Comparative Analysis of Different Definitions of Amyloid-ß Positivity to Detect Early Downstream Pathophysiological Alterations in Preclinical Alzheimer.

Amyloid-ß (Aß) positivity is defined using different biomarkers and different criteria. Criteria used in symptomatic patients may conceal meaningful early Aß pathology in preclinical Alzheimer. Therefore, the description of sensitive cutoffs to study the pathophysiological changes in early stages of the Alzheimer's continuum is critical. Here, we compare different Aß classification approaches and we show their performance in detecting pathophysiological changes downstream Aß pathology. We studied 368 cognitively unimpaired individuals of the ALFA+ study, many of whom in the preclinical stage of the Alzheimer's continuum. Participants underwent Aß PET and CSF biomarkers assessment. We classified participants as Aß -positive using five approaches: (1) CSF Aß42 < 1098 pg/ml; (2) CSF Aß42/40 < 0.071; (3) Aß PET Centiloid > 12; (4) Aß PET Centiloid > 30 or (5) Aß PET Positive visual read. We assessed the correlations between Aß biomarkers and compared the prevalence of Aß positivity. We determined which approach significantly detected associations between Aß pathology and tau/neurodegeneration CSF biomarkers. We found that CSF-based approaches result in a higher Aß-positive prevalence than PET-based ones. There was a higher number of discordant participants classified as CSF Aß-positive but PET Aß-negative than CSF Aß-negative but PET Aß-positive. The CSF Aß 42/40 approach allowed optimal detection of significant associations with CSF p-tau and t-tau in the Aß-positive group. Altogether, we highlight the need for sensitive Aß -classifications to study the preclinical Alzheimer's continuum. Approaches that define Aß positivity based on optimal discrimination of symptomatic Alzheimer's disease patients may be suboptimal for the detection of early pathophysiological alterations in preclinical Alzheimer.

Requirement of protein kinase C zeta for stimulation of protein synthesis by insulin.

The ability of insulin to stimulate protein synthesis and cellular growth is mediated through the insulin receptor (IR), which phosphorylates Tyr residues in the insulin receptor substrate-signaling proteins (IRS-1 and IRS-2), Gab-1, and Shc. These phosphorylated substrates directly bind and activate enzymes such as phosphatidylinositol 3'-kinase (PI3K) and the guanine nucleotide exchange factor for p21Ras (GRB-2/SOS), which are in turn required for insulin-stimulated protein synthesis, cell cycle progression, and prevention of apoptosis. We have now shown that one or more members of the atypical protein kinase C group, as exemplified by the zeta isoform (PKC zeta), are downstream of IRS-1 and P13K and mediate the effect of insulin on general protein synthesis. Ectopic expression of constitutively activated PKC zeta eliminates the requirement of IRS-1 for general protein synthesis but not for insulin-stimulated activation of 70-kDa S6 kinase (p70S6K), synthesis of growth-regulated proteins (e.g., c-Myc), or mitogenesis. The fact that PKC zeta stimulates general protein synthesis but not activation of p70S6K indicates that PKC zeta activation does not involve the proto-oncogene Akt, which is also activated by PI3K. Yet insulin is still required for the stimulation of general protein synthesis in the presence of constitutively active PKC zeta and in the absence of IRS-1, suggesting a requirement for the convergence of the IRS-1/PI3K/PKC zeta pathway with one or more additional pathways emanating from the IR, e.g., Shc/SOS/p21Ras/mitogen-activated protein kinase. Thus, PI3K appears to represent a bifurcation in the insulin signaling pathway, one branch leading through PKC zeta to general protein synthesis and one, through Akt and the target of rapamycin (mTOR), to growth-regulated protein synthesis and cell cycle progression.

Influence of dietary fat and indomethacin on the growth of transplantable mammary tumors in rats.

