Sensitive detection of hydrophobic antigens using a novel lipid-aggregate based ELISA

Antibodies against hydrophobic antigens are common in several autoimmune diseases. However, detection of such antibodies by standard immune-assays, such as ELISA, is problematic, in part because of the problems with coating hydrophobic molecules onto polystyrene multi-well plates. We describe a novel method of stably associating hydrophobic antigens to ELISA plates. By mixing the antigen with a hydrophobic molecule containing a hydrophilic anchor, we generate mixed lipid aggregates that can attach to ELISA plates, and are resistant to detergent wash. Using the ganglioside GM-1 and phosphatidylethanolamine conjugated to the hapten DNP (dinitrophenyl) as model antigens, we show that hydrophobic antigens incorporated into mixed lipid aggregates expose their antigenic determinants in a correct configuration. The detection limit of both GM-1 and DNP-PE was considerably improved compared to when these antigens were coated on ELISA plates using organic solvents. Furthermore GM-1 incorporated into mixed lipid aggregates can be detected by specific antibodies in patient serum. The method of incorporating hydrophobic antigens into mixed lipid aggregates for stable association to ELISA plates can presumably be applied to a vast array of hydrophobic antigens, and may well be developed into a large scale screening system for serum reactivity towards different hydrophobic antigens.     

Two populations of ovine bone marrow-derived dendritic cells can be generated with recombinant GM-CSF and separated on CD11b expression

Whereas studies on dendritic cells in rodents rely largely on bone marrow-derived dendritic cells (BM-DCs), no data are available about BM-DCs in sheep, a species that is largely used for immunology and transplantation studies. We have developed a culture protocol to produce ovine BM-DCs, using 6x(His)-tagged recombinant GM-CSF which was purified from baculovirus-infected insect cells. When ovine bone marrow progenitors were cultured in the presence of recombinant GM-CSF, large numbers of CD11c-positive cells were generated after 6-7 days. The phenotypic appearance of BM-DCs was assessed by flow cytometry and electron microscopy. Two DC subsets were identified that expressed different levels of MHC class II molecules, differed in receptor-mediated endocytosis, and could be separated on CD11b expression. When separated cells were incubated with microbial products, they react differently to those that are considered the TLR2 and TLR4 agonists in other species. Indeed, although CD11b^int/hi cells were partially resistant to maturation induced by lipoteichoic acid or lipopolysaccharide, MHC class II upregulation was observed on CD11b^dull cells. Moreover, these cells had strong stimulatory capacity for CD4 T cells when assayed in allogeneic reactions. This protocol will help analyzing ovine DC interactions with pathogens, and enables future studies on the development of vaccines.     

Human papillomavirus 8 E6 disrupts terminal skin differentiation and prevents pro-Caspase-14 cleavage

Expression of the betapapillomavirus (betaPV) E6/E7 genes has been shown to impair both keratinocyte differentiation and apoptosis. Especially late-terminal keratinocyte differentiation shares certain aspects with apoptosis, such as fragmentation of DNA and activation of caspases. Here we investigated the disruption of keratinocyte differentiation in organotypic skin (raft) cultures of primary (PHK) and immortalized (N/TERT) human keratinocytes, in particular by human papillomavirus (HPV)8.Immunohistochemical analysis of HPV5 and HPV8 E6/E7-expressing PHK revealed thickening of the rafts and complete absence of stratum corneum formation, even after 18 days of culture. This phenotype was confirmed in N/TERT raft cultures. When expressed separately, the aberrant morphology was observed only in rafts expressing E6, not E7. Immunofluorescence analysis of HPV8 E6 PHK rafts showed an increase in number and size of Filaggrin- and Caspase-14-positive cells in the granular layer. In raft lysates analyzed by western-blot, the presence of pro-Caspase-14 in the differentiated keratinocytes was confirmed, but in the HPV8 E6 rafts none of the Caspase-14 subunits were detected.In conclusion, in the raft system, HPV8 E6 prevented late-terminal keratinocyte differentiation resulting in an accumulation of Filaggrin and pro-Caspase-14-positive cells in the absence of stratification. This differentiation arrest was accompanied by the failure to express Caspase-14 subunits, suggesting absence of Caspase-14 activation and probable abrogation of Filaggrin maturation in HPV8 E6-expressing keratinocytes.     

Divergent effects of lithium and sodium valproate on brain-derived neurotrophic factor (BDNF) production in human astrocytoma cells at therapeutic concentrations

Mood stabilizers such as lithium (Li) or valproic acid (VPA) are used in the therapy of bipolar disorders, but the mechanisms by which these medicines work is unclear. Recently, neuroprotection has attracted attention as a potential action for VPA and Li. The close spatial relationship of the pre- and post-synapse with an astrocyte process within a 'tripartite synapse' suggests that mood stabilizer actions on astrocytes may be important. Therefore, we examined the effect of Li and VPA, at therapeutic concentrations, on brain-derived neurotrophic factor (BDNF) production in cultured human astrocytoma cells over an extended period of exposure. Released (extracellular) and intracellular BDNF was measured with sandwich-ELISA. Intracellular BDNF mRNA was also quantified using RT-PCR. VPA treatment potentiated the level of extracellular BDNF, whereas Li reduced it. Furthermore, VPA caused increased intracellular levels of BDNF protein and mRNA, while exposure to Li led to no significant differences compared to control cells. We suggest the possibility that VPA and Li have divergent effects on astrocyte BDNF production. Mood stabilizers play an essential role in regulating BDNF not only in neurons, but also in astrocytes. These findings could form the basis of a new astrocyte-targeted approach towards developing effective medications to treat bipolar disorders.     

