HMG-CoA reductase inhibitors (statins) in the treatment of Alzheimer's disease and why it would be ill-advise to use one that crosses the blood-brain barrier.

Increased circulating cholesterol has been long linked to an increased risk of coronary artery disease (CAD), and is now linked to an increased risk of developing Alzheimer s disease (AD). We first showed the neuropathologic link between CAD and AD as increased incidence of cerebral senile plaques in both disorders. We then showed that AD-like neuropathology occurred in the brains of cholesterol-fed rabbits; including increased -amyloid (Ab). Currently there are a number of transgenic mouse models of AD that exhibit enhanced Ab pathology if cholesterol diet is administered. Culture studies clearly show that excess cholesterol enhances beta-metabolism of amyloid precursor protein (APP) and production of -amyloidogenic peptides, and that sufficiently reducing cholesterol levels by inhibition of synthesis completely inhibits all beta-metabolism of APP. Our finding that the elevated levels of Ab in rabbits fed cholesterol diet could be cleared from the brain by resuming a control diet prompted the hypothesis that lowering cholesterol levels in the blood of AD patients may be of some clinical benefit. Pilot data suggests that therapeutically lowering circulating cholesterol may attenuate Ab production in the cholesterol-fed rabbit brain, may stabilize cognitive performance in mildly impaired AD patients, and may reduce the risk of developing AD. Accordingly, we have initiated a double-blind treatment trial evaluating Atorvastatin Na+ among 120 mild-to-moderately impaired AD subjects randomized to one of two groups receiving placebo or active drug once a day. Atorvastatin is one of a general class of HMG-CoA reductase inhibitor drugs called statins that lower cholesterol by inhibition of synthesis. We chose to use Atorvastatin in this AD Treatment Trial because it does not cross the blood-brain-barrier, and believe it would be ill-advised to use a statin that does. This position stems from the observations that excess cholesterol inhibits cholesterol synthesis and increases Ab production, that Ab kills cells in part by inhibiting cholesterol synthesis, and that statins acting at the neuronal level could further exacerbate degeneration in AD by further inhibition of necessary cholesterol synthesis.

Novel inhibitors of carboxypeptidase G2 (CPG2): potential use in antibody-directed enzyme prodrug therapy.

The design and synthesis of potent thiocarbamate inhibitors for carboxypeptidase G2 are described. The best thiocarbamate inhibitor N-(p-methoxybenzenethiocarbonyl)amino-L-glutamic acid 6d, chosen for preliminary investigations of in vitro antibody-directed enzyme prodrug therapy (ADEPT), abrogated the cytotoxicity of a combination of A5B7-carboxypeptidase G2 conjugate and prodrug PGP (N-p-{N,N-bis (2-chloroethyl)amino}phenoxycarbonyl-L-glutamate) toward LS174T cells. This is the first report of a small-molecule enzyme inhibitor proposed for use in conjunction with the ADEPT approach.

A paracrine role for myoepithelial cell-derived FGF2 in the normal human breast.

We have studied separated normal human breast epithelial and myoepithelial cells for the presence of basic fibroblast growth factor (FGF2) and its receptors, both low (heparan sulfate proteoglycans) and high affinity (FGFR1), and for the effects of FGF2 on the proliferation of both cell types. Our results indicate that these cells differ markedly in their synthesis and response to FGF2. We found, using PCR of purified cell populations, mRNA for FGF2 only in the myoepithelial cells, whereas immunostaining and Western blotting results demonstrated the presence of FGF2 protein in both epithelial and myoepithelial cells. FGF2 had no effect on the proliferation of myoepithelial cells, but it did maintain the survival of the separated epithelial cells in low serum and stimulate their growth in 5% and 10% FCS. Immunostainable FGFR1 was present in epithelial cells and, to a lesser extent, in myoepithelial cells. Low-affinity binding sites for FGF2 were synthesized by epithelial and myoepithelial cells, but myoepithelial cells possessed a greater proportion of higher-affinity heparan sulfate proteoglycans. These results indicate that myoepithelial cell-derived FGF2 may be an important paracrine factor controlling epithelial cell survival and growth in the normal human breast.

