Specific binding of aspartic protease and enterocytes promotes Trichinella spiralis invasion of murine intestinal epithelium cells.

Trichinella spiralis is an important foodborne zoonotic parasite and it is necessary to develop vaccine to prevent T. spiralis infection in food animals. T. spiralis aspartic protease-2 (TsASP2) has been demonstrated to play a crucial role in larval invasion of intestinal epithelium cells (IECs). The purpose of this study was to assess the interaction between TsASP2 and IECs and to investigate the immune protection elicited by vaccination with rTsASP2. The results showed that the enzymatic activity of native aspartic protease was detected in crude proteins of all T. spiralis development stages other than NBL stage, the highest activity was observed in the IIL stage. The results of Western blot showed that TsASP2 protein was expressed at ML, IIL and AW but not NBL, and the TsASP2 expression level at IIL stage was significantly higher than those of other three worm stages (P < 0.05). The specific binding between rTsASP2 and IECs was observed by immunofluorescence test (IFT) and confocal microscopy, and the binding site was localized at the IEC membrane and this binding ability was inhibited by aspartic protease specific inhibitor pepstain A. The results of ELISA showed that the binding ability was protein dose-dependent. Vaccination with rTsASP2 triggered a mixed Th1/Th2 humoral and mucosal immune responses, as demonstrated by the elevation levels of Th1/Th2 cytokines (IFN-γ and IL-4) secreted by the spleen and mesenteric lymph nodes (MLNs) of immunized mice. The mice vaccinated with rTsASP2 exhibited a 54.17% reduction in enteral adult worms and a 54.58% reduction in muscle larvae after T. spiralis challenge. The results demonstrated that TsASP2 might be a potential molecular target for anti-Trichinella vaccines.

Influence of dexmedetomidine to cognitive function during recovery period for children with general anesthesia.

OBJECTIVE: We studied the influence of Dexmedetomidine on cognitive function in children during the recovery period of general anesthesia. PATIENTS AND METHODS: Ninety-three children who underwent general anesthesia were selected and randomly divided into (1) the control group, (2) the dexmedetomidine group, and (3) the dezocine group. Fentanyl, propofol, and rocuronium were used in all patients to induce anesthesia, while sevoflurane inhalation and propofol were used to maintain anesthesia. In the control group, 20 ml NS were infused intravenously 10 min before anesthetic induction. In the dexmedetomidine group, 1.0 µg/kg dexmedetomidine in 20 ml was infused for 10 min. In the dezocine group, 0.1 mg/kg dezocine in 20 ml was infused for 10 min. Mean arterial blood pressure, average heart rate, and average oxygen saturation (SaO2) were compared at the following time points: end of surgery (T0), before extubation (T1), during extubation (T2), and 30 min after extubation (T3). The VAS scale, Ramsay sedation score, delirium grading scale and occurrence of adverse reactions at 30 min after extubation were recorded. The occurrence of postoperative cognitive dysfunction (POCD) and the expression of serum neuron-specific enolase (NSE) and IL-6 at postoperative days 1 and 7 were recorded. RESULTS: Comparing mean arterial blood pressure, average heart rate, and average oxygen saturation (SaO2) at the different time points in the dexmedetomidine group, there were no statistically significant differences (p>0.05). The difference in the occurrence of adverse reactions in the different groups was statistically significant (p<0.05). The occurrence of postoperative cognitive dysfunction (POCD) at postoperative day 1 was significantly higher in the control group than the other two groups (p<0.05), and on the postoperative day 7th, the differences were not statistically significant (p>0.05). Regarding the expression of neuron-specific enolase (NSE) and IL-6, the levels were the highest in the control group, followed by the dezocine group (p<0.05). CONCLUSIONS: The dexmedetomidine is safer than dezocine in aspects of hemodynamics, sedation, analgesia, degree of delirium, occurrence of adverse reactions, and postoperative cognitive dysfunction (POCD). The improvement in the occurrence of postoperative cognitive dysfunction (POCD) is related to the levels of serum neuron-specific enolase (NSE) and IL-6.

Induction of human beta-defensin-2 expression in human astrocytes by lipopolysaccharide and cytokines.