Studies were designed to determine if treatment with indomethacin influenced the growth of a transplantable, metastatic, rat mammary tumor. Female, Wistar-Furth inbred rats were fed either a standard chow diet or a semipurified diet containing 2, 5, 10, or 20% stripped corn oil. Indomethacin was given in drinking water, and rats consumed between 2.5 and 3.0 mg indomethacin/kg body weight/day. Feeding of diets and initiation of treatment with indomethacin were started when rats were weaned (21 days old) and continued until they were killed. Approximately 5 X 10(3) mammary tumor cells (DMBA-4) were injected into the fat pad of the sixth mammary gland which is adjacent to the right inguinal lymph node. Each dietary/treatment group consisted of at least 10 rats. Since indomethacin inhibits prostaglandin synthesis, two other groups of non-tumor-bearing rats were used to determine if dietary fat and treatment with indomethacin either influenced prostaglandin E2 production (in vitro) by mononuclear cells from the spleen or altered serum levels of fatty acids. Results indicated that: (a) the rate of tumor growth in untreated rats was significantly greater when the dietary fat content was either 10 or 20% compared to diets containing either 2 or 5% fat; (b) the tumor growth-promoting effects of 10 and 20% fat diets were completely abrogated in rats treated with indomethacin; (c) treatment with indomethacin also inhibited tumor growth in rats fed diets containing either 2 or 5% fat; (d) synthesis of prostaglandin E2 by mononuclear cells from the spleens of untreated rats increased as the dietary fat content increased; (e) in indomethacin-treated rats, prostaglandin E2 synthesis was inhibited in all dietary groups and was not dependent on dietary fat; and (f) in both untreated and indomethacin-treated rats, the serum concentrations of oleic and linoleic acids were influenced to the same extent by dietary fat.

Inhibition of lymphocyte response by prostaglandin-producing suppressor cells in patients with melanoma.

Peripheral blood mononuclear cells (PBMC) from 11 patients with metastatic melanoma (group II) had a significant decrease in blastogenesis to concanavalin A (Con A) (38.7 +/- 7.7 cpm X 10(3); mean +/- SE) compared to 21 patients who were disease free (70.6 +/- 6.7) or 16 healthy controls (83.6 +/- 10.3). If PBMC from patients were preincubated for 72 hr prior to exposure to mitogen, blastogenesis was restored to normal. In group II patients a similar improvement in reactivity of fresh PBMC occurred with indomethacin addition or following rigorous depletion of adherent monocytes. Supernatants from cells cultured with and without Con A in several group II patients contained very high levels of endogenous PGE2. Patients had a greater percentage of T lymphocytes bearing Fc receptors for ox erythrocytes (T gamma) than controls, which appeared to correlate with the increased sensitivity to suppression by exogenously added PGE2. These data suggest that the decreased blastogenesis in certain melanoma patients is due to an increase in PGE2 production by monocytes, along with an increase in lymphocyte sensitivity to its effects.

The influence of dietary fat and non-specific immunotherapy on carcinogen-induced rat mammary adenocarcinoma.

The incidence of mammary adenocarcinoma in Sprague-Dawley female rats, caused by the carcinogen 7,12-dimethylbenz(alpha)anthracene, was influenced by the level of dietary fat fed after exposure to carcinogen. Carcinogen was given by stomach tube to 50-day-old rats, and tumors were evaluated when rats were 9 months old. Rats on diets containing 20% unsaturated fat had a tumor incidence of 97%, while rats changed to a low-fat diet (2% unsaturated fat) three or four weeks after exposure to the carcinogen had an incidence of 45%. Some rats on each diet were given two treatments with the methanol extraction residue of Bacillus Calmette-Guerin, either three and five weeks after carcinogen or four and six weeks after carcinogen. Tumor incidence in the treated group and the untreated group was the same when rats were maintained on the high-fat diet, but tumors in the treated group were larger and the disease was more severe by histological criteria. These tumors were more anaplastic and many were extensively infiltrated with lymphocytes compared to the untreated group. Tumor incidence was significantly lower in rats changed to the low-fat diet (45%) than in those on the high-fat diet (97%), and tumor incidence was reduced to 20% when rats changed to the low-fat diet were treated with methanol extraction residue. The treated group had less severe disease than the untreated group on the low-fat diet. Only half the tumor-bearing rats in this group had malignant tumors, and none were invasive. Methanol extraction residue protected most rats on the low-fat diet against mammary adenocarcinoma, and reduced the severity of disease in those rats that did develop tumors. Methanol extraction residue treatment provided no protection, and even increased the severity of disease, in rats on the high-fat diet.

Inhibition of lymphocyte function in rats fed higher-fat diets.