Trypanosoma brucei brucei: Patterns of glycolysis at 37°C in vitro

In the absence of mammalian cells, freshly isolated monomorphic bloodstream forms of Trypanosoma brucei brucei maintain a constant and high level of aerobic glycolysis in vitro for at least 4 h at 37°C when suspended in RPMI medium 1640 containing 20% heat-inactivated and dialyzed fetal calf serum and 25 mM Hepes at an initial pH of 8. In the absence of nutrients other than glucose, salts and protein, some cell death and a decrease in the rate of glycolysis are observed. In the absence of protein, extensive cell death and a decrease in the rate of glycolysis are seen. These observations may be useful in the design of short-term in vitro metabolic studies with T. b. brucei.     

Severe hypertriglyceridemia is primarily polygenic

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Complexity and content of the DNA and RNA in Trypanosoma cruzi

The content and sequence complexity of the nuclear DNA and messenger RNA for epimastigotes of Trypanosoma cruzi were determined. From analysis of nuclear DNA reassociation studies and microspectrofluorometric measurements of laser induced fluorescence of cellular DNA, T. cruzi is found to be a diploid organism with a nuclear DNA content of 2.5 x 10(8) nucleotide pairs (2.8 x 10(-13) g) and a kinetoplast DNA content of 4.9 x 10(7) nucleotide pairs (5.4 x 10(-14) g). Reassociation kinetics of nuclear DNA of average length 0.4 kb reveals three kinetic components: a moderately repetitive component with a reiteration frequency of 5.1 x 10(3) present in 9% of the fragments, a lowly repetitive component with a reiteration frequency of 32 present in 51% of the fragments, and a single-copy component present in 23% of the fragments. By saturation hybridization of total polysomal RNA to 3H-labeled single-copy DNA, it was determined that 68% of the single-copy DNA was represented in the epimastigote polysomal RNA. This corresponds to ca. 12 000 different mRNA species. Of these, ca. 9000 are present as poly(A)+-RNA, while the remaining 3000 appear not to be polyadenylated. Kinetic analysis of the poly(A)+-RNA population indicates it is composed of at least three classes of RNA's of different abundancy levels: two sequences which occur ca. 3000 per cell, ca. 750 sequences which occur about 20 times per cell, and ca. 15 500 sequences which occur 1-2 times per cell.     

Use of a self-generating percoll gradient and single cell cytotoxicity assay to identify tumor-lytic properties of inflammatory neutrophils

Within a murine model of regional immunotherapy, the cytolytic potential of peritoneal neutrophils could not be confirmed or quantified using routine techniques of cell separation and chromium release assays. We, therefore, developed procedures for the enrichment of neutrophils and estimation of the frequency of killer cells. Peritoneal exudate cells from mice injected with Corynebacterium parvum were fractionated on a self-generating Percoll gradient to enrich for neutrophils and deplete macrophages. A significant enrichment of neutrophils (greater than 90%) was obtained in a band corresponding to a density of 1.088 with a recovery of 35-50% of input. Neutrophil-enriched cell populations were then mixed with tumor cells to examine neutrophil-target interactions at the single cell level. Conjugates of neutrophils and tumor targets were obtained and the majority were lytic. With the aid of trypan blue staining and safranin counterstaining, it was possible to distinguish effector cells from targets and neutrophils from other host cells. The frequency of conjugates was dependent upon the effector to target cell ratio and was not affected by changes in temperature (range 4-30 degrees C). The post-binding lytic events were initiated rapidly after conjugation and tumor lysis was completed within 30 min. The lytic events occurred optimally between 25 degrees and 37 degrees C. The present studies support the role of neutrophils in tumor lysis following administration of an immunoadjuvant. The techniques described are important to further study the role of neutrophils in disease states as well as the underlying mechanisms of neutrophil-mediated tumor cytotoxicity.     

Production and Characterization of Monoclonal Antibodies against Bovine Luteinizing Hormone

Five mouse hybridoma cell lines producing monoclonal antibody against bovine luteinizing hormone (LH) have been established and the respective antibodies characterized by radioimmunoassay, immunofluorescence and immunoelectrophoresis. All antibodies belong to the IgG class and bind to staphylococcus protein A. Intraspecies cross-reactivity studies revealed no reaction with bovine follicle stimulating hormone (FSH). However, all antibodies showed partial cross-reaction with bovine thyroid stimulating hormone (TSH) suggesting a close conformational similarity between bovine LH and TSH. Studies on interspecies cross-reactivity (rat and human) showed that three of these five antibodies strongly react with rat LH but not at all with either rat FSH or rat TSH thus representing monospecific reagents for investigations concerning LH in this species. One of these three antibodies also strongly binds to human LH and to the same extent to human chorionic gonadotropin (CG) but not to human FSH or TSH. It was concluded that at least three different epitopes on the bovine LH molecule are recognized and that they are located on the β-chain of the hormone.