Adjuvant effects of various lipopeptides and interferon-gamma on the humoral immune response of chickens.

The adjuvant effects of various lipopeptides and recombinant chicken interferon gamma (IFN-gamma) on the humoral immune response of laying hens was investigated in four immunization studies. We used the lipopeptide Pam3Cys-Ser-(Lys)4 (PCSL), the conjugate P-Th1 consisting of the lipopeptide P3CS and the T-helper epitope Th1 (FISEAIIHVLHSRHPG), and the conjugate P-Th2 of the lipopeptide P3CSS and the T-helper epitope Th2, which corresponds to the peptide EWEFVNTPPLV, as adjuvants. Human serum albumin (HSA), recombinant bovine somatotropin (RBST), and human immunoglobulin G (IgG) served as antigens in the different experiments. All tested adjuvants enhanced the humoral immune response with various intensities. Chickens showed high antibody titers after the immunization with HSA even without adjuvant, but the adjuvant effects of PCSL and the combination of PCSL and recombinant chicken interferon-gamma (IFN-gamma) were much more pronounced using the antigens RBST and IgG. Especially after the third immunization, higher titers of antibodies were induced by the coadministration of P-Th1 and, to a greater extent, by the combination of PCSL and P-Th1 compared with the use of PCSL. Also, chickens that had received PCSL and P-Th2 showed the highest immune response, even after the second booster. The average concentrations of chicken immunoglobulin Y were significantly higher in 5-mo-old chickens (9.4 mg/mL serum and 10.1 mg/mL egg yolk) compared with 9-mo-old chickens (5.9 mg/mL serum and 5.1 mg/mL egg yolk). The specific serum antibody response was higher in the older chickens than in the younger chickens. Because chicken antibodies are likely to be used increasingly for diagnostic and therapy in the future, lipopeptides and recombinant chicken IFN-gamma may find many applications as adjuvants, thus contributing to the welfare of experimental animals.

Evaluation of various immunisation procedures in laying hens to induce high amounts of specific egg yolk antibodies.

The present study, involving 972 laying hens divided into 162 groups (n = 6), was aimed at the development of an immunisation protocol for laying hens to produce specific egg yolk antibodies. Recombinant bovine somatotropin (rbst), Escherichia coli pilus antigen K88 (K88), human serum immunoglobulin G (IgG), and low density lipoprotein (LDL) were used as antigens, each at four different doses (rbst, K88, LDL : 1µg, 10µg, 100µg, 1mg ; IgG : 0.5µg, 5µg, 50µg, 0.5mg). Three subcutaneous or intramuscular immunisations were performed at intervals of four weeks. The adjuvant used was either the lipopeptide Pam3Cys-Ser-(Lys)4 (PCSL) or Freund ` s incomplete adjuvant (FIA), in two different doses (PCSL: 0.1 and 0.25mg ; FIA: 0.1 and 0.25ml). In the four antigen control groups, hens were immunised without any adjuvant. In two negative control groups, only physiological saline was injected. The mean egg weight and egg yield were not influenced by the immunisation procedures. An antigen dose of 10-100g per injection was sufficient to induce high specific antibody titres in the egg yolk. The adjuvant efficacy of PCSL and FIA was proved to be the same (p < 0.05 versus antigen control). With PCSL as adjuvant, some groups showed a tendency to produce even higher specific antibody titres than did FIA groups. A second booster often caused a further significant increase in the amounts of specific antibodies, especially with PCSL. Subcutaneous administration of the antigen together with 250µg PCSL, resulted in a significantly higher immune response than when FIA was used.

Proline-directed kinase systems in Alzheimer's disease pathology.

Immunohistochemical analysis was used to assess the distribution of the proline-directed kinase, cdc2, in Alzheimer's disease (AD) pathology. A robust signal was most prominent in the neurofibrillary tangle (NFT) of affected neurons that also contained abnormally phosphorylated tau protein. Biochemical analysis identified a pool of cdc2 in bovine brain microtubules that contain normal tau. These results strongly support the hypothesis that cdc2 is involved in the abnormal phosphorylation of tau in AD pathology and they raise important issues regarding regulation of tau phosphorylation in normal and diseased neurons.

Demonstration of cdc2 kinase in mature neuronal nuclei.

