to 2000 bp. The average length of the cDNA library fragment was about 1000 bp, and the recombination rate was approximately 100%. Four single-domain antibody sequences were selected, and proteins expressed in the supernatant were obtained. The four VHHs were shown to have biological activity, close affinity to PPRV, and no cross-reaction with common sheep diseases. All four VHHs had neutralization activity, and VHH2 was specific to the PPRV M protein. @*Conclusions@#The results of this preliminary research of PPRV VHHs showed that four screened VHH antibodies could be useful in future applications. This study provided new materials for inclusion in PPRV research.
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Objective@#To understand the status and related factors of quality of life (QOL) among children aged 4-5 years old in rural areas of Anhui Province, and to provide a reference for improving the quality of life among children in rural areas.@*Methods@#A total of 4 457 preschool children aged 4-5 years old were selected from rural areas in five counties of Anhui Province by cluster sampling method. Parents of children were surveyed using the Pediatric Quality of Life Inventory Measurement Models 4.0.@*Results@#The total QOL score of children aged 4-5 years old in rural areas of Anhui Province was (79.44±12.51). The scores of emotional function, school performance and psychosocial summary were higher in left-behind children than that in non-left-behind children(t=2.99, 3.51, 3.22, P<0.05). Multivariate Logistic regression analysis showed that the older children (OR=0.82, 95%CI=0.71-0.95) and the bigger size of households (OR=0.85, 95%CI=0.73-0.98) were positively associated with quality of life of children, while the higher father’s educational level(OR=1.40, 95%CI=1.21-1.62), the lower father’s income, mothers doing housework or unemployment and children suffering from illness in the past two weeks (OR=1.76, 95%CI=1.50-2.06) were negatively associated with quality of life of children(P<0.05).@*Conclusion@#The quality of life of children aged 4-5 year old in rural areas of Anhui Province is relatively low. The children’s age, the father’s education level, the father’s annual income, the mother’s occupation, the size of households, and children suffering from illness in the past two weeks were the related factors that affectchildren’s quality of life.
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Objective To analyze the mRNA expression level of lymphoid enhance factor 1 (LEF-1), and to investigate its clinical significance in bone marrow mononuclear cells of patients with chronic myeloid leukemia chronic-phase (CML-CP) after initial diagnosis and chemotherapy, and to analyze its clinical significance. Methods The real-time fluorescence quantitative polymerase chain reaction was used to measure the expression level of LEF-1 gene in 38 CML-CP patients after initial diagnosis and chemotherapy and 20 persons without blood system diseases and neoplastic diseases as normal control. The difference of LEF-1 expression level between the patients and healthy control was compared, and the effect of imatinib on the main molecular response (MMR) was analyzed. Results The expression of LEF-1 mRNA in 38 newly diagnosed patients [0.00214 (0.00020 - 0.02120)] was significantly higher than that in normal controls [0.00101 (0.00009 - 0.00233)] (U= 163.0, P 0.05). The level of LEF-1 mRNA expression of non-MMR group was also higher than that of the normal control group (U= 14.0, P<0.01). The rate of acquiring MMR was significantly higher in high LEF-1 mRNA expression group [84.2 %(16/19)] than that in low expression group [47.4%(9/19)] (χ2=4.209, P<0.01). The time of acquiring MMR was significantly shorter in the high LEF-1 mRNA expression group [(10.0 ± 4.5) months] than that in the low expression group [(14.6 ± 3.8) months] (t= 2.705, P< 0.01). Conclusions LEF-1 may be involved in the occurrence and development of CML, and reflects the tumor burden. It may be one of the indicators to predict the efficacy of imatinib.
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It has long been one significant focus in the field of surgery to reduce postoperative incisional complications.Recently,incisional negative pressure wound therapy (iNPWT),which was designed to reduce the incidence of incisional complications,has gradually been applied for primary incision closure.This article reviews the relevant basic and clinical studies to elucidate the mechanism of iNPWT and its clinical safety and efficacy,and answers some fundamental questions regarding clinical application of iNPWT.
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Objective To quantitatively analyze the mRNA expression level of lymphoid enhance factor 1 (LEF-1) in bone marrow mononuclear cells of patients with acute myeloid leukemia (AML) at intermediate-risk after initial diagnosis and chemotherapy, and to analyze its clinical significance. Methods The real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was used to measure the expression level of LEF-1 gene in AML patients at intermediate-risk after initial diagnosis and chemotherapy, and its relationship with effectiveness and survival were analyzed. Results The LEF-1 mRNA level in preliminarily diagnosed patients with AML was significantly higher than that in control arm [0.00519 (0.00015-0.09207) vs. 0.00101 (0.00009-0.00233)], and the difference was statistically significant (u=134.50, P<0.01). The LEF-1 mRNA level in patients after chemotherapy was significantly declines as compared to that in patients before chemotherapy [0.00107 (0.00008 - 0.00744) vs. 0.00519 (0.00015 - 0.09207)], and the difference was statistically significant (u= 317.00, P< 0.01) and LEF-1 mRNA expression level before chemotherapy in complete remission (CR) patients was significantly higher than that in non-CR patients [(0.01108 (0.00164 - 0.09207) vs. 0.00110 (0.00015 - 0.00916)], and the difference was statistically significant (u=19.00, P<0.01). High LEF-1 expression predicted a significantly better overall survival in AML patients with intermediate-risk cytogenetics (χ2= 4.549, P= 0.033). Conclusions LEF-1 may be involved in the development and progression of AML at intermediate-risk patients and is closely related to tumor burden and treatment efficacy. LEF-1 may be a good predictor of better prognosis and a novel target for therapeutic effect.