Concanavalin A-induced blastogenesis of spleen lymphocytes was significantly inhibited when lymphocytes from rats on a high-polyunsaturated-fat diet were compared to lymphocytes from rats on a low-fat diet. Responsiveness was dependent on source of serum since lymphocytes from rats fed a low-fat diet were suppressed in serum from rats fed a high-polyunsaturated-fat diet. Alternatively, lymphocytes from rats on a high-polyunsaturated-fat diet were more responsive in serum from low-fat-fed rats compared to their response in autologous serum. One of the inhibiting factors in serum was the lipoprotein fraction; however, rats on a high-polyunsaturated-fat diet probably had additional inhibitors in their serum. While tumor incidence was highest in rats with the least responsive lymphocytes was highest in rats with the least responsive lymphocytes and lowest in rats with the most responsive lymphocytes, the significance of the observation is not known.

Correlations between humoral immunity and successful chemotherapy-immunotherapy.

Experiments were designed to evaluate the characteristics of the humoral immune response induced by active immunotherapy, both specific (neuraminidase-treated tumore cells) and nonspecific (Bacillus Calmette-Guérin organisms), in the L1210-C57BL/6 X DBA/2F tumor-host system. Tumor burden was minimized with chemotherapy (1,3-bis-(2-chloroethyl)-1-nitrosourea) prior to immunotherapy. A marked increase in the concentration of serum immunoglobulins (immunoglobulin M, immunoglobulin G, and immunoglobulin G) was observed following successful therapy. The highest concentration of these immunoglobulins was found in mice given both Vibrio cholerae neuraminidase-treated cells and B. Calmette-Guérin after chemotheraphy. Tumor-specific immunoglobulin M and immunoglobulin G, as measured by indirect immunofluorescnece, were detected in the sera during the course of successful therapy. Positive immunofluorescence was not observed with progressive sera. Complement-dependent cytotoxic activity against L1210 cells was first detected 5 days after immunotherapy, and increased for several weeks. A high level of cytotoxic activity correlated with successful therapy, whereas low levels were foun in treated mice with recurring tumors. Serum cytotoxicity was not detected in untreated mice with progressively growing tumor.

Humoral reponse to neuraminidase-treated tumor cells.

L1210 leukemia cells grew progressively and caused tumor deaths in all recipient mice. However, when these cells had been treated with Vibrio cholerae neuraminidase (VCN) prior to injection, tumor deaths did not occur. Both untreated and VCN-treated L1210 cells elicited a humoral response, as manifested by an increasing percent of cells in the spleen and peritoneal cavity, with various types of membrane-associated immunoglobulins. Progressive tumor growth was associated with a large percent of peritoneal exudate (PE) cells bearing membrane-associated IgG, a late increase in the percent of PE cells with IgG, and only a small percent of PE cells with IgM on their surfaces. Conversely, PE cells from mice given VCN-treated L1210 cells were characterized by a small percent with IgG, an early increase in percent of cells with IgG, and a large percent with membrane-associated IgM. An injection of VCN-treated L1210 cells into mice with progressively growing L1210 tumors caused frequent tumor remissions, with a corresponding alteration of the ongoing humoral responses. Both the degree of alteration and the number of cures depended on the tumor burden at the time VCN-treated tumor cells were injected. The humoral response in mice with tumor remission following immunization was comparable to the response detected after an injection of VCN-treated cells only.

Immunotherapy with neuraminidase-treated cells and bacillus Calmette-Guerin.

Experiments were designed to determine the effectiveness of active immunotherapy, both specific (neuraminidase-treated cells) and nonspecific [bacillus Calmette-Guerin (BCG) organisms] in the L1210-BDF1 tumor-host system. Tumor burden was minimized with chemotherapy (1,3-bis-(20chloroethyl)-1-nitrosourea) prior to immunotherapy. The effectiveness of immunotherapy was dependent on the amount of drug used to minimize tumor burden. An interval 36 hours between chemotherapy and immunotherapy produced the maximum number of survivors. A single immunization with 10(4) neuraminidase-treated cells was superior to other single or multiple immunizations. BCG was most effective when mice were given 393 X 10(5) organisms. Beneficial effects of immunotherapy were observed only when immunizations were given by an intraperitoneal route. All mice cured of tumor developed tumor-specific immunity. The highest levels of immunity were observed in mice given both neuraminidase-treated cells and BCG organisms after chemotherapy.