OBJECTIVETo observe the effects of recombinant fusion protein interleukin (IL)-18 on the expression of immune-inflammatory factors in the mice infected with Staphylococcus aureus (SA), and to investigate the mechanism of action of IL-18 in defense of SA infection in vivo.

METHODSA total of 40 specific pathogen-free female BLAB/c mice were randomly divided into four groups: control, SA infection, immunized, and intervention. A mouse model of SA infection was established by nasal inoculation with SA liquid. The immunized group and the intervention group were intranasally given IL-18 before SA modeling, and then the SA infection group and the intervention group received the nasal inoculation with SA liquid; the control group was treated with phosphate buffered saline instead. The levels of IL-4, interferon (IFN)-γ, tumor necrosis factor (TNF), granulocyte colony-stimulating factor (G-CSF), IgM in the serum and bronchoalveolar lavage fluid (BALF) of mice were measured by enzyme-linked immunosorbent assay. The expression of macrophage inflammatory protein (MIP)-1α mRNA and MIP-2β mRNA in the lung tissue of mice were determined by real-time fluorescent quantitative PCR.

RESULTSCompared with the control group, the SA infection group and the immunized group had significantly higher levels of IL-4, G-CSF, and IgM in the serum and BALF and expression of MIP-1α mRNA and MIP-2β mRNA in the lung tissue (P<0.05); the SA infection group had a significantly lower level of IFN-γ and a significantly higher level of TNF in the serum and BALF (P<0.05); the immunized group had a significantly higher level of IFN-γ in the serum and BALF (P<0.05). Compared with the SA infection group, the intervention group had significantly higher levels of IL-4, IFN-γ, G-CSF, and IgM in the serum and BALF and expression of MIP-1α mRNA in the lung tissue. In contrast, the intervention group showed a significantly lower level of TNF in the serum and BALF and expression of MIP-2β mRNA in the lung tissue (P<0.05). All the above indicators in the intervention group were significantly higher than those in the control group (P<0.05), except the serum level of IFN-γ.

CONCLUSIONSIn the mice infected with SA, the recombinant fusion protein IL-18 by mucosal immunity can affect inflammatory factors in the serum and BALF and the expression of MIP-1α mRNA and MIP-2β mRNA in the lung tissue to promote the anti-infective immune response and enhance the ability to clear pathogens.

Animals ; Chemokine CCL3 ; analysis ; Female ; Granulocyte Colony-Stimulating Factor ; blood ; Interferon-gamma ; blood ; Interleukin-18 ; therapeutic use ; Interleukin-4 ; blood ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; pharmacology ; therapeutic use ; Staphylococcal Infections ; drug therapy ; immunology

OBJECTIVETo explore the effect of infusing G-CSF mobilized recipient spleen cells at different time after haploidentical stem cell transplantation(HSCT) on graft-versus-host disease (GVHD) in mice and its possible mechanism.

METHODSForty mice after HSCT were randomly divided into 4 groups (n=10): GVHD positive control group (control group), 1st d recipient cell infusion group after transplantation (+1 d group), 4th d recipient cell infusion group after transplantation(+4 d group), 7th d recipient cell infusion group after transplantation(+7 d group). The mice in control group were injected the normal saline of same equivalent with experimental group which were given the same amount of G-CSF-mobilized recipient spleen cells. The general manifestation and pathological change of GVHD were observed. The expression changes of CD3CD4, CD3CD8cell subsets and FasL in peripheral blood were detected by flow cytometry.

RESULTSThe incidence of GVHD was significantly decreased in +4 d group and the median survival time was longer than 60 days, which was significantly higher than that of control group (24 d), +1 d group (21 d), +7 d group (28 d). (P<0.01, P<0.01, P<0.01). The Fasl expression of peripheral blood T lymphocytes in +4 d group were significantly lower than that in the other 3 groups(P<0.05).

CONCLUSIONThe +4 d infusion of G-CSF mobilized recipient spleen cells on 4th day after haploidentical HSC transplantation can inhibit the expression of FasL in donor T lymphocytes, and significantly reduce the incidence of GVHD.

OBJECTIVETo investigate the effects of microvesicles(MV) isolated from human peripheral blood hematopoietic stem cells(PB-HSC) on immune regulation and hematopoiesis.