Separated human breast epithelial and myoepithelial cells have different growth factor requirements in vitro but can reconstitute normal breast lobuloalveolar structure.

In order to investigate the specific factors controlling the growth of normal breast cell types, purified populations of human breast epithelial and myoepithelial cells from reduction mammoplasties were grown in primary culture in three defined media and their response to foetal calf serum (FCS), epidermal growth factor (EGF) and basic fibroblast growth factor (FGF2) measured using MTT growth assays. Epithelial and myoepithelial cells differed markedly in their growth requirements. Whereas epithelial cell survival was dependent on the presence of FCS, myoepithelial cell growth was dramatically inhibited by serum. EGF and FGF2 were mitogenic for epithelial cells but not myoepithelial cells, the addition of insulin being the only essential supplement required for myoepithelial cell growth. Heparin inhibited FGF2-stimulated epithelial cell growth but also basal myoepithelial cell proliferation and this inhibition could be overcome by the addition of EGF. Neutralizing antibodies to EGF also inhibited basal myoepithelial cell growth. This suggests the possibility of an autocrine role for a heparin-binding member of the EGF family in the growth of myoepithelial cells. Purified cells combined to form lobuloalveolar structures when incubated in a reconstituted basement membrane matrix (Matrigel) in the presence of EGF and FGF2.

The location of acidic fibroblast growth factor in the breast is dependent on the activity of proteases present in breast cancer tissue.

Acidic fibroblast growth factor (FGF1) and two of its receptors, FGFR1 and FGFR4, were localized in cryostat sections of normal, benign and malignant human breast tissue by immunohistochemistry. Without pretreatment, FGF1 staining was mainly seen in normal epithelial cells. However, polymerase chain reaction (PCR) analysis and immunoblotting of isolated normal epithelial and myoepithelial cells showed FGF1 mRNA and protein to be present in both cell types. Following incubation of frozen sections at 37 degrees C in phosphate-buffered saline, FGF1 staining was also revealed in myoepithelial cells and basement membrane adjacent to carcinoma cells. Treatment with protease inhibitors demonstrated that this effect was due to the activity of an endogenous protease. In contrast, FGF1 staining was found to be associated with the stroma adjacent to malignant cells only in the presence of protease inhibitors. FGFR1 and FGFR4 immunostaining was localized to both normal and malignant epithelial cells and to a lesser extent to myoepithelial cells. There was no difference in the staining intensity for the FGF receptors between normal and cancer samples. The change in location of FGF1 between normal and malignant tissues and the sensitivity of stored FGF1 to the action of endogenous proteases raises the possibility of both autocrine and paracrine roles for FGF1 in the normal and malignant human breast.

Isolation of pure populations of epithelial and myoepithelial cells from the normal human mammary gland using immunomagnetic separation with Dynabeads.

Monoclonal antibodies to epithelial membrane antigen and common acute lymphoblastic leukemia antigen were bound to second antibody-coated magnetic microspheres. These specific antibody/bead complexes were then used to directly isolate purified epithelial and myoepithelial cells from normal breast organoid cell preparations by magnetic separation. Near homogeneous cell populations were selected with yields of 10-20 x 10(6) epithelial and myoepithelial cells per organoid preparation. Repeated purification steps allowed almost complete depletion of myoepithelial cells for RNA studies. Tissue culture of separated cell populations in appropriate defined media further ensured purity of cell type and was unimpeded by Dynabead attachment.

A multi-center evaluation of a new immunoenzymometric assay for human choriogonadotropin.

A new, rapid, monoclonal immunoenzymometric assay for human choriogonadotropin (HCG), the Enzymun-Test HCG (Boehringer Mannheim GmbH), was evaluated by 15 laboratories in 10 European countries. Inter-assay percent CVs ranged from 5.4 to 40.5, 2.4 to 15.4, and 2.3 to 17.1 for low, medium, and high dose sera, respectively. Mean assay sensitivity was 0.34 mIU/mL HCG. Analytical recovery in serum ranged from 75 to 119%. Serial dilutions of HCG in normal human serum and in assay zero standard were found to be linear. Cross-reactivity with lutropin, follitropin, and thyrotropin was negligible. Serum reference values were established for normals and all stages of pregnancy. Comparative studies with nine other HCG immunoassays were carried out using a range of normal, pregnancy, and tumour sera. We conclude that this is an acceptable assay for monitoring HCG-producing tumours and pregnancy.