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Objective To investigate the mRNA level of lymphoid enhancing factor-1 ( LEF-1) in bone marrow mononuclear cells after the initial diagnosis and chemotherapy of patients with multiple myeloma (MM) and its clinical significance. Methods The LEF-1 mRNA of target gene in 42 MM patient was detected by real-time fluorescence quantitative polymerase chain reaction (RTQ-PCR), and 20 patients without hematological disease were enrolled as the healthy controls. Results The LEF-1 mRNA median level in previously diagnosed MM patients was significantly higher than that in the healthy controls [0.01068 (0.00017 - 0.14100) vs. 0.00101 (0.00009 - 0.002326)], and the difference was statistically significant (U = 91.00, P< 0.001); The LEF-1 mRNA median level in MM patients after chemotherapy was declined compared with the patients before chemotherapy [0.00011 (0.00001 - 0.01548) vs. 0.01068 (0.00017 -0.14100)], and the difference was statistically significant (U = 343.0, P< 0.001). The LEF-1 mRNA median level of MM patients after chemotherapy in progression of disease (PD) group was higher than that in the non-PD groups [0.08386 (0.00288 - 0.14100) vs. 0.003454 (0.000156 - 0.05660)], and the difference was statistically significant (U = 343.0, P< 0.001). The overall survival (OS) rate in the high LEF-1 expression group was shorter than that in the low LEF-1 expression group for MM patients in the initial diagnosis (47.6%vs. 65.5 %, χ2 = 3.931, P= 0.0414). Conclusion LEF-1 may be involved in the occurrence and development of MM, which has a potential to become an indicator of evaluating the poor prognosis and PD of MM patients, and could be served as a novel therapy target for the treatment of MM.
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<p><b>OBJECTIVE</b>To explore the function of nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammsomes in liver damage after allogeneic hematopoietic stem cell transplantation (allo-HSCT).</p><p><b>METHODS</b>The study presented a murine (BALB/c-based) model of allo-HSCT. Chimera rate was measured by flow cytometry. The hematoxylin-eosin, Masson's trichrome, immunohistochemistry staining were used to observe the pathology changes in liver, then measured the degree of liver damage. Inflammation cells and NLRP3 were measured by Western blot, cytokines IL-1β, IL-18 and NLRP3 related genes were tested with real-time quantitative polymerase chain reaction (q-PCR).</p><p><b>RESULTS</b>Hematopoietic stem cells had been successfully transplanted, the chimera rate was geater than 97% on the 10th day. Liver damage occurred after allo-HSCT and suffered infiltration of inflammation cells, which reached the peak on day 15, then moved to moderate; the cytokines IL-1β, IL-18 had the similar trend with liver injury, and reached the highest level on day 15, their mRNA expressions increased by (1.19 ± 0.40) fold and (1.64 ± 0.76) fold, respectively; Meanwhile, caspase-1 had the similar trend, its mRNA expression increased by (3.51 ± 0.46) fold on day 15; the inflammasomes NLRP1, NLRP3, NLRC4 and NLRP5 expressed in liver on day 15 of post-allo-HSCT, and NLRP3 inflammasome expressed highest among them. The mRNA and protein level of NLRP3 inflammasomes were kept with the serious degree of the liver damage, its mRNA expression increased by (2.91 ± 0.41) fold on day 15.</p><p><b>CONCLUSION</b>NLRP3 inflammsome expressed in liver injury during allo-HSCT in mice, and may be one of the important factors contributed to liver injury.</p>
Subject(s)
Animals , Male , Mice , Carrier Proteins , Metabolism , Hematopoietic Stem Cell Transplantation , Inflammasomes , Metabolism , Liver , Metabolism , Pathology , Mice, Inbred BALB C , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein , Postoperative PeriodABSTRACT
Objective To evaluate the safety and efficacy of infliximab (IFX) combined with conventional therapy in the treatment of Crohn's disease (CD) with incomplete intestinal obstruction.Methods From 2007 to 2013,22 cases of CD with incomplete intestinal obstruction were enrolled and were divided into IFX treatment group and conventional therapy group,each 11 cases.In conventional therapy group,patients were fasting or liquid food intake,received conventional therapy such as anti-infection and fluid supplement therapy to maintain water and electrolyte balance,long-term oral mesalazine,metacortandracin and imuran.In IFX treatment group,on the basis of conventional therapy patients received intravenous IFX 5 mg/kg at the 2nd,6th,14th,22nd and 30th week after admission.Regular blood test,liver function,erythrocyte sedimentation rate (ESR),C reaction protein (CRP) and CD active index (CDAI) of two groups were compared before and after treatment.Patients underwent endoscopy examination on admission day and the 30th week after treatment to evaluate the efficacy.The side effects were recorded during the period of