Defensins are cationic peptides with broad-spectrum antimicrobial activity. They are members of a supergene family consisting of alpha and beta subtypes and each subtype is comprised of a number of different isoforms. For example, human alpha-defensin (HAD) has six isoforms, which are expressed by polymorphonuclear leukocytes and Paneth cells. In contrast, human beta-defensin (HBD) has two isoforms that are expressed by epithelial cells of the skin, gut, respiratory and urogenital tracts. Recently, HBD-1 was detected in human brain biopsy tissue. However, little is known about the expression of HBD-1 or HBD-2 in the CNS and whether neural cells can secrete these peptides. For the present study, human astrocyte, microglial, meningeal fibroblast and neuronal cultures were probed for the expression of HBD-1 and HBD-2 mRNA and protein. Each cell type was either maintained in tissue culture medium alone or in medium containing lipopolysaccharide (LPS) at concentrations ranging from 0.1 to 1 microgram/mL, interleukin-1 beta (IL-1beta) at 1-50 ng/mL, or tumor necrosis factor alpha (TNF-alpha) at the same concentrations. The expression of HBD-1 and HBD-2 mRNAs was monitored by RT-PCR. The cDNA products were sequenced to characterize the gene product. HBD-2 protein was detected by immunoblot, immunoprecipitation and immunocytochemistry. Results of these studies showed that HBD-1 mRNA was detected in all cell cultures except in those enriched for neurons. In contrast, HBD-2 mRNA was detected only in astrocyte cultures that were treated with LPS, IL-1beta or TNF-alpha. The detection of the respective proteins correlated positively with the mRNA results. As such, these data represent the first demonstration of HBD-2 expression by astrocytes and suggest that this peptide may play a role in host defense against bacterial CNS pathogenesis.

An efficient method for the cryopreservation of fetal human liver hematopoeitic progenitor cells.

The use of human hematopoietic progenitor cells (HPC) for transplantation requires efficient recovery methods and cryopreservation procedures. The purpose of this study was to determine cryopreservation techniques for fetal human liver (FHL) CD34(+) cells. We assessed FHL HPC recovery efficiency after freezing and thawing by viability testing, fluorescence-activated cell sorting analysis, and colony-forming ability under different conditions. We also determined optimal cell freezing concentrations and the effect of rate-controlled freezing on cell recovery. Lastly, cell recovery after varying freezing time periods was examined. Our results indicated that optimal cell recovery occurs when: A) cryopreservation medium consists of either 5% dimethylsulphoxide (DMSO) or 10% DMSO in combination with either 20% fetal bovine serum (FBS) or 70% FBS and when Iscove's modified Dulbecco's medium consists of not more than 10% DMSO; B) a rate-controlled freezing device container is used; C) CD34(+) cells are frozen at a concentration of 1 x 10(6)/ml, and D) a thawing temperature of 37 degrees C is used. These observations indicate that cryopreservation of FHL HPC is possible for up to 18 months in optimal conditions without losing hematopoietic activity.

Transplantation of human fetal brain cells into ischemic lesions of adult gerbil hippocampus.

OBJECT: The goal of this study was to establish whether transplanted cells derived from fetal human brain can survive in an ischemic lesion. METHODS: Sixteen adult male Mongolian gerbils underwent transient bilateral common carotid artery occlusion. One week later, cell suspensions prepared from fetal human brain were injected using stereotactic guidance into the CA1 region of the hippocampus on one side. On the contralateral side injection of the cell suspension medium only was performed. One week after transplantation, the animals were perfusion fixed and their brains were processed for histological studies as well as expression of neuron and glia-specific antigens. Data from ischemic animals were compared with eight nonischemic gerbils that served as sham-operated controls. Last, the in vivo data were correlated with observations made from matching in vitro cultures of the fetal brain cell suspension. The in vivo data indicated that transplanted human fetus-derived brain cells survived in ischemic lesions of gerbil hippocampus after 1 week, provided that the host animal underwent adequate immunosuppression and the transplanted cells were not incorporated into the scar caused by the transplantation procedure. Unlike their in vivo counterparts, after 1 week, most cultured fetal brain cells expressed either neuron- or astrocyte-specific antigens. CONCLUSIONS: This work demonstrates that xenotransplanted fetal human brain cells are able to survive in an ischemic lesion in a rodent model. These data might be useful for future neural transplantation studies of treatments for cerebrovascular ischemia in humans.