METHODSPB-HSCs were separated by density-gradient centrifugation and cultrued. The supernatants of PB-HSC at 48 h were harvested for isolation and purification of MV by using ultracentrifugation. The electron microscopy was used to observe the morphology of MV. The protein level in MV was quantified through bicinchoninic acid(BCA) protein assay. Flow cytometry was used to detect the immunophenotype of MV. Human peripheral blood mononuclear cells(PB-MNC) were isolated from healthy donor and treated with isolated MV. After being co-cultured for 12 h, confocal microscopy was used to observe the action mode of MV on PB-MNC. After being co-cultured for 48 h, the levels of IL-2, IL-6, IL-8, IL-10, IFN-γ and TNF-α were detected by ELISA. Flow cytometry was used to detect the changes of T cell subsets and the activation of T cell subsets as well as intracellular cytokine staining after co-culture for 48 h. The methylcellulose was used to assess the hematopoiesis-supportive function of MV as well as co-cultured supernatants.

RESULTSThe eletron microscopy revealed that MV were elliptical membrane vesicles. The protein amount in MV ranges from 29 to 110 µg. Flow cytometry showed that MV expressed mix markers on the surface, especially highly expressed MV specific marker CD63(85.86%) and hematopoietic stem cell marker CD34(33.52%). After being co-cultured for 12 h, confocal microscopy showed that MV were merged with PB-MNC. After being co-cultured for 48 h, ELISA showed that the secretion of cytokines IL-6,IL-8, IL-10 as well as TNF-α was increased while the level of IL-2 and IFN-γ was not changed much. The results of flow cytometry showed that there was no significant change in T cell subsets and T cell activation. Staining of intracellular factor showed that IL-8 was increased significantly in CD11ccells. The colony-forming experiments revealed that MV and the co-cultured supernatants could facilitate the colony formation.

CONCLUSIONMV isolated from PB-HSC have immune-regulatery function and can prornote hematopoiesis.

OBJECTIVETo establish a new mouse model of H-2 haploidentical stem cell transplantation from double donors (DHSCT) and compare with conventional haploidentical hematopoietic stem cell transplantation (HSCT) so as to alleviate transplant-related complications.

METHODSThe recipients CB6F1 of conventional HSCT group were pretreated by 8 Gy total body irradiation(TBI), and received 3×10donor (male C57) spleen mononuclear cells (spMNC) mobilized by G-CSF within 2 hours after TBI. Recipients CB6F1 of D-HSCT groups accepted 2 Gy TBI, and received total 12×10spMNC mobilized by G-CSF from 2 donors within 2 hours after TBI, each donor donated 6×10cells. According to the different strains and sex of donors, DHSCT were divided into 3 groups: in group A, the stem cells were from male C57 and female BALB/c; in group B, stem cells were from male C57 and male BALB/c, while the stem cells in group C were from male C57 and male C3H. Hematopoietic reconstruction, engraftment, GVHD and survival were observed among these 4 groups.

RESULTSThe nadir of white blood cell count after conventional HSCT were lower than 1×10/L and lasted for 3 to 5 days, while not less than 3×10/L after D-HSCT among either group A, B or C. The complete chimerism (CC) in conventional HSCT group was achieved quickly within only 1 week in peripheral blood. Mixed chimerism (MC) in peripheral blood was found within the first week after DHSCT among either group A, B or C, and transformed into stable CC within the second week eventually. Both GVHD morbidity and mortality of conventional HSCT were 100% at 34th day after transplantation.Among DHSCT groups,the overall GVHD morbidity and mortality at 34th day after transplant were 49.6% and 50%(P<0.01,P<0.05), respectively,and 60.4% and 81.2% at 50th day after transplant. Overall survival of 50 days was 50.9% that indicated a long survival in such mice DHSCT. The differences of hematopoietic reconstruction, donor cell engraftment, GVHD incidence, GVHD mortality and OS were not statistically significant among group A, B and C(P>0.05).

CONCLUSIONA new mouse model of H-2 haploidentical peripheral blood stem cell transplantation from double donors (DHSCT) has been successfully established by reducing conditioning intensity and increasing graft cell numbers from double haploidentical donors without GVHD prophylaxis. DHSCT successfully achieved stable complete chimerism, less GVHD morbidity and mortality and longer OS without hematopoietic suppression. This study provides experimental evidence for clinical application of HLA haploidentical peripheral blood stem cell transplantation from double donors.

OBJECTIVETo investigate the relationship between the clinical and pathological findings in IgA nephropathy with or without IgG deposition in the glomerular mesangial area.

METHODSThe data were collected from 122 patients with a diagnosis of IgA nephropathy by renal biopsy in the Third Affiliated Hospital of Southern Medical University between November, 2009 and February, 2016. All the samples were examined by light microscopy, immunofluorescence and electron microscopy. According to the results of immunofluorescence assay, the patients were divided into IgA group (n=63) and IgA-IgG group (n=59). The pathological classification of IgA nephropathy was analyzed according to Oxford classification and Lee's classification. The clinical and pathological findings were compared between the two groups.