treatment.The t test and Chi squaretest were used for comparison between groups.Results Compared with conventional therapy group,the average intestinal obstruction remission time of IFX treatment group shortened ((10.53±1.28) day vs (16.82±1.97) day) and the difference was statistically significant (t =2.985,P＜0.05).At the end of 30th week after treatment,clinical total effective rate of IFX treatment group and conventional therapy group was 9/11 and 7/11 (x2 ＝22.35,P＜0.05).Under endoscopy,total effective rate of these two groups was 7/11 and 5/11 (x2＝21.93,P＜0.05).At the end of 30th week after treatment,ESR,CRP and CDAI of IFX treatment group ((11.4±7.5) mm/1 h,(13.2±6.6) mg/L and 125.4±26.9) were all significantly lower than those on admission day ((31.3±5.7) mm/1 h,(45.3±7.6) mg/L and 240.5±35.2) (t=2.650,3.022,2.719,all P＜0.05).No serious side effects were observed in IFX treatment group and conventional therapy group during the treatment period.Conclusion For CD patients with incomplete intestinal obstruction,on the basis of conventional therapy,the addition of IFX could achieved better efficacy than conventional therapy alone.
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Postweaning multisystemie wasting syndrome (PMWS) is an important swine disease that is closely associated with porcine circovirus type 2 (PCV2). The capsid protein (Cap protein) is a major structural protein that has at least three immunoreactive regions, and it can be a suitable candidate antigen for detecting the specific antibodies of a PCV2 infection. In the present study, an indirect enzyme-linked immunosorbent assay (TcELISA)based on a truncated soluble Cap protein produced in Escherichia coli (E.coli) was established and validated for the diagnostic PCV2 antibodies in swine. The TcELISA was validated by comparison with an indirect immunofluorescence assay (IIFA). The diagnostic sensitivity (DSN), specificity (DSP), and accuracy of the TcELISA were 88.6%, 90.7% and 89.4%, respectively. The agreement rate was 89.38% between results obtained with TcELISA and IIFA on 113 field sera. A cross-reactivity assay showed that the method was PCV2-specific by comparison with other sera of viral disease. Therefore ,the TcELISA will be helpful for the development of a reliable serology diagnostic test for large scale detection of PCV2 antibodies and for the evaluation of vaccine against PCV2 in swine.
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The bacteriophage T7 RNAP gene was amplified via PCR from -lysogen DE3, and the gene was cloned into pBABEpuro retrovial vector, a recombinant plasmid named as pT7BABEpuro was constructed and sequenced. Then the pT7BABEpuro and pVSV-G plasmids were cotransfected into GP2-293 packaging cells by liposomese, some pseudotype viruses were ingathered and transfected into IBRS-2 cell under polybrene. The IBRS-2 cell was propagated in DMEM with puromycin. The genome extraction from the cells transfected different times, the T7 RNAP gene was amplified from the genome by PCR, the mRNA of T7 RNAP protein expressed in IBRST7 cells was analyzed by RT-PCR, respectively, the results showed the T7 RNAP gene had been integrated into the chromosome of IBRS-2 cell and expressed stably at high level. To study whether T7 RNAP is of transcriptional activity in the established IBRST7 cell line, a plasmid pIERS-EGFP-ET with a reporter gene (EGFP) under control of the T7 promoter was constructed. IRES element from FMDV (for CAP-independent translation) was cloned into plasmid pET-43.1a-c(+) downstream of the T7 promoter sequence, then EGFP gene was cloned in frame downstream of the AUG codon of the FMDV IRES, resulting in the plasmid. IBRST7 cells were transfected with plasmid pIERS-EGFP-ET using lipfection, EGFP was expressed, the results showed the T7 RNAP in IBRST7 cells has transcriptional activity. IBRST7 cell line was directly transfected with linearized full-length cDNA of swine vesicular disease virus (SVDV) HK/70, infectious SVDV was efficiently recovered from the cDNA. The reverse genetic procedure is simplified to a faster, one step protocol to recover RNA virus and will be useful to understand the mechanisms of molecular pathology of RNA virus and develop effective vaccines.
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Objective:To investigate the immunogeneicity of a subunit vaccine of capsid protein precursor(P1) of swine vesicular diseas(SVD).Methods:In this study,the guinea pigs were immunized with the home-made antigen,T-lymphocyte proliferation response,blocking ELISA and micro-neutralization assay were used to detect the effect of the immunized responses in guinea pigs.Results:The results indicated that a retroviral-based vaccine carrying the capsid protein precursor(P1) of SVD was able to elicit strong SVDV-specific humoral immune responses in guinea pigs.Conclusion:It encourages further work towards the development of a vaccine against SVDV infection.