OBJECTIVETo investigate the feasibility and clinical application value of selecting viable spermatozoa by noncontact diode laser.

METHODSWe obtained immotile spermatozoa from 2 infertile men with obstructive azoospermia or severe asthenospermia and selected viable spermatozoa using a single laser shot at the sperm tail. Those that responded to the laser shot by a curling reaction of the tail were regarded as presumably viable and used for intracytoplasmic sperm injection (ICSI).

RESULTSThe mean fertilization rate was 88.89% after ICSI with the laser-selected viable spermatozoa. Both of the embryo transfers resulted in a single pregnancy.

CONCLUSIONNoncontact diode laser is a useful alternative for the assessment of sperm viability, which may help to achieve successful pregnancy.

Embryo Transfer ; Female ; Fertilization ; Humans ; Infertility, Male ; therapy ; Male ; Pregnancy ; Pregnancy Outcome ; Sperm Injections, Intracytoplasmic ; Sperm Motility ; Sperm Tail ; physiology

To optimize the culture environment and protocol of hematopoietic cells' expansion, avoiding the fluctuation caused by medium changing in stirred culture and concentration gradient in static culture, the hematopoietic cells from cord blood (CB) were cultured in a stirred bioreactor connected with a cell retention system, which is a gravity sedimentation settler designed for hematopoietic cell. Total cells expanded 11.5 and 18.6 fold respectively in the twice perfusion stirred cultures, in which CFU-Mix was expanded 23.2 and 20.4 fold, CFU-GM 13.9 fold and 21.5 fold, BFU-E 8.0 fold and 6.9 fold, CD34+ cells 17.1 fold and 15.4 fold. After 12-day culture, it was obtained that 1082 x 10(6) total cells, 6.31 x 10(6) CFU-GM, 6.2 x 10(6) CFU-Mix and 23 x 10(6) CD34+ cells from 267 x 10(6) CB mononuclear cells (MNC) in the first culture, and 1080 x 10(6) total cells, 4.65 x 10(6) CFU-GM, 11.0 x 10(6) CFU-Mix, and 25.0 x 10(6) CD34+ cells from 180 x 10(6) CB MNC. These two cultures met to the clinical scale. Due to the optimized dissolved oxygen (DO) and stable culture environment, the rate of stem/progenitor cells to total cells in the perfusion culture was higher than that in T-flask cell-retention feeding culture. But the cell growth was inhibited in the later phase of perfusion culture, when the cell density is high. The inhibition should be attribute to the high cell density itself. The perfusion culture environment in bioreactor with optimal DO and pH controlling is more favorable for stem/progenitor cells' maintenance and expansion, and the expanded cells' number has reached a clinical scale. But the high cell density in the later phase of perfusion culture caused inhibition to mature hematopoietic cell's growth.
Bioreactors ; Cell Culture Techniques ; methods ; Cells, Cultured ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans

OBJECTIVETo explore the effects of different fertilization methods on the outcomes of elective blastocyst culture.

METHODSWe retrospectively analyzed the outcomes of elective blastocyst culture for 1 153 cycles of IVF and 205 cycles of ICSI performed between january 2009 and December 2012.

RESULTSA total number of 14 748 embryos in the IVF group and 2 655 embryos in the ICSI group underwent sequential blastocyst culture, with 7 871 blastocysts formed in the former and 1 210 in the latter. No cycles were canceled for no blastocyst formation in either of the two groups. The rates of quality embryos, blastocyst formation and embryo utilization were significantly higher in the IVF than in the ICSI group (64.77 vs 58.72%, 53.37 vs 45.57%, and 60.06 vs 52.17%, all P < 0.05), but the rates of implantation, clinical pregnancy and abortion showed no significant differences between the two groups (48.94 vs 51.43%, 49.03 vs 52.02%, and 11.69% vs 15.56, all P > 0.05).

CONCLUSIONWith the same inclusion criteria of selective blastocyst culture, IVF has a lower risk of cycle cancellation due to no blastocyst formation and therefore may effect higher rates of blastocyst formation and embryo utilization than ICSI. Our study suggested that appropriate inclusion criteria of selective blastocyst culture should be laid down according to different fertilization methods.

Adult ; Blastocyst ; Embryo Transfer ; Female ; Fertilization in Vitro ; methods ; Humans ; Pregnancy ; Retrospective Studies ; Sperm Injections, Intracytoplasmic

OBJECTIVETo explorer the effectiveness of enriched bone marrow stem cells technique for lumbar fusion.