Immunocytochemistry has been used to assess the distribution of the mitosis-associated protein kinase, cdc2, in mature neurons of rat brain. A robust signal is apparent in most, but not all neurons in cerebellum, cortex and hippocampus. The signal is concentrated in neuronal nuclei with lower levels of immunoreactivity in neuronal cytoplasm. High resolution analysis indicates that the amount of cdc2 is uneven, suggesting that regulated amounts of enzyme may reflect different functional status of the neurons. In many cases, the kinase is concentrated at the nuclear periphery, implying that it may be particularly involved in phosphorylating substrates in this subnuclear compartment. These results indicate additional roles for cdc2 in terminally differentiated neurons which have undergone final mitosis.

Tyrosine phosphorylation systems in Alzheimer's disease pathology.

Immunohistochemical techniques have been used to assess the distribution of phosphotyrosine-containing compartments in Alzheimer's disease (AD) pathology. Elevated levels of phosphotyrosine are apparent in the somatodendritic compartment of tangle-bearing neurons, in the neuritic plaque (NP) and in dystrophic neurites coursing through the neuropil. The only neuronal staining observed in non-AD tissue is in developing neurites. This suggests that some neuronal elements involved in AD pathology may be recapitulating a developmental profile or, alternately, that elevated phosphotyrosine levels may reflect a role for tyrosine kinase/phosphatase systems in the degeneration process directly. Cells in the neuritic plaque which strongly resemble microglia also contain elevated levels of phosphotyrosine compared to non-activated ramified microglia in the same tissue section. Thus, tyrosine phosphorylation systems may be involved in the response of microglia to degeneration in AD pathology. Implications of these results are discussed.

Immunohistochemical evidence for reorganization of tau in the plaques and tangles in Alzheimer's disease.

Cytochemical and biochemical techniques have been used to assess the relationship of epitopes on the microtubule-associated protein, tau, to the cytoskeletal pathology of Alzheimer's disease. The main probes were Tau-1 and Alz-50, two monoclonal antibodies which recognize tau and a potentially related 68 kDa protein. Sequential treatment of tissue slices with combinations of the antibodies showed that each blocked the binding of the other to neurofibrillary tangles and neuritic plaques but not to normal axons. Western blot analysis of tau proteins isolated from Alzheimer's disease brains did not reveal such blocking patterns. The issue of steric hindrance affecting antibody binding in tissue sections was addressed by using Alz-50 in combination with Tau-2, another monoclonal antibody recognizing tau on blots and in Alzheimer's disease pathology. Neither antibody blocked the binding of the other to neurofibrillary tangles and neuritic plaques. These data suggest that the Alz-50 and Tau-1 epitopes are selectively organized in the tangles and plaques to be in close proximity which supports the hypothesis that in Alzheimer's disease pathology, tau is modified.

Age and cell type influences on nerve--non-nerve contact interactions.

Eight-day ciliary ganglion neurons respond in a significantly different fashion to contact with dorsal root ganglia non-neurons than do 8-day dorsal root neurons. The ciliary neuron-dorsal root non-neuron interactions result in contact inhibition of both cells, whereas in the dorsal root neuron-dorsal root non-neuron case no such inhibition is observed. In addition, contact of 8- and 14-day ciliary neurons with heart fibroblasts results in inhibition of locomotion. However, the response of the fibroblast to contact with these neurons of different ages varies in a predictable fashion.

Differential response to contact during embryonic nerve-nonnerve cell interactions.

The outcome of contact interactions involving neurons and nonneurons varies depending on the cell types involved. When neuronal growth cones from either ciliary (motor) or dorsal root (sensory) ganglia directly contact the lamellipodium of an embryonic heart fibroblast, both neurite elongation and fibroblast locomotion are inhibited. This occurs in spite of the fact that cell-surface activity in both cells continues unabated. Such contact inhibition is not observed when homologous ganglionic nonneurons are involved in the interaction. In fact, these cells become intimately associated with growth cones and/or neuritic shafts as a result of the contact. The detailed nature of the response to contact exhibited by nerves and nonnerves varies not only with cell type but also with the portion of the cell involved in the contact. Growth cone filopodia tend to actively palpate the fibroblast surface, whereas spread regions, termed "veils," form areas of apposition with fibroblast lamellipodia. This latter situation resembles the "typical" contact inhibition of locomotion that occurs following embryonic heart fibroblast-fibroblast interactions. Growth cones also frequently exhibit contact guidance when interacting with nonruffling lateral surfaces of heart fibroblasts.