Induction of vitellogenin in primary monolayer cultures of cockerel hepatocytes.

A primary monolayer culture system from cockerel hepatocytes was established. The cultures synthesize and secrete proteins that comigrate with authentic serum proteins on polyacrylamide gels and are found in the same relative abundance. Addition of estradiol increased the synthesis of apoprotein B, found in very low density lipoprotein, under all culture conditions. Vitellogenin synthesis could not be induced directly by estradiol. However, when serum was obtained from cockerels injected with estradiol 4 days before blood collection and included in the culture medium, the cultures secreted a protein identified immunologically as vitellogenin by affinity chromatography. Furthermore, addition of growth hormone or prolactin to cultured cockerel hepatocyte monolayers resulted in the synthesis and secretion of a polypeptide that comigrates with authentic vitellogenin on polyacrylamide gels.     

Human monoclonal antibodies specific to hepatitis B virus generated in a human/mouse radiation chimera: the Trimera system.

An approach to develop fully human monoclonal antibodies in a human/mouse radiation chimera, the Trimera system, is described. In this system, functional human lymphocytes are engrafted in normal strains of mice which are rendered immuno-incompetent by lethal total body irradiation followed by radioprotection with severe combined immunodeficient (SCID) mouse bone marrow. Following transplantation, human lymphocytes colonize murine lymphatic organs and secrete human immunoglobulins. We have established this system as a tool to develop fully human monoclonal antibodies, and applied it for the generation of monoclonal antibodies specific for hepatitis B virus surface antigen. A strong memory response to hepatitis B surface antigen was elicited in Trimera engrafted with lymphocytes from human donors positive for antibodies to hepatitis B surface antigen. The human specific antibody fraction in the Trimera was 10(2)-10(3)-fold higher as compared with that found in the donors. Spleens were harvested from Trimera mice showing high specific-antibody titres and cells were fused to a human-mouse heteromyeloma fusion partner. Several stable hybridoma clones were isolated and characterized. These hybridomas produce high-affinity, IgG, anti-hepatitis B surface antigen antibodies demonstrating the potential of the Trimera system for generating fully human monoclonal antibodies. The biological function and the neutralizing activity of these antibodies are currently being tested.     

Everyday memory and laboratory memory tests: general function predictors in schizophrenia and remitted depression

This study was designed to compare neuropsychological memory measures ("laboratory memory tests") and an everyday memory measure in patients with schizophrenia, patients with major depression, and normal controls. Patients with schizophrenia ( N= 68) treated with typical (N = 33) or atypical ( N= 35) neuroleptics, patients with major depression (N = 30), and a control group (N = 36) were evaluated with clinical measures (Positive and Negative Syndrome Scale and Hamilton Depression Rating Scale), laboratory memory tests (Digit-Span, Paired-Associates, Rey Complex Figure Test, and Digit-Symbol), everyday memory test (RMBT), and the Global Assessment of Functioning (GAF). The schizophrenia group had a significantly lower level of performance in everyday memory and general function but not in laboratory memory tests. Verbal and everyday memory measures were correlated with general function. The diagnosis rather than current symptoms (in remission) contributed to test variance and was correlated with performance on everyday memory and general function tests. Everyday memory and verbal memory were good predictors of general function in schizophrenic and depressive patients in the remitted phase. However, the advantages of these tests over laboratory memory tests need to be further investigated in larger and more representative samples.     

Spongiform encephalopathy in transgenic mice expressing a point mutation in the β2-α2 loop of the prion protein

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases attributed to misfolding of the cellular prion protein, PrP(C), into a β-sheet-rich, aggregated isoform, PrP(Sc). We previously found that expression of mouse PrP with the two amino acid substitutions S170N and N174T, which result in high structural order of the β2-α2 loop in the NMR structure at pH 4.5 and 20°C, caused transmissible de novo prion disease in transgenic mice. Here we report that expression of mouse PrP with the single-residue substitution D167S, which also results in a structurally well ordered β2-α2 loop at 20°C, elicits spontaneous PrP aggregation in vivo. Transgenic mice expressing PrP(D167S) developed a progressive encephalopathy characterized by abundant PrP plaque formation, spongiform change, and gliosis. These results add to the evidence that the β2-α2 loop has an important role in intermolecular interactions, including that it may be a key determinant of prion protein aggregation.