METHODSWith the randomization and control principles, 2 graft materials [Enrichment bone marrow mesenchymal stem cells hybridized with beta-tri calcium phosphate (composite graft group), autologous iliac crest bone graft (autograft group)] were compared in posterior lumbar fusion procedures. 56 patients with degenerative disc disease, lumbar instability or spinal stenosis, were included. The volume of cells suspension in pre- and post-enrichment and the number of nucleated cells (NCs) were identified. The number of osteoprogenitor cells was estimated by counting the colony-forming units which express alkaline phosphatase (CFUs/ALP+). Then the efficiency of the enrichment was evaluated. Clinical follow-up with roentgenogram and Oswestry scale scores was performed for outcome evaluation.

RESULTS(249 +/- 31) ml bone marrow per patient from bilateral iliac crests was aspirated peri-operatively. About (43 +/- 11) ml enriched bone marrow was collected. The number of NCs was concentrated from (15.9 +/- 3.3) x 10(6)/ml to (44.1 +/- 10.8) x 10(6)/ml, CFUs/ALP+ was significantly increased from (118 +/- 86)/ml to(486 +/- 305)/ml. The follow-up was about (26.3 +/- 7.5) months. There was no significant differences in age, gender, disease and fusion segments between the two groups. The fusion rate was 93.3% and 96.2% for composite graft group and autograft group, respectively (chi2 = 0.2146, P = 0.6432). There was no difference in operation time between the two group (t = 0.5243, P = 0.6022), but blood loss in composite graft group was more than that in autograft group (t = 6.4664, P < 0.01). Cell salvage for auto-transfusion could transfuse back half of the blood loss during operation. No hematoma or chronic soreness in the bone marrow donor sites of composite graft group occurred, but a little exudation or moderate swelling in the wound happened in 4 cases which disappeared under medical treatment. Meanwhile, 15.4% patients had hematoma in the iliac bone donor site and 26.9% patients had chronic soreness, but no case had wound problem in autograft group. As for Oswestry scale scores, there was no significant difference between the two groups.

CONCLUSIONSThe enrichment technique of autologous bone marrow stem cells can greatly increase the concentration of MSCs. It is a rapid and safe method used peri-operatively. The composite material of enriched MSCs and porous beta-TCP is a good bone substitute in posterior spinal fusion.

Adult ; Aged ; Bone Substitutes ; Bone Transplantation ; methods ; Calcium Phosphates ; Female ; Follow-Up Studies ; Humans ; Ilium ; transplantation ; Lumbar Vertebrae ; surgery ; Male ; Middle Aged ; Spinal Fusion ; instrumentation ; methods ; Stem Cell Transplantation ; Transplantation, Autologous ; Treatment Outcome

BACKGROUNDThe vaccination of mice with DNA encoding single candidate antigens has failed to induce significant protection against Schistosoma japonicum (S. japonicum) challenge infections. In this study, we evaluated the feasibility of using a multivalent DNA vaccine which co-expressed S. japonicum integral membrane protein Sj23 and murine cytokine IL-12 to induce protective immune responses.

METHODSThe plasmid pVIVO2-IL12-Sj23, a eukaryotic expression vector expressing Sj23 and murine IL-12 simultaneously, was constructed, identified, and tested for expression in vitro. Its ability to protect against S. japonicum challenge infections was analyzed according to worm reduction rate and egg reduction rate after vaccination of BALB/c mice. The serum levels of specific IgG antibody were determined by enzyme-linked-immuno sorbent assay (ELISA) and Western blot analysis. Using cultured spleen cells, IFN-gamma and IL-4 post-stimulation were quantified by ELISA. The phenotypes of splenocyte populations were analyzed by flow cytometry (FCM).

RESULTSThe plasmid DNA pVIVO2-IL12-Sj23 was proven to express well in vitro by transient transfection of HEK-293 cells. Immunization resulted in a worm reduction rate of 45.53% and egg reduction rate of 58.35%. ELISA and Western blot analysis indicated that immunized mice generated specific IgG against Sj23. Spleen cells showed significant increases in IFN-gamma but decreases in IL-4. No significant differences in CD4+ and CD8+ subgroup ratios were observed after the challenges.