RESULTSCompared with the patients with IgA nephropathy but without IgG deposition, patients with IgA nephropathy with IgG deposition had higher serum creatinine, higher 24-h urine protein, higher blood uric acid, higher triglyceride levels (P<0.05) and lower eGFR (P<0.05); more of these patients were in Lee's grade IV-V, had renal tubular atrophy and/or interstitial fibrosis, and had MEST scores more than 3 (P<0.05).

CONCLUSIONPatients with IgA nephropathy with IgG deposition in the glomerular mesangial have severer clinical symptoms and more serious pathological changes. Measures should be taken to control IgG deposition in patients with IgA nephropathy to delay the progress of the disease.

In recent twenty years, there are a lot of studies about the effect of borneol on permeability of blood-brain barrier(BBB); however, it isDODOrt of regular conclusions of effect factors and in-depth analysis of functional mechanisms. The current researching data were collected and analyzed in this paper for illuminating the effect factors and mechanisms of borneol on permeability of BBB.The following conclusions were obtained: five factors about borneol influencing the permeability of BBB. First, opticity activity of borneol had no significant effect on action effects. Second, dose of borneol in the range of 50.00-200.00 mg•kg⁻¹, did not affect the effect direction, but only affect its action intensity either with use alone or combination use. Third, the borneol can increase the permeability of physiological BBB, and decrease the permeability of pathological BBB. Fourth, regardless of using singly or using compatibility with musk, borneol can decrease the permeability of BBB in different brain disease models. Fifth, when used with astragalus, catalpol or puerarin, borneol can increase the permeability of BBB and promote the drugs through BBB in pathological conditions. The target spots and mechanisms of borneol's bidirectional regulation on the permeability of BBB are related to the structure and function of cerebral endothelial cells, the exocytosis effects of P-gp and low pinocytosis internal transport effects. On one hand，borneol can down-regulate P-gp by inhibiting NF-κB to reduce the exocytosis effects of P-gp and promote the blood brain barrier pinocytosis to increase the permeability of BBB; On the other hand，borneol can reduce the degradation of basement membrane of blood vessel and tight junctions by inhibiting the expression of IL-1β, MMP-9 to decrease the permeability of BBB；moreover，borneol has bidirectional regulation effects on blood-brain barrier permeability by influencing the signaling pathways of Ca2+-eNOS-NO, VEGF-eNOS-NO. However, the detailed mechanisms that borneol regulates and controls the permeability of BBB are so complicated, so they shall be further proved and clarified.

OBJECTIVETo study the effect of Bushen Huoxue decoction on calcification of cartilage endplate in lumbar vertebrae.

METHODSSix healthy male gerbils with 2-month-old were selected as normal control group, and 24 7-month-old healthy male gerbils were fed to 12-month-old to establish the aged gerbil model. Thirty gerbils were randomly divided into five groups as follow: the normal control group (=6), model group (=6, normal saline 4 ml/kg, intragastric 30 d), Bushen Huoxue low dose group(=6, 1.9× 10⁻ ³ ml/g given Bushen Huoxue recipe orally, 30 d), Bushen Huoxue middle dose group(=6, 3.8× 10⁻ ³ ml/g given Bushen Huoxue recipe orally, 30 d), Bushen Huoxue high dose group(=6, 7.6× 10⁻ ³ ml/g given Bushen Huoxue recipe orally, 30 d), the intervention group administered for 1.36 g from 7-month-old age, 30 d. The animals were sacrificed at the age of 2 months in the normal control group and 12 months of age in the other groups. The morphology of the lumbar vertebral cartilage endplate, the area of vascular bud, the ratio of non-calcified/calcified layer were analysis by HE chromosome visual method. The expression of type X collagen and BMPs in cartilage endplates were detected by rabbit monoclonal immunohistochemical staining.

RESULTSThe relative area of the vascular buds cartilage endplate measurements showed that compared with the model group, middle dose group and normal control group increased (<0.05), high and low dose groups all had different degrees of increase, but no statistical significance(>0.05). The ratio of cartilage endplate thickness of non-calcified/calcified showed that compared with the model group, Bushen Huoxue middle dose, normal control group increased, with statistical significance(<0.05), and high and low dose groups all had different degrees of increase, but there were no statistical significance(>0.05). Compared with the model group, the expression of type X collagen in the cartilage endplate of the normal group, the Bushen Huoxue low, middle and high dose groups decreased, and had statistical significance(<0.01); compared with the model group, the expression of BMPs in the normal group, Bushen Huoxue middle dose group increased, with statistically significant(<0.01), while the high and low dose groups increased in different degrees, but there was no statistical significance(>0.05).

CONCLUSIONSBushen Huoxue prescription can delay the calcification of cartilage endplate in the process of aging, suggesting that it can be used as a preventive medicine for early disc degeneration.