Mianserin in the treatment of depressive illness: A comparison with amitriptyline.

In a double-blind comparative trial 71 depressive in-patients were treated with either mianserin or amitriptyline for 3 weeks.Mianserin was found to be as effective as amitriptyline. No significant difference in side-effects was found between the 2 drugs.

Hypertrophy or dilatation? A vectorial analysis of echocardiographically determined left ventricular enlargement.

Echocardiograms (ECHO) and Frank vectorcardiograms (VCGs) were obtained in three groups of patients: Group I (n = 16), concentric left ventricular hypertrophy (LVH) with increased interventricular septal (IVS) and left ventricular posterior wall (LVPW) thickness in the presence of a normal left ventricular internal dimension (LVID); Group II (n = 17), left ventricular dilatation (LVD) with an enlarged LVID, normal IVS and LVPW thickness, and Group III (n = 22), no catheterization evidence of heart disease with normal IVS, LVPW and LVID. VCGs were analyzed with respect to magnitude of the QRS maximal deflection vector (MDV) and +/- 10 msec QRS vectors, horizontal plane (HP) maximal posterior force, time of HP MDV inscription, distal and proximal HP loop areas and HP loop configuration utlizing criteria of Varriale et al. The results indicate that: 1) HP QRS vector magnitude cannot reliably differentiate concentric LVH from isolated LVD and 2) proximal-distal loop area relationships and pattern of the HP QRS loop, when reviewed together, are superior to other criteria for distinguishing whether ECHO determined LVH or LVD is the primary correlate of an enlarged left ventricle.

Eelectrocardiographic correlates of ultrasonically increased septal, left ventricular posterior wall and left ventricular internal dimensions.

The electrocardiograms (ECG) of 64 subjects who exhibited an echocardiographically demonstrable increase in thickness of the interventricular septum and left ventricular posterior wall (Group 1, 22 patients), isolated left ventricular internal dimension (Group 2,26 patients), combined wall thickness and chamber diameter (Group 3, 2 patients), and septal thickness, (Group 4, asymmetric septal hypertrophy, 14 patients) were reviewed in order to determine sensitivity of ECG criteria for the diagnosis of left ventricular hypertrophy (LVH) proposed in 1949 by Sokolow and Lyon (13), in 1968 by Romhilt and Estes (14), and in 1973 the New York Heart Association (15). Relative sensitivity of the three methods was as follows: Total group, NYHA (77%) greater than Sokolow and Lyon (67%) greater than Romhilt and Estes (58%); Group 1, NYHA (91%) greater than Sokolow and Lyon (73%) greater than Romhilt and Estes (54%); Group 2, NYHA and Sokolow and Lyon (65%) greater than Romhilt and Estes (61%); Group 4, NYHA (79%) greater than Sokolow and Lyon (64%) greater than Romhilt and Estes (57%). We conclude that 1)ECG criteria of the NYHA for the diagnosis of LVH correlate best with an increase of ultrasonically determined septal, left ventricular posterior wall or left ventricular internal dimensions when compared with voltage criteria of Sokolow and Lyon and the point score system of Romhilt and Estes; and 2) isolated increase of left ventricular internal dimension, in the absence of thickened septum or posterior left ventricular wall, frequently results in ECG criteria compatible with the diagnosis of LVH.

The echocardiographic correlates of left ventricular hypertrophy diagnosed by electrocardiography.

Echocardiography was performed in 28 consecutive patients who manifested accepted criteria for left ventricular hypertrophy on their electrocardiograms. Four groups of patients were identified: Group 1, nineteen (68%) who had an increase in both interventricular septal and left ventricular posterior wall thickness; Group 2, three patients (11%) with isolated enlargement of the left ventricular internal dimension; Group 3, two subjects (7%) with increased septal thickness, left ventricular posterior wall thickness and left ventricular internal dimension and Group 4, four patients (14%) with normal echocardiographic measurements. It is concluded that increases in both septal and left ventricular wall thickness are the primary echocardiographic correlates of left ventricular hypertrophy as diagnosed on the electrocardiogram.