CONCLUSIONSThe multivalent DNA vaccine pVIVO2-IL12-Sj23 is sufficient to elicit moderate but highly significant levels of protective immunity against challenge infections. Cytokine IL-12, as a gene adjuvant, was able to enhance the Th1 responses and, hence, the protective immunity.

Animals ; Antibodies, Helminth ; blood ; Antigens, Helminth ; genetics ; immunology ; CD4-CD8 Ratio ; Cytokines ; biosynthesis ; Helminth Proteins ; genetics ; immunology ; Interleukin-12 ; genetics ; immunology ; Male ; Membrane Proteins ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Schistosoma japonicum ; immunology ; Th1 Cells ; immunology ; Vaccination ; Vaccines, DNA ; immunology

BACKGROUNDRecently congenital infection with Schistosoma japonicum (S. japonicum) has been demonstrated in pigs, rabbits, mice and dogs. We explored the rabbit as an animal model for the congenital infection of schistosomiasis japonica and assessed the effect of a congenital S. japonicum infection on the resistance of rabbit kittens to a postnatal challenge infection.

METHODSSixteen pregnant New Zealand white rabbits were infected with a single dose of S. japonicum cercariae. The exposed animals were divided into three groups according to the gestation age at the time of infection. Diagnosis of prenatally acquired S. japonicum infection in the rabbit kittens was primarily based on serological tests in combination with parasitological and histopathological findings. Congenitally infected kittens were challenged percutaneously with 100 S. japonicum cercariae to assess the effect of a congenital S. japonicum infection on kitten resistance to a postnatal challenge infection.

RESULTSThe overall prevalence of congenital infection in offspring of infected mothers was 20% (12/60). The congenital infection rate in group L (late gestation) was much higher than in group E (early gestation) and group M (mid-gestation) (P <0.05). After a postnatal challenge infection, prenatally infected kittens had a 54.66% worm reduction rate, 41.45% egg reduction rate, and 51.76% granuloma size reduction rate compared to naïve kittens.

CONCLUSIONSThis study demonstrates the possibility of congenital infection of S. japonicum in rabbits and the resistance of congenitally infected kittens to a postnatal challenge infection. These results have important implications not only for epidemiological investigations, but also in designing government control programs for schistosomiasis.

Animals ; Antibodies, Helminth ; blood ; Female ; Immunoglobulin M ; blood ; Infectious Disease Transmission, Vertical ; Male ; Pregnancy ; Rabbits ; Schistosomiasis japonica ; congenital ; immunology ; parasitology

Three-dimensional finite elemennt method (3-D FEM) is an approach to simulate the intraoral environment by fabricating virtual model in computer software, to provide various options of restorations according to the requirements of study, and finally to give relevant mechanic data for clinical reference by stress analysis. 3-D FEM has been applied in stomatological research more and more widely in recent years. This review has summarized the applications of 3-D FEM in various kinds of dental restorations.

OBJECTIVETo evaluate the diagnostic utility of Warthin-Starry silver stain, immunohistochemistry and transmission electron microscopy in the detection of human Bartonella henselae infection and pathologic diagnosis of cat scratch disease (CSD).

METHODSThe paraffin-embedded lymph node tissues of 77 histologically-defined cases of cat scratch disease collected during the period from January, 1998 to December, 2008 were retrieved and studied using Warthin-Starry silver stain (WS stain) and mouse monoclonal antibody against Bartonella henselae (BhmAB stain). Five cases rich in bacteria were selected for transmission electron microscopy.

RESULTSUnder electron microscope, the organisms Bartonella henselae appeared polymorphic, round, elliptical, short rod or bacilliform shapes, ranged from 0.489 to 1.110 microm by 0.333 to 0.534 microm and often clustered together. Black short rod-shaped bacilli arranged in chains or clumps were demonstrated in 61.0% (47/77) of CSD by WS stain. The organisms were located outside the cells and lie mainly in the necrotic debris, especially near the nodal capsule. In 72.7% (56/77) of the cases, dot-like, granular as well as few linear positive signals were observed using BhmAB immunostain and showed similar localization. Positive results for both stains were identified in 59.7% (46/77) of the cases. When applying both stains together, Bartonella henselae was observed in 74.0% (57/77) of the case. The difference between the results obtained by WS stain and BhmAB immunostain was of statistical significance (P < 0.05).