Cytokine regulation of CD40 expression in fetal human astrocyte cultures.

CD40 can participate in inflammatory processes after binding its cognate ligand (CD40L). We found that fetal human astrocytes constitutively express CD40 mRNA and protein. Upon incubating cultures with proinflammatory cytokines (TNF-alpha, IL-1beta and IFN-gamma) or with lipopolysaccharide (LPS), CD40 expression was increased. No change in CD40 expression was noted in astrocyte cultures incubated with IL-6, HIV or gp41. Astrocytes also showed increased release of proinflammatory cytokines TNF-alpha, IL-1beta and IL-6 after incubation with CD40L peptide. These observations suggest a role for CD40 in central nervous system (CNS) inflammation and that CD40/CD40L autocrine or paracrine pathways may mediate this role.

HIV infection of fetal human astrocytes: the potential role of a receptor-mediated endocytic pathway.

HIV infects microglia and astrocytes both in vivo and in vitro. Although there is a significant amount of information about microglial infection, data regarding astrocytes are more limited. For example, little is known about the initial membrane events occurring between HIV and astrocytes. Also, the mechanism by which HIV enters these cells remains to be determined. To address these questions, we exposed human astrocyte cultures to either HIV or to the HIV glycoprotein gp120. The cultures were analyzed for viral infection and gp120 binding to cultured cells by light and electron microscopy (EM) with and without immunocytochemistry, respectively; ligand-receptor biochemistry; and, Western, Northern and Southern blot analyses. The results of these studies showed that HIV binds to astrocytes via gp120 and a cell surface molecule weighing approximately 65 kDa that is neither CD4 nor galactocerebroside. Furthermore, binding of gp120 to astrocytes was concentration dependent and displayed a curve consistent with ligand-receptor binding. Additionally, radiolabeled gp120 binding was displaced by unlabeled gp120 but not by deglycosylated gp120, suggesting that the binding was specific. By EM, HIV virions were seen in clathrin-coated pits and in cytoplasmic vacuoles. This suggests linkage, in astrocytes, between a plasma membrane-associated protein that can act as a receptor for HIV and an endosomal pathway.

HIV infection of human fetal neural cells is mediated by gp120 binding to a cell membrane-associated molecule that is not CD4 nor galactocerebroside.

HIV infection of central nervous system (CNS) tissue is a common finding in both adult and pediatric AIDS. Because most children are believed to be infected perinatally, we have developed a model of HIV CNS infection that utilizes explant organotypic cultures of human fetal CNS tissue. Using this model we previously reported that both lymphocytotropic and monocytotropic HIV isolates infect microglia and astrocytes. However, the mechanism by which HIV infects these cells remains to be elucidated. We have observed that neural cell infection in these cultures may be the result of receptor-mediated endocytosis. In order to confirm this observation and to determine the ligand responsible for this process, organotypic cultures were exposed to untreated HIV, HIV pretreated with soluble CD4 (sCD4) or, as a control, heat-inactivated HIV. To address the question of a putative receptor for HIV infection, CNS cultures were either untreated or pretreated with gp120 or with the deglycosylated form of this protein. Other cultures were treated with antibodies to CD4 (anti-T4A) or to galactocerebroside (GC). Results demonstrate that pretreatment of either HIV with sCD4 or CNS cultures with gp120 significantly inhibits HIV infection. The inhibition of infection was demonstrated by a reduction in the number of cells positive for HIV proteins and by decreases in HIV proviral DNA and p24 production. Pretreatment of CNS cultures with deglycosylated gp120, anti-T4A or anti-GC antibodies did not inhibit HIV infection. These data suggest that HIV gp120 is needed for binding to a surface molecule on CNS cells that is not CD4 nor GC and that this molecule may function as a receptor and lead to infection of neural cells.