OBJECTIVETo investigate the difference of postoperative drainage and systemic trauma response between endoscopic and traditional near total thyroidectomy to provide the basis for selecting appropriate operative methods.

METHODSIn this prospective clinical controlled study, 80 patientsscheduled for near total bilateral thyroidectomy for the first time were divided equally into endoscopic surgery group (group A) and open surgery group (group B). The total drainage volume after operation, postoperative extubation time, and postoperative daily drainage volume were recorded after the operation. The contents of triglyceride (TG) and total protein (TP) were determined in the postoperative drainage fluid onthe first day. The levels of interleukin 6 (IL6), high sensitive C reactive protein (HSCRP), alpha 1 acid glycoprotein (AAG), ceruloplasmin (CER) and haptoglobin (HPT) in venous blood were tested before the operation and on the first day after surgery.

RESULTSCompared with those in group B, the postoperative drainage volumein group Aincreased significantly (P=0.000) and the postoperative extubation time was significantly prolonged (P=0.000); the mean postoperative daily drainage volume was significantly larger ingroup A than in group B (P=0.000) and tended to decrease with time in both groups. There was no significant difference in the content of triglycerideortotal protein in the drainage fluid between the two groups on the first day after operation (P=0.429 and 0.324, respectively). In both groups, the contents of AAG, ceruloplasmin and haptoglobin on the first postoperative day were all similar with those measurement before operation (P>0.05), but significant variations occurred in the levels of IL6 and HSCRP on the first postoperative day (P=0.000). The serum levels of IL?6 or HS?CRP did not differ significantly between the two groups on the first day after operation (P=0.054 and 0.066, respectively).

CONCLUSIONCompared with open surgery, endoscopic near total bilateral thyroidectomyis associated with an increased the volume of postoperative drainage and a prolonged time of extubationbut not an increased systemic trauma response. Therefore, endoscopic surgery can serve as one of the routine options for patients who are concerned with neckscars resulting from open surgeries.

OBJECTIVETo explore the biological characteristics of microvesicles(MV) derived from bone marrow mesenchymal stem cells (BM-MSC) and their capability supporting ex vivo expansion of hematopoietic stem cells(HSC).

METHODSThe MV from cultured BM-MSC supernatant were isolated by multi-step differential velocity contrifugation; the morphological characteristics of MV were observed by electron microscopy with negative staining of samples; the protein level in MV was detected by using Micro-BCA method; the surface markers on MV were analyzed by flow cytometry. The peripheral blood HSC(PB-HSC) were isolated after culture and mobilization; the experiment was diveded into 2 group: in MV group, the 10 mg/L MV was given, while in control group, the same volume of PBS was given; the change of PB-HSC count was observed by cell counting; the change of surface markers on PB-HSC was detected dynamically by flow cytometry; the cell colony culture was used to determin the function change of PB-HSC after co-culture with MV.

RESULTSMSC-MVs are 20-100 nm circular vesicles under electron microscope. About 10 µg protein could be extracted from every 1×10MSC. The flow cytometry showed that CD63 and CD44 were positive with a rate of 96.0% and 50.2%, while the HLA-DR, CD34, CD29 and CD73 etc were negative. When being co-cultured with GPBMNC for 2 days, the cell number of MV groups was 1.49±0.15 times of the control group (P>0.05). When being co-cultured for 4 days, the cell number of MV groups was 2.20±0.24 times of the control group(P<0.05). The CD34cell number of MV groups was 1.76±0.30 times the control group after culture for 2 day and 1.95±0.20 times after culture for 4 day.

CONCLUSIONThe MV has been successfully extracted from MSC culture supernatant by multi-step differential velocity centrifugation. MSC-MV can promote HSC expansion in vitro.

Enterovirus 71 is a neuroinvasive virus that is associated with severe neurological complications. We had earlier suggested that the replication capacity of a severe strain was higher than that of a mild strain. The recombinant 3CRV and 3CDRV virus strains were successfully rescued in our previous study. In the present study, we found no difference in virulence between 3CRV and severe strains. However, the capacity of replication and to cause cell injury of 3CDRV strain decreased in vitro, especially at 39.5 °C. Replacement of 3CD region in the severe strain led to milder symptoms, less body weight loss, and lower viral load in ICR mice. Histopathological findings indicated less severe injury in mice infected with 3CDRV strain. This study suggests that the 3CD region contributes to the attenuation of the severe strain, including its replication capacity and temperature sensitivity.
Animals ; Cytopathogenic Effect, Viral ; Enterovirus A, Human ; genetics ; pathogenicity ; Enterovirus Infections ; pathology ; virology ; Gene Expression Regulation, Viral ; Mice ; Mice, Inbred ICR ; Mutation ; Viral Load ; Viral Proteins ; genetics ; metabolism ; Virulence ; Virus Replication