CONCLUSIONSBartonella henselae is the causative pathogen of cat scratch disease. WS stain, BhmAB immunostain and transmission electron microscopy are helpful in confirming the histologic diagnosis. Immunostaining using BhmAB can be a better alternative than WS stain in demonstrating the organisms.

Adolescent ; Adult ; Aged ; Antibodies, Bacterial ; blood ; Bartonella henselae ; immunology ; isolation & purification ; ultrastructure ; Cat-Scratch Disease ; diagnosis ; microbiology ; pathology ; Child ; Child, Preschool ; Humans ; Immunohistochemistry ; methods ; Infant ; Lymph Nodes ; pathology ; ultrastructure ; Microscopy, Electron, Transmission ; Middle Aged ; Paraffin Embedding ; Staining and Labeling ; methods ; Young Adult

OBJECTIVETo investigate the feasibility and efficacy of laparoscopic Roux-en-Y gastric bypass(LRYGB) for super obesity(BMI≥50 kg/m(2)).

METHODClinical data of 42 patients undergoing LRYGB in the First Affiliated Hospital of Jinan University between 2004 and 2008 were analyzed retrospectively.

RESULTSAll the LRYGB procedures were successfully performed with no conversion to open surgery. Average operation time was 145.1 minutes, volume of blood loss during the surgery was 25.0 ml, and length of postoperative hospital stay was 9.9 days. The cases were followed up for 1 month to 30 months. Body weight and BMI decreased significantly 1 month after the operation and reached a minimum level after 2 years then became stable while excess body weight loss rate(EWL) increased(P<0.05). All the obese-related symptoms were relieved significantly. Four cases(9.5%) showed complications during perioperative period including 1 case of respiratory failure, 2 cases of gastrojejunal anastomotic bleeding, 1 case of umbilical wound infection, and 11 developed long-term complications. All of them were cured by conservative treatment.

CONCLUSIONSTreatment of super obesity by LRYGB is feasible with significant short-term results. But due to the difficulty of the operation and postoperative complications, comprehensive treatment from experienced bariatric surgical team is needed. The long-term outcome needs for further observation.

Adolescent ; Adult ; Female ; Gastric Bypass ; methods ; Humans ; Laparoscopy ; Male ; Middle Aged ; Obesity, Morbid ; surgery ; Retrospective Studies ; Treatment Outcome ; Young Adult

The aim of this study was to investigate the effects of polarization program on the ability of macrophages to regulate iron metabolism. M1 and M2 macrophages were propagated in vitro from porcine alveolar macrophages 3D4/2 and polarized by cytokines. The 3D4/2 macrophages were treated with 20 ng/mL interferon gamma (IFN-γ) and 10 ng/mL interleukin-4 (IL-4) combined with 10 ng/mL macrophage colony-stimulating factor (M-CSF) to induce polarization to M1 and M2, respectively. After incubation for 24 h, the expression levels of inflammatory factors and iron-metabolism genes were determined using real-time qPCR, Western bot and immunofluorescence. The M1/M2 macrophages culture media supernatant was collected and used to treat porcine intestinal epithelial cells IPEC-J2. The proliferation ability of IPEC-J2 was detected using CCK-8 assay kit. Following exogenous addition of ammonium ferric citrate (FAC) to M1/M2 macrophages, the phagocytic function of macrophages was detected using fluorescein isothiocyanate-dextran (FITC-dextran) and flow cytometry. The results showed that, compared with control, M1 macrophages had higher mRNA levels of iron storage proteins (ferritin heavy and light polypeptide, i.e. FtH and FtL), hepcidin and lipocalin-2, as well as iron content. Moreover, iron enhanced the ability of M1 macrophages to phagocytize FITC-dextran. There was no significant change in these mRNA expression levels in M2 macrophages, but the mRNA expression levels of ferroportin and transferrin receptor were up-regulated. In addition, the conditioned media supernatant from M2 macrophages promoted cell proliferation of IPEC-J2. These findings indicate that M1 macrophages tend to lock iron in the cell and reduce extracellular iron content, thereby inhibiting the proliferation of extracellular bacteria. While M2 macrophages tend to excrete iron, which contributes to the proliferation of surrounding cells and thus promotes tissue repair.
Animals ; Cytokines ; Ferritins ; Iron/metabolism* ; Macrophages/metabolism* ; Macrophages, Alveolar/metabolism* ; Swine